GENERAL MICROBIOLOGY

STRUCTURE OF ARCHAEL, EUBACTERIAL AND EUKARYOTIC MICROBIAL CELLS

Prof. Anjana Sharma Professor

Bacteriology Laboratory Department of Biosciences

Rani Durgavati Vishwavidyalaya Jabalpur - 482001 (MP)

30-Jun-2006 Revised 11-Jan-2007

CONTENTS

Introduction Prokaryotic Cells Eukaryotic cells EndosymbiosisArchaebacterial Cell Archaen Phylogeny Archaebacterial cell walls Archael Cell Membranes Genome OrganizationEubacterial Cell: Structure and Functions Surface Structures-Appendages Prosthecae and Stalks Cell Envelope (cell wall and plasma membrane)Eukaryotic Microbial Cell: General Structure and FunctionSpecialized structures Algae Protozoa Fungi Keywords Archea, extremophiles, thermophiles, methanogens, halophiles, Pseudomurein, Eubacteria, bacteria, eukaryotic cell, microorganisms, organelles, algae, fungi, protozoa

Introduction

Both living and nonliving things are composed of molecules made from elements such as carbon, hydrogen, oxygen, and nitrogen. The organization of these molecules into cells is one feature that distinguishes living things from all other matter. The CELL is the smallest unit that can carry out all the processes of life. It is the basic living unit of organization for all organisms. The simplest forms of life are solitary cells, and in more complex organisms, groups of cells perform specialized functions and are linked by intricate systems of communication. Cells can be divided into two groups based on membrane complexity.

• Prokaryotic Cells • Eukaryotic Cells

Historical background

When life arose on Earth about 4 billion years ago, the first types of cells to evolve were prokaryotic cells. For approximately 2 billion years, prokaryotic-type cells were the only form of life on Earth. They are the oldest known fossils, 3.5 billion years in age, found in Western Australia and South Africa. The nature of these fossils, and the chemical composition of the rocks in which they are found, indicate that lithotrophic and fermentative modes of metabolism were the first to evolve in early prokaryotes. Photosynthesis developed in bacteria at least 3 billion years ago. Anoxygenic photosynthesis (bacterial photosynthesis, which is anaerobic and does not produce O2) preceded oxygenic photosynthesis (plant-type photosynthesis, which yields O2). But oxygenic photosynthesis also arose in prokaryotes, specifically in the cyanobacteria, which existed millions of years before the evolution of plants. Larger, more complicated eukaryotic cells did not appear until much later, between 1.5 and 2 billion years ago. The Universal Tree of Life

On the basis of small subunit ribosomal RNA (ssrRNA) analysis the Woesean tree of life gives rise to three cellular domains of life,- Archae, Bacteria and Eukarya. Bacteria (formerly known as eubacteria) and Archae (formerly called archaebacteria) share the prokaryotic type of cellular configuration, but otherwise are not related to one another any more closely than they are to the eukaryotic domain, the Eukarya. Between the two prokaryotes, Archae are apparently more closely related to Eukarya than are the Bacteria. Eukarya consists of all eukaryotic cell types, including protista, fungi, plants and animals. Prokaryotic Cells

The first evidences of study of these prokaryotic cells date back to the early 17th century. Antony van Leeuwenhoek (1632–1723), a Dutch student of natural history and maker of microscopes has been credited for his pioneering work in this field. His use of lenses in examining cloth as a draper's apprentice probably led to his interest in lens making. He assembled over 247 microscopes, some of which magnified objects 270 times. In the course of his examination of innumerable microorganisms and tissue samples, he gave the first complete descriptions of the bacteria, which he called animalcules. By definition the Bacteria are a group of single-cell microorganisms with prokaryotic cellular configuration. The genetic material (DNA) of prokaryotic cells exists unbound in the cytoplasm of the cells. There is no nuclear membrane, which is the definitive characteristic of eukaryotic cells such as those that make up plants and

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animals. Until now, bacteria are the only known type of prokaryotic cell, and the discipline of biology related to their study is called bacteriology.

Figure 1: Woesean tree of life A typical prokaryotic cell is about the size of a eukaryotic mitochondrion. Since prokaryotes are too small to be seen except with the aid of a microscope, it is usually not appreciated that they are the most abundant form of life on the planet, both in terms of biomass and total numbers of species. For example, in the sea, prokaryotes make up 90 percent of the total combined weight of all organisms. In a single gram of fertile agricultural soil there may be in excess of 109 bacterial cells, outnumbering all eukaryotic cells there by 10,000: 1. About 3,000 distinct species of bacteria and archae are recognized, but this number is probably less than one percent of all the species in nature. These unknown prokaryotes, far in excess of undiscovered or unstudied plants, are a tremendous reserve of genetic material and genetic information in nature that awaits exploitation. Prokaryotes are ubiquitous organisms as they are found in all of the habitats where eukaryotes live, but, as well, in many natural environments considered too extreme or inhospitable for eukaryotic cells. Thus, the outer limits of life on Earth (hottest, coldest, driest, etc.) are usually defined by the existence of prokaryotes. Where eukaryotes and prokaryotes live together, there may be mutualistic associations between the organisms that allow both surviving and flourishing. The organelles of eukaryotes (mitochondria and chloroplasts) are thought to be remnants of Bacteria that invaded, or were captured by, primitive eukaryotes in the evolutionary past. Numerous types of eukaryotic cells that exist today are inhabitated by endosymbiotic prokaryotes. Prokaryotic Characteristics

“Prokaryotic” means “pre-nucleus.” This name reflects the idea that prokaryotes are believed to have been the first cells to evolve. Eukaryotic cells are believed to have evolved from

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prokaryotic ancestors. Prokaryotic cells are much simpler than eukaryotic cells. Microorganisms having prokaryotic cells include the eubacteria (“true bacteria”) and the archaebacteria. Prokaryotic cells are characterized by the following properties.

• Prokaryotic cells lack extensive internal membrane systems. Like all cells, prokaryotic cells are surrounded by a plasma membrane. Cell walls may be absent /present depending on cell type. In most prokaryotes, the cell wall is rigid and is composed of polysaccharides and peptides. The cell wall is porous and relatively easy to penetrate so the plasma membrane must regulate the movement of molecules into and out of the cell. However, there is very little internal membrane structure. In some prokaryotes, there are internal folds of the plasma membrane that are believed to carry out specific functions.

• Prokaryotic cells lack extensive internal compartmentalization. They lack nucleus, rather possess a nucleoid.

• Prokaryotic cells lack membrane-enclosed organelles. Specialized regions of the plasma membrane in prokaryotes perform the functions of organelles (such as respiration, protein secretion, and photosynthesis).

• Prokaryotic DNA is not contained within a membrane-bound nucleus. Instead it is found within a specialized region of the cell called the "nucleoid." Nucleoid is of indefinite outline, less dense than the surrounding cytoplasm, containing tangled masses of fibers 3-5 nm in thickness representing a single molecule of DNA irregularly folded into a compact mass. There is no nuclear membrane. Prokaryotic cells have much less DNA than eukaryotic cells.

Eukaryotic cells

Each cell possesses a nucleus that contains the genetic material, external cytoplasm containing discrete compartments and organelles, bounded by the plasma membrane that marks the periphery of the cell. They are larger (10 - 50 µm in diameter) in size; red blood cell, 6-8 µm in diameter. First eukaryotic cells originated approximately 2 billion years ago, following 1.5 billion years of prokaryotic evolution. Eukaryotic Characteristics

“Eukaryotic” means “true nucleus.” Eukaryotic cells are characterized by the following properties.

• Eukaryotic cells have extensive internal membrane systems. All cells are surrounded by a plasma membrane. Cell walls may be absent / present depending on cell type. In eukaryotic cells, the interior of the cell there exist additional membrane systems that carry out specialized tasks.

• Eukaryotic cells exhibit internal compartmentalization. Membranes divide the interior of the eukaryotic cell into different regions, or compartments. They possess a distinct nucleus (isolating the DNA from the rest of the cell).

• Eukaryotic cells have membrane-enclosed organelles. The compartments within the eukaryotic cell form structures called organelles. Each organelle performs specific functions, such as cellular respiration (in the mitochondria), photosynthesis (in the

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chloroplasts), site of modification of proteins with carbohydrates, lipids, etc. (in the golgi bodies) and other functions.

• Eukaryotic DNA is contained within a membrane-bound nucleus. This allows eukaryotic cells to have a greater amount of DNA than prokaryotic cells, permitting greater evolutionary diversity. The nucleus contains usually more than one chromosome.

• Eukaryotic cells have an extensive cytoskeleton. It is a network of protein filaments extending throughout the cytoplasm providing the structural framework of the cell, determining cell shape, allowing for motility intracellular transport and positioning of organelles and other structures, including the chromosomes.

A typical bacterial cell is about 1 micrometer in diameter while most eukaryotic cells are from 10 to 100 micrometers in diameter. Eukaryotic cells have a much greater volume of cytoplasm and a much lower surface: volume ratio than prokaryotic cells.

Figure 2: Major difference between prokaryotic and eukaryotic cell The organelles of eukaryotic cells are thought to have evolved from a symbiotic association with prokaryotes. Endosymbiosis

During the 1980s, Lynn Margulis proposed endosymbiont theory, that organelles are derived from ancient colonization of larger bacteria (became the eucaryotic cell) by smaller bacteria (became the mitochondria, chloroplast, etc.). Eventually, organelles lost their ability to exist as separate organisms, cannot be separated from cell. This theory is particularly well supported by studies of mitochondria and chloroplasts, which are thought to have evolved from bacteria living within large cells. They are similar to bacteria in size and they reproduce by binary fission dividing into two. In addition, they both contain their own DNA without nucleus and their own ribosomes. The ribosomes (70S) and ribosomal RNAs of these organelles are more closely

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related to those of bacteria than to those encoded by the nuclear genome of eukaryotes. In addition, a double membrane surrounds both organelles. Interestingly, the inner membrane of these organelles has properties, which resemble prokaryotic characteristics. The endosymbiotic theory is now widely accepted with mitochondria thought to have evolved from aerobic bacteria and chloroplasts from photosynthetic bacteria, such as cyanobacteria

Figure 3: The basic events in endosymbiosis

Archaebacterial Cell

The term archaebacteria refers to the ancient origins of this group, which exist today in extreme environments. In the late 1970s, Dr. Carl Woese spearheaded a study of evolutionary relationships among prokaryotes based on RNA sequences and discovered that the prokaryotes were actually composed of two very different groups -- the Bacteria and a newly recognized group that he called Archae. As a group, the archaebacteria (Greek archois, ancient and bacterion, a small rod) are quite diverse, both in morphology and physiology. They are considered ancient because they probably resemble the FIRST FORMS of LIFE on Earth. They can stain either Gram positive or Gram negative and may be spherical, rod-shaped, spiral, lobed, plate-shaped, and irregularly shaped or pleomorphic. Some are single celled whereas others filamentous or in aggregation. They range in diameter from 0.1to 15 µm and some filaments can grow upto 200 µm in length. Approximately 200 species have been discovered. Multiplication may be by binary fission, budding, fragmentation or other mechanisms. They can be aerobic, facultatively anaerobic, or strictly anaerobic. Nutritionally they range from chemolithoautrophs to organotrophs. Some are mesophiles; others are hyperthermophiles that can grow above 100oC. Archeabacteria usually prefer restricted or extreme aquatic and terrestrial habitats and can be found in bogs and oceans, inside other organisms, hot springs, salt flats and deep-sea vents. It appears that they constitute up to 34% of the prokaryotic biomass in coastal Antarctic surface waters. Archaen Phylogeny

The phylogeny of archaens, based on molecular sequences in their DNA reveals three distinct groups within the Archae-

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(a) Euryarcheota are probably the best known, including many methane-producers (methanogens) and salt-loving archeans (halophiles).

(b) Crenarcheota include those species that live at the highest temperatures of any known living things, though a wide variety has recently been discovered growing in soil and water at more moderate temperatures (thermophiles).

(c) Korarcheota not much is known about them except for their DNA sequences as they have only recently been discovered.

(a) a coccus

(b) a lobed coccus (c) a short bacillus

Figure 4: Archael cell shapes

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Figure 5: Three sub-groups of Archae

Figure 6: Phylogenetic tree of Archae Archaebacterial cell walls

Although archeabacteria can stain either Gram positive or Gram negative, their wall structure and chemistry differ from that of the eubacteria. There is considerable variety in archeabacterial wall structure. Many Gram positive archeabacteria have a wall with a single thick homogenous layer like Gram positive Eubacteria. Gram negative archeabacteria lack the outer membrane and complex peptidoglycan network, and have a surface layer of protein or glycoprotein subunits.

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Figure 7: Generalized Gram positive archael cell wall

Figure 8: Generalized Gram negative archael cell wall Archael cells have more variations in their cell wall chemistries and some do not contain cell walls (e.g.Thermoplasma). None have the muramic acid and D-amino acids characteristic of eubacterial peptidoglycan. All archaebacterial cellwalls resist attack by lysozyme and beta (β) –lactam antibiotics such as penicillin.

(b)

(a)µ m

µ m

Figure 9: Cell envelopes of Archaebacteria Schematic representations and electron micrographs of (a) Methanobacterium formicicum, a typical gram-positive organism (b) Thermoproteus tenax, a gram-negative

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archaeobacteria. CW- cell wall; SL- surface layer; CM- cell membrane or plasma membrane; CPL- cytoplasm

Cell Walls of Methanogens

At least three different types of cell wall are found among the methanogens. The most chemically complex cell wall is that of the group I methanogens, which is rigid and composed of Pseudomurein, a peptidoglycan similar to the murein of eubacteria. Pseudomurein contains:

• N-acetyl-talosaminouronic acid (NAT) instead of N – acetylmuramic acid (NAM) and N-acetyl glucosamine (NAG) joined by a β - 1,3 glycosidic bond

• L-amino acids and lacks D- amino acids. In appearance the wall of group I methanogens resemble those of Gram positive eubacteria; in Methanobacterium species it is thick and homogenous; in Methanobrevibacter species it is triple layered. The inner layer of the Methanobrevibacter wall contains the pseudomurein.

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N-acetyl talosaminouronic acid

L-glutamate

L-lysine (ε)

L-alanine

L-glutamate (γ)

NH

CH2

NH

HC CH3

C O

NH

C O

(CH2)2

NH

C O

CH COO-

O O

NH

NH

CH3

CH2 OH

CH3C O

O

C O

OH

O

N-acetyl glucosamine

O

HC (CH2)3

NH O C

C (CHHC 2)2

COO-

Figure 10: Structure of the repeating unit of Pseudomuerin

Methanococcus, the sole representative of group II methanogens, has a flexible cell wall composed of proteins with traces of glucosamine. Methanospirillum, one of the genera of group III methanogens, has the most complex cell wall. Its wall is flexible envelope composed of at least two layers; an inner, electron dense one of unknown chemical composition and an outer which appears membrane like in cross section but is composed entirely of protein. This protein is resistant to hydrolysis by proteinases (e.g. trysin), and to solubilization by detergents (e.g. sodium dodecyl sulphate, SDS). The outer layer does not participate in septum formation; rather it is a sheath that envelops the individual cells forming a helical trichome. Methanosarcina, the other member of group III, contains a thick, rigid, lamellar wall composed of an acidic heteropolysaccharide, the principal constituents of which are galactosamine, neutral sugars and uronic acids.

Table 1: Major structural differences among Methanogens

Group Representative Genera

% G+C

Characteristic lipid

Cell Wall structure Gram Reaction

Gram Reaction

I Methanobacterium

Methanobrevibacter

32-50

27-32

C20 diethers Or

C40 tetraethers

Pseudomurein

+

-

II Methanococcus 30-32 C20 diethers Protein (trace of glucosamine)

- +

III Methanospirillum

Methanosarcina

45-47

38-51

C20 diethers and

C40 tetraethers C20 diethers

Proteinaceous sheath, unknown structural material Heteroploysaccharide

-

+

+ -

Cell wall of Halophiles

The principal structural constituent of the cell wall of Halobacterium is a large, acidic glycoprotein (MW = 20,000 daltons), the removal of which either by dilution of the suspending medium or by proteinase treatment results in loss of cell shape and in dilute media, osmotic lysis. Its glycan component consists:

• 22 to 24 disaccharides (glucosylgalactose) linked via O- glycosidic bonds to threonine residues.

• 12 to 14 trisaccharides also O- linked to threonines. • a single hetero oligosaccharide in N- glycosidic linkage to asparagine.

In addition to the glycoprotein, the cell envelope of Halobacteria contains nonglycosylated protein and glycolipid.

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The cell wall of Halococcus is chemically and structurally quite different from that of Halobacterium. It resembles the wall of Gram positive eubacteria and unlike the proteinaceous wall of Halobacterium; it retains its integrity when the salt concentration of the suspending medium is reduced. The principal structural component of its cell wall is a complex sulfated hetero polysaccharide composed of:

• Several neutral sugars, • Uronic acids • Amino sugars (many of which are acetylated).

The glycan strands are probably cross- linked by glycine residues that bridge the amino groups of amino sugars and the carboxyl groups of uronic acids.

(a)

(b)

Figure 11: Halophiles (a) Halobacterium salinarium (b) Halococcus morrhae

Cell wall of Thermoacidophiles

The cell wall of Sulfolobus is composed of lipoprotein and carbohydrate (containing both neutral sugars and amino sugars), which form a distinct layer outside the cell membrane. Thermoplasma lacks a cell wall while the cell wall of Thermoproteus consists of glycoprotein. Archael Cell Membranes

There are four fundamental differences between the archeal membrane and all other cells: (1) chirality of glycerol, (2) ether linkage, (3) isoprenoid chains, and (4) branching of side chains. (1) Chirality of glycerol: The basic unit from which cell membranes are built is the

phospholipid. This is a molecule of glycerol, which has a phosphate added to one end, and two side chains attached at the other end. When the cell membrane is put together, the glycerol and phosphate end of the molecules hang out at the surface of the membrane, with

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the long side chains sandwiched in the middle. This layering creates an effective chemical barrier around the cell and helps maintain chemical equilibrium. The glycerol used to make archeal phospholipids is a stereoisomer of the glycerol used to build bacterial and eukaryotic membranes. While bacteria and eukaryotes have D-glycerol in their membranes, Archaens have L-glycerol in their membranes.

Figure 12: Basic Archeal Structure - The three primary regions of an archaeal cell are the cytoplasm, cell membrane, and cell wall. Above, these three regions are labelled, with an enlargement at right of the cell membrane structure. Archaeal cell membranes are chemically different from all other living things, including a "backwards" glycerol molecule and isoprene derivatives in place of fatty acids.

(2) Ether linkage: When side chains are added to the glycerol, most organisms bind them

together using an ester linkage. The side chain that is added has two oxygen atoms attached to one end. One of these oxygen atoms is used to form the link with the glycerol, and the other protrudes to the side when the bonding is done. By contrast, archael side chains are bound using an ether linkage, which lacks that additional protruding oxygen atom. This gives the resulting phospholipid different chemical properties from the membrane lipids of other organisms.

(3) Isoprenoid chains: The side chains in the phospholipids of bacteria and eukaryotes are fatty acids, chains of usually 16 to 18 carbon atoms. Archae do not use fatty acids to build their membrane phospholipids. Instead, they have side chains of 20 or 40 carbon atoms (phytanyl, diethers or biphytanyl, tetraethers) built from isoprene.

(4) Branching of side chains: The side chains have a different physical structure. Because isoprene is used to build the side chains, there are side branches off the main chain. The fatty acids of bacteria and eukaryotes do not have these side branches. For example, the isoprene side chains can be joined together. This can mean that the two side chains of a single phospholipid can join together, or they can be joined to side chains of another phospholipid on the other side of the membrane. No other group of organisms can form such transmembrane phospholipids.

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Another interesting property of the side branches is their ability to form carbon rings. This happens when one of the side branches curls around and bonds with another atom down the chain to make a ring of five carbon atoms. Such rings are presumed to provide structural stability to the membrane, since they are found common among species that live at high temperatures. In Sulfolobus, the cell membrane is a monolayer, consisting of a long chain branched hydrocarbon. Unusual structure of cell membrane is also found in Thermoplasma and Halobacterium, which live in extreme habitats, and their cell membrane makes them resistant to the environmental conditions.

(b)

(a)

Figure 13: Archeabacterial membranes (a) A membrane composed of integral proteins and a bilayer of C20 diethers (b) A rigid monolayer composed of integral proteins and C40

tetraethers

Genome Organization

Nucleoid or nuclear region: It usually consists of a single negatively supercoiled circular dsDNA molecule. It may be mesosome bound. Genome

• Size varies from 0.58 Mbp to 4.4 Mbp • G + C contents vary between 28 to 72%.

The DNA molecules wrap around the protein molecules known as histone-like proteins (HTa). In Thermoplasma, the core protein is made up of four molecules of HTa and 40 nucleotides surrounds it. The genome size of some archeabacteria is significantly smaller than the normal eubacterium:

• E.coli DNA has a size of about 2.5x 109 daltons

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• Thermoplasma acidophilum DNA is about 0.8x 109 daltons. DNA duplication (40') is slower than cell division through binary fission (20') and therefore the cell initiates new rounds of synthesis even though the previous copy has not fully replicated. Some Archae genes (23S, 16S rRNA and tRNA) contain introns and the transcription systems are orthologous to Eukarya ones (TBP, more kind of RNA polymerases). Nanoarchaeum equitans has the smallest genome to date, a circular chromosome only 490 kb long, with 552 coding sequences covering 95% of the genome and containing little noncoding or pseudogene sequence that would have suggested reductive evolution. Function has been assigned to 66% of the genes; 18% have homologues of unknown function, and the remainder represent archael-specific sequences. RNA

Archaebacterial mRNA appears similar to that of eubacteria rather than to eukaryotic mRNA. Polygenic mRNA has been discovered and there is no evidence for mRNA splicing. Archaebacterial promoters are similar to those in eubacteria. Unlike both eubacteria and eukaryotes, the TC arm of archeabacterial tRNA lacks thymine and contains pseudouridine or I-methylpseudouridine. The archeabacterial tRNA carries methionine like the eukaryotic initiator tRNA in contrast to the formylmethionine in eubacteria. RNA Polymerase

Archael RNA polymerases are of several types and are structurally more complex than those of eubacteria. The RNA polymerases of Methanogens and Halophiles contain:

• 8 polypeptides, 5 large and 3 small ones Hyperthermophlic archea contain even more complex RNA polymerase consisting

• at least 10 distinct polypeptides None of the archeal RNA polymerases are affected by the antibiotic rifampicin, a known inhibitor of the bacterial RNA polymerase. The RNA polymerase in the archea is similar to the RNA polymerase (termed RNA polymerase II) of the eukaryotes consisting of 12 proteins. RNA polymerases from all organisms recognize a variety of start sequences or promoters. A promoter for mRNA transcription in bacteria is recognized by the σ (sigma) protein and has two recognition zones about 10 and 35 bases before the transcription start site. The exact sequence recognized by RNA polymerase depends upon the σ factor that has bound to the enzyme, and cells have different numbers of σ factors that bind to different types of promoter sequences. This allows regulation of mRNA expression by changing the levels of σ factors inside the cell. In archea and eukaryotes, the transcription recognition sequence is a TATA sequence (TATA box) an A-T rich sequence at –32 to –25 bp upstream of transcription at start and transcription is regulated by various protein transcription factors that bind to regions near the TATA box and then recruit RNA polymerase. Ribosomes

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Archaebacterial ribosomes are 70S like eubacterial ribosomes but electron microscopic techniques have shown that their shape is quite variable and sometimes differs from that of both eubacterial and eukaryotic ribosome. They resemble eubacterial ribosomes in

• sensitivity to Ansiomycin • insensitivity to Chloramphenicol and Kanamycin.

Plasmids

Archael self-transmissible plasmids have been found only in diverse strains of the hyperthermophilic genus Sulfolobus, where they occur in about 3% of isolated strains. The two which have already been characterized are:

• pNOB8 from the Japanese strain Sulfolobus NOB8H2 • pING1 from Sulfolobus islandicus strain HEN2P2.

Comparative sequence analyses reveal minimal significant sequence similarity between open reading frames (ORFs) of archeal and bacterial conjugative plasmids. The exceptions are two large archael proteins that show significant sequence similarity to limited sections of the bacterial TraG and TrbE proteins, both carrying Walker A and B, and other motifs. In bacteria, both proteins are involved in coordinating the transport of single- stranded DNA through membrane pores. There is also evidence from electron microscopic studies of conjugating Sulfolobus cultures that extensive cellular contact occurs, suggesting that DNA is transferred directly through cell membranes. Intercellular transfer of chromosomal genes has been demonstrated in Sulfolobus acidocaldarius and likely occurs via conjugation. An archeal intron encoding a homing enzyme was shown to transfer between cells, inserting into the single chromosomal 23S rRNA gene, and various marker genes have been shown to exchange intercellularly between chromosomes. Proteins encoded in a conjugative plasmid encaptured in the S. acidocaldarius genome may facilitate these phenomena. Flagella

Flagellation is widespread in both bacteria and archae. Flagellation occurs throughout all the main groupings of the Archae:

• Extreme halophiles, • Methanogens, • Various sulfur-dependent thermophiles • Hyperthermophiles and even in Thermoplasma species which lack a cell wall.

The range of environmental conditions or niches where flagellated Archea are found: • saturated salt solutions • strictly anaerobic environments • extremely low pH (pH < 2), and • extremely high temperatures ( >1000C).

Recent research on archeal flagellins has revealed a number of unusual traits, such as the presence of short signal sequences, a sequence similarity to type IV pilins and not bacterial flagellins, and often-posttranslational modifications such as glycosylation. The assembly of

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archael flagella is novel and is different from that of eubacteria. Electron microscopy of isolated archael flagella has shown that there are no ring-like or other substructures in the basal body as seen on bacterial flagella instead, knob shaped structures have been noted on flagella isolated from methanogens and other archea by phase separation with Triton X- 114 or detergent of envelope fragments. Hook regions, which act as flexible couplers between the basal body and filament of bacterial flagella, are also poorly defined or absent in isolated archeal flagella. Purified flagellar filaments from the Archae are distinctively more narrow (10 to 15 nm) than the bacterial counterpart (generally around 20 nm)

Figure 14: Distribution of flagellation throughout the Domain Archae. (+) at least some members of the genus are flagellated; (-) no members of the genus are flagellated

Ultra structure Filament Flagella filaments have been characterized from many archea, including species from the genera Halobacterium, Methanococcus, Methanoculleus, Methanospirillum, Methanothermus, Natrialba (formerly Natronobacterium), Pyrococcus, Sulfolobus, Thermococcus and Thermoplasma. Archeal filaments differ from bacterial filament in following aspects: (a) The diameter of the filament - typically, the archael filaments are thinner than their bacterial

counterparts (10-14 nm in archae compared to about 20 nm in bacteria). Filaments thinner than usual have been reported under certain conditions in flagella preparations of Natrialba (formerly Natronobacterium) magadii and Thermoplasma volcanium. These so-called protofilaments appear to be intermediate structures in the process of dissociation. Generally, archael flagella filaments are featureless when observed under the electron microscope after 2% uranyl acetate staining. An exception is Sulfolobus shibatae flagella, which demonstrate well-defined subunits within the filament.

(b) The rotation of filament - typical bacterial flagellar filaments generally possess a left-handed helix, with swimming motility resulting from counter-clockwise rotation. This rotation

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results in the flagella filaments forming a bundle in peritrichously flagellated bacteria, such as Escherichia coli. It is during the `tumble' or clockwise rotation that the bundles fly apart. In comparison to this, Halobacterium salinarum possesses unusual flagella, in that the filament forms a right-handed helix. The halophiles flagella, and presumably all archeal flagella, are rotating structures as observed by direct light microscopy. In H. salinarum, a clockwise rotation of the flagellar bundle pushes the cell, while a counter-clockwise rotation pulls the cell, so the cell appears to swim with the flagella in front, and the flagellar bundle is never seen to fly apart. It was found that the switching behavior of the flagellar bundle and rotation are synchronous in H. salinarum, which is not the case in the peritrichously flagellated E. coli. This `pushpull' motility feature is unique to H. salinarum or commonly found in archea is not known. There are very few reports that measure archeal swimming speed. A rare exception is the case of H. salinarum where the speed was measured at 2-3 µm s-1, about 10% of the speed reached by E. coli under similar conditions of observation. Cells were shown to swim almost twice as fast when the flagella are rotating clockwise as opposed to counter-clockwise.

Hook and anchoring structure To date, the best methods for isolating archael flagella with some attached basal structure have been with phase separation using Triton X-114 detergent or by solubilization of whole cells or flagellated cell envelopes with Triton X-100. The basal structure has been reported as knob like in several Methanococcus species and Halobacterium while a Gram positive like basal body with two rings has been reported for Methanococcus thermolithotrophicus and M. hungatei. Other researchers have shown that M. hungatei basal structure lacks a well-defined hook and rings. It is possible that a typical basal body is present in archeal flagella but it is sensitive to the treatments used thus far to try to isolate `intact' archeal flagella. However, when the phase separation technique was used to isolate flagella from three different Gram negative bacterial species, all four rings and rod of the basal bodies were clearly seen. If the archea do possess a bacterial- like basal body, the proteins comprising such a structure must be distinct from their bacterial equivalents as no homologues to basal body proteins are seen in the analysis of complete archael genomes. The best-studied archea with regard to flagellation are Methanococcus and Halobacterium, which have a wall profile not seen in bacteria. This is an S layer that over layers the cytoplasmic membrane with no other intervening wall layer. Perhaps the insertion of archael flagella into these structurally unique walls has resulted in the development of a different anchoring mechanism. Due to this relative simplicity, additional anchoring may be needed, and it has been speculated that some Archae may stabilize the flagella with a sub-cytoplasmic membrane layer, such as the polar cap of H. salinarum. However, it is reminiscent of polar membrane like structures often associated with flagellar insertion and near the site of septum formation in bacteria. Similar polar membrane-like structures have been observed in many-flagellated archea including M. voltae. The end plug is a complex structure composed of several proteinaceous matrices possessing hexagonal symmetry and pores (15 nm) large enough to accommodate the diameter of the flagella (10 nm). These layers in the end plug may act as a bushing for the flagella. The hook region of archeal flagella has been considered in the past to be poorly defined, and was identified as a slight thickening of the filament located next to the knoblike end when staining of

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flagella was done with uranyl acetate. A clearly defined hook region on M. voltae flagella has also been noted by phase separation when these samples were stained with phosphotungstic acid at neutral pH. In the case of M. voltae flagella, the hook length is almost twice as long as that of Salmonella flagella, which is 55 nm. Hooks of varying length (72-265 nm) located near the anchoring structures were seen in preparations of flagella isolated from M. thermolithotrophicus. This is a much larger variability in length than usually reported for bacterial hooks.

Figure 15: Negatively stained preparation of flagella from M. voltae showing hook regions showing the curved hook structure present at the end of many flagella

Assembly of flagella It is believed that flagellin-specific chaperones bind to the preflagellins in the cytoplasm, possibly to prevent their aggregation via their hydrophobic N-termini. Chaperone escorted preflagellin monomers are delivered to the cytoplasmic membrane where their leader peptide is cleaved by a membrane-located preflagellin peptidase, an enzyme thought to be specific for the flagella system. A preflagellin peptidase activity has been demonstrated in M. voltae membranes and the sequence immediately surrounding the cleavage site has been shown to be important for processing. Whether this transport involves an archeal Sec system is unknown. If the flagellins are to be glycosylated as many archeal flagellins are, this modification would occur on the exterior of the cytoplasmic membrane, as for H. salinarum. This could occur with the flagellins embedded in the cytoplasmic membrane via their N-termini with much of the protein exposed on the outer leaflet. These data point to a novel mode of assembly of flagella in the archea where new subunits would be added at the base and not at the distal tip after passing through the hollow structure as is the case for bacterial flagella. There is evidence that not all flagellins are equal. Mutants in the minor flagellin FlaA of M. voltae assemble normal-looking flagella when viewed in the electron microscope, but the cells are less motile by semi-swarm plate analysis. Transcription data suggest that FlaB3 is present in much smaller amounts than FlaB1 and FlaB2 as well. Recent analysis of mutant flagellins of H. salinarum suggests that in the case of this extreme halophile, the A flagellins make up the bulk of the filament (corresponding to the FlaB1 and FlaB2 flagellins of M. voltae and M. vannielii).

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Figure 16: Structure and assembly model of M.voltae flagella Eubacterial Cell: Structure and Functions

Unlike plants and animals, bacteria are unicellular organisms that do not develop or differentiate into multicellular forms. Some bacteria grow in filaments or masses of cells, but each cell in the colony is identical and capable of independent existence. The cells may be adjacent to one another because they did not separate after cell division or because they remained enclosed in a common sheath or slime secreted by the cells, but typically there is no continuity or communication between the cells. Phylogenetic analysis of the Eubacteria commonly termed as bacteria has demonstrated the existence of at least 13 distinct groups, but many groups consist of members that are phenotypically and physiologically unrelated, and sometimes phylogenetically unrelated. The current edition of Bergey's Manual of Systematic Bacteriology (2001) recognizes 23 distinct phyla of Bacteria (Phylum is the highest taxon in a Domain), but there may still be great variation in phenotype among members. Bacterial cells have three architectural regions:

• Appendages (proteins attached to the cell surface) in the form of flagella, pili and glycocalyx.

• A cell envelope consisting of a cell wall and plasma membrane • A cytoplasmic region that contains the cell genome (DNA) and ribosomes and various

sorts of inclusions.

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Figure 17: Phylogenetic tree of Bacteria

Pili These short hair like structures are involved in

Ribosome The ribosomes are used for translation of mRNA into

d i d

Cell Membrane Materials move between the cytoplasm and environment by crossing

Flagella Flagellar rotations create currents that

h b i

Cell Wall Protects the cell and forms the basis for classification

Plasmids It is a small circular DNA that replicates independently of

Chromosomes Bacteria have a single circular

Figure 18: The major structural components and their functions of a typical bacterium

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Surface Structures-Appendages

Surface structures originate outside the cell membrane, sometimes being attached to it, and extend into the environment. Important structures include flagella, pili, fimbriae, and glycocalyx. Flagella

Flagella are filamentous protein structures attached to the cell surface that provide swimming movement for most motile prokaryotic cells. A motor apparatus in the plasma membrane allowing the cell to swim in fluid environments rotates the flagellar filament. Bacterial flagella are powered by proton motive force (chemiosmotic potential) established on the bacterial membrane, rather than ATP hydrolysis that powers eukaryotic flagella. Prokaryotes are known to exhibit a variety of types of tactic behavior, i.e., the ability to move (swim) in response to environmental stimuli. For example, during chemotaxis a bacterium can sense the quality and quantity of certain chemicals in their environment and swim towards them (if they are useful nutrients) or away from them (if they are harmful substances). There is a loose correlation between cell shape and the presence of flagella. Almost all spirillum, half of all rods, and rarely cocci are motile via flagella.

Figure 19: Vibrio cholerae with single polar flagellum Structure

Flagella are like semi-rigid whips that are free at one end and attached to a cell at the other. The diameter of a flagellum is thin, 20 nm, and long with some having a length 10 times the diameter of cell (about 100 nm). Due to their small diameter, flagella cannot be seen in the light microscope unless a special stain is applied. Bacteria can have one or more flagella arranged in clumps or spread all over the cell. It is helical and has a sharp bend just outside the outer membrane called the hook, which allows the helix to point directly away from the cell

Chemical Structure

Flagella are mostly composed of flagellin (a protein) that is bound in long chains and wraps around itself in a left handed helix. The number of units, the wavelength and diameter of a single helix of the flagella are determined by the protein subunits.

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Figure 20: Flagellar Arrangements (Polar or Monotrichous, Lophotrichous, Amphitrichous and Peritrichous respectively)

Hook and Basal Body

The hook and basal body of the flagella help in its attachment to the cell. A shaft runs between the hook and the basal structure, passing through protein rings in the cell membrane that act as bearings. Gram positive organisms have two rings, one in the cell wall and one in the cell membrane. Gram negative organisms have four rings, two in the cell wall and two in the cell membrane.

In Gram negative bacterium In Gram positive bacterium

Figure 21: The flagellar structure in Gram positive and Gram negative bacteria Flagellar synthesis

If a flagellum is cut off, it will regenerate until reaches a maximum length. As this occurs, the growth is not from base, but from tip. The filament is hollow and subunits travel through the filament and self-assemble at the end.

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Movement

The flagellum is a rigid structure and rotates like a propeller. Rings in the basal body rotate relative to each other causing the flagella to turn. The energy to drive the basal body is obtained from the proton motive force. How protons drive the rotation of the flagella is unclear. The average motility of a bacterial cell is 50 m/sec, which is about 0.0001 miles/hr.

Table 2: Relative Speeds of Organisms

Organism Kilometers per hour Body lengths per second Cheetah 111 25 Human - Michael Johnson 37.5 5.4 Bacterium 0.00015 10

Typically microbes that live in aqueous environments continually move around looking for nutrients. Sometimes this movement is random, but in other cases it is directed toward or away from something. Bacteria are capable of a tactic response to various stimuli.

o Chemotaxis – (towards or away from a chemical stimulus). Sensing the environment and adjusting the rotation of the flagella in response to stimuli accomplish chemotaxis.

o Phototaxis – (towards or away from light). Seen in case of photosynthetic bacteria. They adjust to the intensity of light for maximum photosynthesis.

o Aerotaxis – (towards or away from oxygen). Seen in aerobic bacteria. They tend to move towards air sources while the anaerobes move away from it.

o Magnetotaxis – (orientation in a magnetic field). An example is Aquaspirillum magnetotacticum, which has magnetosomes. These structures orient themselves in a magnetic field (The earth's magnetic field under natural conditions). The microbe uses this to determine which way is up and that helps it to find nutrients or adjust its depth in an aquatic environment.

Flagella rotation

Flagella can rotate clockwise or counterclockwise. When flagella rotate counterclockwise this creates a force pushing on the bacterium. In the case of E. coli the peritrichous flagella bunch together and all push from one side. This causes the bacterium to move in a straight line, called a run. When flagella rotate clockwise, they all pull on the microbe. With all these forces pulling in different directions, it causes the bacterium to tumble or twiddle. When the twiddling is over, the bacterium starts out a new run in a completely random direction. Neutral conditions: In plain medium containing no attractant or repellent, the length of runs is random and the bacterium moves about the solution aimlessly. Attractant: When cells are put in an environment containing an attractant such as glucose, they move toward the source of the attractant. The microbes sense the change in concentration of the attractant, the concentration gradient, as they move through solution. If they move up the gradient to higher attractant concentrations, the length of the run increases. If they move down the gradient, the length of the run is much shorter. Thus, a bacterium eventually moves to the source of the attractant.

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Repellent: When the compound is harmful, such as HCl (an acid), the microbe shortens runs that go up the gradient and lengthen those that take it away from the repellent. It is noteworthy that cells do not detect absolute concentration of chemicals but detect a change in gradient. After being exposed to a stimulus for a period of time, the cells quit response to it. This is actually a type of chemical memory. They detect the gradient by using a complex molecular mechanism. The attractant or repellent binds to receptors in the membrane and this signal is passed through the cell, eventually regulating the direction of flagellar rotation. Rhodobacter sphaeroides moves using single polar flagellum. It runs when the flagella rotates, but does not tumble. During chemotaxis, if conditions become unfavorable, flagellar rotation ceases and the microbe stops. During stops the microbe slowly changes direction due to Brownian movement and then off it runs in a new direction. R. sphaeroides is also able to regulate the speed of rotation, moving faster toward an attractant or away from a repellent.

Figure 22: Directed motility in bacteria (a) flagellated cell (b) Run and Tumble movement (c) Effect of attractant and repellant

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Different modes of motion - Gliding

Not all bacteria get around by using bacterial flagella. Other bacteria use gliding motility that depends upon contact with a solid surface. Under the microscope it seems as if they are sliding along the surface. The exact mechanism for gliding motility is unknown, but the best hypothesis suggests that little circular motors (similar to the basal body of flagella) are spinning underneath the microbe. The circular force causes the bacterium to glide across a surface in a manner similar to a floor buffer while waxing a floor. Fimbriae

Fimbriae and Pili are interchangeable terms used to designate short, hair-like structures on the surfaces of prokaryotic cells. Fimbriae are shorter and stiffer than flagella, slightly smaller in diameter and are more numerous. Like flagella, they are composed of protein and are not synthesized by all bacteria. Fimbriae do not function in motility, but are usually involved in adherence (attachment) of bacteria to surfaces in nature. In medical situations, they are major determinants of bacterial virulence because they allow pathogens to attach to (colonize) tissues and to resist attack by phagocytic white blood cells.

Figure 23: Fimbriae of Neisseria gonorrhoeae allow the bacterium to adhere to tissues. Pili

Pili are longer than fimbriae and there are only a few per cell. They are known to be receptors for certain bacterial viruses. There are two basic functions for pili, gene transfer and attachment. A specialized type of pilus the F or sex pilus, mediates the transfer of DNA between mating bacteria, but the function of the smaller, more numerous common pili is quite different. The sex pilus (or F-pilus) is involved in sexual reproduction of certain bacteria. A donor bacterium will attach to a recipient via the sex pilus. Then a copy of part of the donor bacteria's genome passes through the sex pilus into the recipient. This is a mechanism of genetic exchange between bacteria. Interestingly, transfer of genes this way is not restricted to species. It is possible for E. coli to transfer information to many different Gram negative species. This is termed as conjugation and is one explanation for the rapid occurrence of drug resistance in many different species of bacteria. Pili have also been shown to be important for the attachment of some pathogenic species to their host. Neisseria gonorrheae, the causative agent of gonorrhea, has special pilus that helps it

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adhere to the urinogenital tract of its host. The microbe is much more virulent when able to synthesize pili.

Figure 24: Transmission electron micrograph of Aquaspirillum hydrophila showing flagella (thick, long structures) and pili (thinner fibers)

Glycocalyx

Many, but not all bacterial cells have an external coating excreted onto the outside of the cell. There are two types of glycocalyx, capsules and slime layers, but the difference between the two is somewhat arbitrary.A glycocalyx is a general term for any network of polysaccharide or protein containing material extending outside of the cell. A capsule is closely associated with cells and does not wash off easily. A slime layer is more diffuse and is easily washed away. Structure

Glycocalyx contains polysaccharide, although some may also contain protein(s), typically glycoproteins. There are many different types of polysaccharides plus some polyalcohols and amino sugars in glycocalyx and the exact makeup is species specific. The structure can be thick or thin, rigid or flexible. Glycocalyx is identified by staining cells with India ink, which does not penetrate the structure. When observed in the microscope the cells appear dark with a clear outline around them. This outline is the capsule or slime layer.

Figure 25: Capsule-surrounding cells of Streptococcus species

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Functions of Fimbriae, pili and glycocalyx (capsules and slime)

• Attachment: These structures help cells to attach to their target environment. Streptococcus mutans produces a slime layer in the presence of sucrose. This result in dental plaque and many bacteria can stick to tooth surfaces and cause decay once S. mutans forms a slime layer. Vibrio cholerae, the cause of cholera, also produces a glycocalyx that helps it attach to the intestinal villi of the host.

• Protection from phagocytic engulfment: Bacterial pathogens are always in danger of being "eaten" by phagocytes. (Host cells that protect you from invaders.) Streptococcus pneumoniae, when encapsulated is able to kill 90% of infected animals, when non-encapsulated no animals die. The capsule has been found to protect the bacterium by making it difficult for the phagocyte to engulf the microbe.

• Resistance to drying: Capsules and slime layers inhibit water from escaping into the environment.

• Reservoir for certain nutrients: Glycocalyx will bind certain ions and molecules. These can then be made available to the cell.

• Depot for waste products: Waste products of metabolism are excreted from the cell, and will accumulate in the capsule. This binds them up, and prevents the waste from interfering with cell metabolism.

Prosthecae and Stalks

Prosthecae are semi-rigid extensions of the cell wall and cytoplasmic membrane and have a diameter that is always less than that of the cell. They are characteristic of a number of aerobic bacteria from fresh water and marine environments. Some bacterial genera such as Caulobacter have a single prostheca while others such as Stella have several. Prosthecae increase the surface area of the cells for nutrient absorption. Some prosthecate bacteria may form a new cell at the end of a prostheca; others have an adhesive substance at the end of a prostheca that aids in attachment to surfaces. The term stalk is used for certain nonliving ribbon-like or tubular appendages that are secreted by the cell, such as those found in genera Gallionella or Planctomyces. These stalks also help in attachment of the cells to surfaces. Cell Envelope (cell wall and plasma membrane)

Eubacterial cell wall Most prokaryotes have a rigid cell wall. The cell wall is an essential structure that protects the delicate cell protoplast from osmotic lysis. This is because in the inside of the bacterial cell there is a high solute concentration and a great pressure on the membrane (75 lb/in2). Outside of the cell there is a low solute concentrate. A fundamental law of physics is that water will tend to flow into a cell to equilibrate the amount of water inside and outside of the cell. It is also a fact that membranes prevent most other molecules from crossing them, but water can. Without something supporting the membrane the cell would swell and burst. A cell wall protects bacteria from osmotic lysis. The cell wall also determines the shape of the cell. Any cell that has lost its cell wall, either artificially or naturally becomes amorphic, without a defined shape.

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The cell wall of Bacteria consists of a polymer of disaccharides cross-linked by short chains of amino acids (peptides). This molecule is a type of peptidoglycan, which is called murein. However there are certain differences in the cell walls of various bacteria. On the basis of the cell wall composition, the bacteria are divided into two types- Gram positive and Gram negative.

Figure 26: A comparison of cell wall types In gram staining, a bacterial smear is first stained with crystal violet, followed by Lugol’s iodine, decolorized with alcohol and then counterstained with safranin. The bacteria, which retain the color of crystal violet and appear purple, are termed as Gram positive bacteria whereas those, which are destained by alcohol and are counterstained by safranin and appear pinkish red in color, are termed as Gram negative bacteria.

Gram positive Bacteria Gram negative Bacteria

Figure 27: Gram’s character of Gram positive (Staphylococcus) and Gram negative (E. coli) cells

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Gram positive cell wall

A thick peptidoglycan layer constitutes most of the Gram positive wall. It accounts for 50% or more of the dry wt. of the wall of some Gram positive species. In addition to the peptidoglycan, there may also be other substances present in the cellwall of Gram positive bacteria, like, cell wall of Streptococcus pyogenes contains polysaccharides that are attached to the peptodoglycan. Another structure in the Gram positive cell wall is teichoic acid. It is a polymer of glycerol or ribitol joined by phosphate groups. Amino acids, such as D-alanine are attached. Teichoic acid is covalently linked to muramic acid and links various layers of the peptidoglycan mesh together. The walls of most Gram positive bacteria contain very little lipid, but those of Mycobacterium and Corynebacterium are rich in lipids.

Figure 28: Gram positive cell wall As a result, the Gram positive cell wall is very sensitive to the action of lysozyme and penicillin, or its derivatives. Penicillin is often the antibiotic of choice for infections caused by Gram positive organisms. An example being Streptococcus pyogenes, which causes strep throat. This is almost always treated with some type of penicillin.

Gram negative cell wall

Gram negative cell walls have a more complicated structure. There are two separate areas with an additional membrane besides the cellular membrane. Outside of the cytoplasmic membrane (CM) is an open area called the periplasmic space. Beyond this is a thin layer of peptidoglycan. Finally, external to the peptidoglycan is an additional membrane, the outer membrane (OM). The outer membrane serves as a barrier to prevent the escape of important enzymes from the space between the cytoplasmic membrane and the outer membrane; to various external chemicals and enzymes that could damage the cell.

Chemical Structure of cell wall

Chemically the cell wall is composed of peptidoglycan, lipopolysaccharides and certain proteins.

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Figure 29: Structure of teichoic acid

Figure 30: Gram negative cell wall Peptidoglycan

It is a thick rigid layer that is found in both Gram positive and Gram negative cells. It is composed of an overlapping lattice of 2 sugars that are cross-linked by amino acid bridges. The exact molecular makeup of these layers is species specific. The two sugars are N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM). NAM is only found in the cell walls of bacteria and nowhere else. Attached to NAM is a side chain

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generally of four amino acids. Many bacterial cell walls have been looked at and the cross bridge is most commonly composed of

• L-alanine • D-alanine* • D-glutamic acid* • diamino pimelic acid (DPA)

Figure 31: The chemical structure of a peptidoglycan subunit The D-amino acids are different than the L-amino acids found in proteins. D-amino acids have the identical structure and composition as L-amino acids except that they are mirror images of the L amino acids. Most biological systems have evolved to commonly handle only the L form of compounds. Bacteria however use the D-amino acids in their cell walls and have enzymes called racemases to convert between D and L forms.

Figure 32: A comparison of L and D amino acids The NAM, NAG and amino acid side chain form a single peptidoglycan unit that can link with other units via covalent bonds to form a repeating polymer. The polymer is further strengthened by cross links between amino acid 3 (D-glutamic acid above) of one unit and amino acid 4 (DPA) of the next glycan tetrapeptide thereby forming a mesh. In some Gram positive microbes there is often a peptide composed of glycine, serine and threonine in between the crossbridges. The degree of cross-linking determines the degree of rigidity. In Gram positive cells the

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peptidoglycan is a heavily cross-linked woven structure that wraps around the cell. It is very thick with peptidoglycan accounting for 50% of weight of cell and 90% of the weight of the cell wall. Electron micrographs show the peptidoglycan to be 20-80 nm thick. In Gram negative bacteria the peptidoglycan is much thinner with only 15-20% of the cell wall being made up of peptidoglycan and this is only intermittently cross-linked. In both cases the peptidoglycan is a strong, woven mesh that holds the cell shape. It is not a barrier to solutes, the openings in the mesh are large and all types of molecules can pass through them.

Figure 33: A diagrammatic representation of the peptidoglycan mesh The cell wall is the site of action of many important antibiotics and antibacterial agents. Penicillin inhibits cells wall synthesis. Lysozyme an enzyme found in tears and saliva-attacks peptidoglycan. It hydrolyzes the NAG - NAM linkage.

Table 3: Difference between Gram positive and Gram negative cell wall

Property Gram positive Gram negative Thickness of wall 20-80 nm 10 nm Number of layers in wall 1 2 Peptidoglycan content >50% 10-20% Teichoic acid in wall + - Lipid and lipoprotein content 0-3% 58% Protein content 0% 9% Lipopolysaccharide 0 13% Sensitive to penicillin + - (not as much) Digested by lysozyme + - (not as much)

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Outer membrane

The Outer Membrane has been extensively studied due to its large role in the virulence (ability to cause disease) of Gram- negative bacteria. It is a Lipid bilayer similar to the cell membrane, containing lipids and proteins, but also contains lipopolysaccharides. The membrane has distinctive sides, with the side that faces the outside containing the lipopolysaccharide. Lipopolysaccharide (LPS)

LPS is composed of two parts, Lipid A and the polysaccharide chain that reaches out into the environment. Lipid A is a derivative of 2 NAG units with up to 7 fatty acids connected to it that anchor the LPS in the membrane. Attached to Lipid A is a conserved core polysaccharide that contains, heptose, glucose and glucosamine sugars. The rest of the polysaccharide consists of repeating sugar units and this is called the O-antigen. The O-antigen gets its name from the fact that it is exposed to the outer environment and host defenses will often raise antibodies to this structure. The O-antigen varies between species and even between various isolates of a species. Bacteria protect themselves against the host’s defenses by varying the make-up of the O-antigen.

O-Antigen

Figure 34: Lipopolysaccharide

LPS confers a negative charge and also repels hydrophobic molecules. Some Gram- negative species live in the gut of mammals and LPS will repel fat solubilizing molecules such as bile that the gall bladder secretes. The O-antigen is also involved in recognition by certain bacteriophage (viruses that infect bacteria). LPS is biologically very important because it has activities in humans. Free LPS in solution is toxic and is called endotoxin. The compound, when release from bacterial cells is toxic to mammals creating a wide spectrum of physiological reactions such as:

• The induction of a fever. Endotoxins are said to be pyrogenic. • Changes in white blood cell counts • Disseminated intravascular coagulation • Tumor necrosis

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• Dropping blood pressure leading to vascular collapse and eventually shock • At high enough concentrations endotoxin is lethal

Outer membrane proteins

There are fewer different types of outer membrane proteins when compared to the cytoplasmic membrane, but they are in higher abundance. Porins are proteins that form pores in the outer membrane wide enough to allow passage of most small hydrophilic molecules. This allows migration of these molecules into the periplasmic space for transport across the cytoplasmic membrane. Larger or hydrophobic molecules cannot penetrate the outer membrane. Periplasm This is the space in between the peptidoglycan and the outer membrane that contains many different proteins. These proteins function to detect the environment and transport needed nutrients into the cell. Some examples of periplasmic enzymes include:- 1. Hydrolytic enzymes-

phosphatases - degrade phosphate containing compounds proteases - degrade proteins and peptides endonucleases - degrade nucleic acids

2. Binding proteins - recognize specific solutes and transport across membrane: sugars amino acids inorganic ions vitamins

3. Chemoreceptors - helps cell interpret chemical composition of its environment 4. Detoxifying enzymes - alter harmful agents before they get into cell

Beta-lactamases (e.g. penicillinase) Aminoglycoside-phosphorylating enzymes

5. Osmotic protection - When the cell is put in high osmolarity (high solute concentration) it causes water to flow out of the cell. To protect themselves, bacteria synthesize small molecules to balance the osmotic stress. These are called compatible solutes.

Bacteria Lacking cell walls For most bacterial cells, the cell wall is critical to cell survival, yet there are some bacterial cells which do not have cell walls. Mycoplasma species are one example and they are very wide spread. They are obligate intracellular pathogens (they can only survive inside of their host). Some of this dependency is based on the lack of cell walls. As an example, they are quickly killed if placed in an environment with very high or very low salt concentrations. Due to the lack of a cell wall, Mycoplasma has unusually tough membranes that are more resistant to rupture than other bacteria. The presence of sterols in the membrane contributes to their durability. Mycoplasma is also pleomorphic. Some bacteria may mutate or change because of extreme nutritional conditions to form a cell wall-less form or L-forms. This phenomenon is observed in both G+ and G- species. These forms may result from partial or complete loss of the cell wall. L-forms have a varied shape and are sensitive to osmotic shock.

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Functions of the cell wall

1. The cell wall confers a shape to the bacterium. If removed, the cell will become an amorphous blob. Certain bacteria have long appendages that increase the surface area of the cell. This allows the cell to live in very dilute environments, yet still scavenge what it needs.

2. The cell wall is also directly in contact with the environment. Its interaction with the outside world may determine the successful survival of the cell. Two examples are:

a. Interaction with a host cell in the intestine to begin attachment b. The binding of a virus that infects the bacterial cell.

3. The cell wall is also involved in many pathogenic properties of the bacterium. In both Gram positive and Gram negative species, the cell wall is very important in attachment to specific host cells during the infection stage. The cell wall can even be a pathogenic determinate, the outer membrane of Gram negative cells contains toxic components (endotoxin). Endotoxins are actually derived from the LPS of the cell and are released when the cell lyses and dies. These endotoxins induce fever in the host and can even kill at high concentrations.

4. The cell wall can act as a barrier to some molecules. Gram positive cell walls have a negative charge and are hydrophilic due to the presence of teichoic acid. This acts as a barrier to molecules with a positive charge.

5. Gram negative cells walls are very hydrophilic due to the presence of LPS in their outer membrane. The LPS act, as a barrier to hydrophobic molecules and that is why these organisms are resistant to hydrophobic compounds like Crystal Violet and bile.

Cellular Membrane (Plasma Membrane)

General properties

The membranes of Bacteria are structurally similar to the cell membranes of eukaryotes, except that bacterial membranes consist of saturated or monounsaturated fatty acids (rarely polyunsaturated fatty acids) and do not normally contain sterols. The cytoplasmic membrane is that area of the cell immediately surrounding the cytoplasm and is perhaps the most conserved structure in living cells. Every living thing on this planet has some type of membrane. Membranes are thin structures, measuring 8 nm thick. They are the major barrier in the cell, separating the inside of the cell from the outside. It is this structure, which allows cells to selectively interact with their environment. Membranes are highly organized and asymmetric having two faces with different topologies and different functions. Membranes are also dynamic, constantly adapting to changing environmental conditions. Membranes in bacteria are composed of phospholipids and proteins. Structure of components

Phospholipids Phospholipids contain a charged or polar group (often phosphate, hence the name) attached to a 3 carbon glycerol back bone. There are also two fatty acid chains dangling from the other carbons of glycerol. The phosphate end of the molecule is hydrophilic and is attracted to water. The fatty acids are hydrophobic and are driven away from water.

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Proteins Typically 20-30% of membrane associated protein is soluble in water and loosely associated. The other 70-80% is tightly bound to the membrane, often spanning both sides. These proteins are also often amphipathic molecules (contain both hydrophobic and hydrophilic portions) with stretches of hydrophilic amino acids and stretches of hydrophobic amino acids. Most of them are placed in the membranes so that the hydrophobic amino acids associate with the lipids in the membrane and the hydrophilic amino acids are outside the membrane interacting with either the cytoplasm or the periplasm. Physical structure

Bilayer formation Because phospholipids have hydrophobic and hydrophilic portions, they do remarkable things. When placed in an aqueous environment, the hydrophobic portions stick together, as do the hydrophilic. A very stable form of this arrangement is the lipid bilayer. This way the hydrophobic parts of the molecule form one layer, as do the hydrophilic. Lipid bilayers form spontaneously if phospholipids are placed in an aqueous environment. These are known as membrane vesicles and are used to study membrane properties experimentally.

Figure 35: Schematic representation of the structure of plasma membrane Some proteins span the membrane while others are found on the outside or the inside. Many of the membrane spanning proteins are involved in transport or energy generation. Stabilization of the membrane The cytoplasmic membrane is stabilized by hydrophobic interactions between neighboring lipids and by hydrogen bonds between neighboring lipids. Hydrogen bonds can also form between membrane proteins and lipids. Further stability comes from negative charges on proteins that form ionic interactions with divalent cations such as Mg2+ and Ca2+ and the hydrophilic head of lipids

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Some proteins may move within the plane of the membrane while others are anchored to structures in or near the membrane. The result is that the membrane is actually fluid and has the consistency of a light grade oil. It has been termed a fluid mosaic, mosaic because there is a definite pattern to it, fluid because the lipids are free to move about on each side of the membrane. Lipids do not generally switch sides, moving from outside to inside or inside to outside. This arrangement confers a number of properties on the membrane that allow it to perform many functions. Functions of the Cytoplasmic Membrane

1) Retains the cytoplasm: The concentrations of solutes, sugars, ions etc. are much higher within the cell than outside. A fundamental principle of nature however is that solute concentrations will tend to equilibrate and in this case, causing water to flow into the cell (a process known as osmosis) and the solutes to flow out. The cell membrane prevents free flow of material and thus serves as an osmotic barrier.

2) Selective barrier: Since the cell is separated from its environment and needs to get nutrients in and waste out, the membrane must be able to accommodate this. It acts as a selective barrier. Some molecules can cross the membrane without assistance, most cannot. Water, non-polar molecules and some small polar molecules can cross. Non-polar molecules penetrate by actually dissolving into the lipid bilayer. Most polar compounds such as amino acids, organic acids and inorganic salts are not allowed entry, but instead must be specifically transported across the membrane by proteins.

3) Transport: Many of the proteins in the membrane function to help carry out selective transport. These proteins typically span the whole membrane, making contact with the outside environment and the cytoplasm. They often require the expenditure of energy to help compounds move across the membrane.

There are four basic types of transport systems Passive diffusion For these molecules, laws of simple diffusion direct transport. There is no transport protein, it is nonspecific, and energy is not required. A concentration gradient of these molecules cannot be generated. Compounds capable of passive diffusion must be soluble both in the lipid membrane and aqueous environments (cytoplasm, outside). Facilitated Diffusion This involves a protein that binds the molecule to transport and is therefore specific. However, solutes are not concentrated against a gradient nor are energy required. It is not a widely used strategy in prokaryotes as far as we know. Group translocation A protein specifically binds the target molecule and during transport a chemical modification takes place. No actual concentration of the transported substance takes place, since as it enters the cell it is now chemically different. Most group translocation requires energy.

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Active transport In active transport the target is not altered and a significant accumulation occurs in the cytoplasm with the inside concentration reaching many times its external concentration. Active transport proteins are molecular pumps that pump their substrates against a concentration gradient. As in all pumps, fuel is necessary and in the case of cells, this fuel comes in two forms, ATP or the proton motive force (PMF). Both pmf and ATP are made by central metabolism. Active transport proteins may be highly specific and carry only one molecule or may be more general and carry one class of molecules. An example of a general transport protein is the branch chain amino acid transporter of Pseudomonas aeruginosa, which transports leucine, valine, and isoleucine.

PASSIVE TRANSPORT Osmosis Cytoplasm

Solute

Carrier molecule

Cytoplasm

Carrier molecule

ATP or protonmotive force

Solute

GROUP TRANSLOCATION

FACILITATED DIFFUSION

ACTIVE TRANSPORT

Figure 36: Types of transport across membranes

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4) Energy generation: The membrane is site for many reactions that lead to the generation of energy. Many cells use respiratory processes to obtain their energy. During respiration, organic or inorganic compounds that contain high energy electrons are broken down, releasing those electrons to do work. These electrons find their way to the membrane where they are passed down a series of electron acceptors. During this operation, protons are transported outside the cell. The outside of the membrane becomes positively charged; the inside becomes negatively charged.

This proton gradient energizes the membrane, much like a battery is charged. The energy can then be used to do work directly, a process known as the proton motive force, or can be channeled into a special protein known as ATP synthase: ATP synthase can convert ADP to ATP and the ATP can do work itself. Photosynthetic cells also have a membrane system. Here light excites electrons and the electrons are again passed down through a series of electron carriers, a proton motive force is generated and ATP is synthesized. All the photosynthetic machinery is situated in the membrane. 5) Synthesis: Membranes also contain specialized enzymes that carry out many biosynthetic

functions. These functions include: Membrane synthesis Cell wall assembly Secretion of many proteins.

Mesosomes and Infoldings of the Membrane

Mesosomes

Mesosomes are membrane invaginations found in the form of systems of convoluted tubules and vesicles. They are found in both Gram positive and Gram negative organisms. Central mesosomes penetrate deeply into the cytoplasm, are located near the middle of the cell and involved in DNA replication and cell division. Peripheral mesosomes show only a shallow penetration into the cytoplasm, are not restricted to a central location and not associated with nuclear material. They are involved in export of exocellular enzymes such as penicillinase.

e

Figure 37: An electron micrograph and schematic of a mesosome

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Other infoldings

Although the width of the cell membrane is fixed, the area is not. Some bacteria have complex infoldings. These are attached to the cytoplasmic membrane but serve to increase its area. These infoldings are found in photosynthetic and rapidly respiring bacteria. The infolding provides additional surface area and contact with the environment needed for high metabolic rates.

Figure 38: The intercytoplasmic membrane (ICM) of Rhodobacter sphaeriodes

Importance of Surface Components

All of the various surface components of a prokaryotic cell are important in its ecology since they mediate the contact of the cell with its environment. The only "sense" that a bacterium has results from its immediate contact with its environment. It must use its surface components to assess the environment and respond in a way that supports its own existence and survival in that environment. The surface properties of a prokaryote are determined by the exact molecular composition of its plasma membrane and cell wall, including LPS, and the function of surface structures such as flagella, fimbriae and capsules. Some important ways that bacteria use their surface components are:

1. As permeability barriers that allow selective passage of nutrients and exclusion of harmful substances;

2. As "adhesins" used to attach or adhere to specific surfaces or tissues; 3. As enzymes to mediate specific reactions on the cell surface important in the survival of

the bacteria; 4. As "sensing proteins" that can respond to temperature, osmolarity, salinity, light, oxygen,

nutrients, etc., resulting in a signal to the genome of the cell that will cause a beneficial response to the new environment.

Cytoplasm

The cytoplasm or protoplasm is the portion of the cell that lies within the cytoplasmic membrane. The cytoplasmic matrix is defined as substances within the plasma membrane, excluding the genetic material. It is relatively featureless by electron microscope - although small granules can be seen. However, the cytoplasm carries out very important functions for the cell.

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RibosomesPlasma membraneChromosome

Protein Fibrils

SeptumCell wall

Figure 39: The complex structure of Streptococcus pyogenes Characteristics

• Gel-like consistency • Compartmentalization

o The glycolysis enzymes are organized into a unit and degrade substrate in an assembly line fashion.

o Many DNA synthesis enzymes congregate at the replication fork. • Constituents

o Ribosomes o Proteins including enzymes o Vitamins o Ions o Nucleic acids and their precursors o Amino acids and their precursors o Sugars, carbohydrates and their derivatives o Fatty acids and their derivatives

Function

The cytoplasm holds many cellular constituents, including the cell pools. It is within the cytoplasm that many of the functions for cell growth, metabolism and replication are carried out. Cytoplasmic Constituents

The cytoplasmic constituents of bacteria invariably include the prokaryotic chromosome and ribosomes. The chromosome is typically one large circular molecule of DNA, more or less free in the cytoplasm. Prokaryotes sometimes possess smaller extrachromosomal pieces of DNA called plasmids. The total DNA content of a cell is referred to as the cell genome. During cell growth and division, the prokaryotic chromosome is replicated in the usual semi-conservative fashion before for distribution to progeny cells. However, the eukaryotic processes of meiosis and mitosis are absent in prokaryotes. Replication and segregation of prokaryotic DNA is coordinated by the membrane, possibly by mesosomes.

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General Function of Cytoplasmic Constituents

Nucleic acids, Ribonucleic acid (RNA) and Deoxyribonucleic acid (DNA), serve as storage units for hereditary information. DNA can be thought of as a large cookbook with recipes for making every protein in the cell. RNA helps the ribosome translate the information in DNA into protein. Specific regions of interest in the cytoplasm

Genetic regions - The regions containing the hereditary information for the cell. Proteins - Found throughout the cell either as reaction catalysts or in various structures

of the cell. Ribosomes - The protein synthesis machinery. Mesosomes- involved with segregation of newly replicated chromosomes.

The Genetic Regions

Bacterial cells lack a membrane defined nucleus. However a discrete region in the bacterial cytoplasm seems to contain the genetic material and this nucleoid region can be made visible under the light microscope by Feulgen staining and by electron microscopy it appears as a light area with a delicate fibrillar structure. Most cells have only one main chromosome although a few species do have 2 or more. A: The Bacterial Chromosome The bacterial chromosome consists of a single, circle of deoxyribonucleic acid termed as the nucleoid. Structure of the building blocks of life Despite their importance in cellular function, nucleic acid structure is surprisingly simple. RNA and DNA are long polymers of only 4 nucleotides, adenine, guanine, cytosine and thymine (or uracil for RNA).

Figure 40: The structure of nucleotides The nucleotide structure can be broken down into 2 parts - the sugar-phosphate backbone and the base. All nucleotides share the sugar-phosphate backbone. Nucleotide polymers are formed by

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linking the monomer units together using oxygen on the phosphate, and a hydroxyl group on the sugar. A, T, G and C are capable of being linked together to form a long chain. The 3'-hydroxyl group on the ribose unit, reacts with the 5'-phosphate group on its neighbor to form a chain.

Figure 41: A schematic of the bonding of one DNA strand to another The base on each nucleotide is different, but they still show similarities. Adenine (A) and Guanine (G) are purines, having two ring structures, with the differences in the molecules coming in the groups attached to the ring. Likewise, Cytosine (C) and Thymine (T) and Uracil (U) are pyrimidines and share a similar structure, but differ in their side groups. Base Pairing If two strands of nucleic acid are adjacent to one another. The bases along the polymer can interact with complementary bases in the other strand. Adenine is capable of forming hydrogen bonds with thymine and cytosine can base pair with guanine. Adenine forms two hydrogen bonds with thymine, cytosine forms 3 with guanine. The G to C pair is 33% stronger than the A to T pair due to the extra hydrogen bond.

Figure 42: Base pairing between nucleic acids Cells contain two strands of DNA that are exact mirrors of each other. When correctly aligned, A can pair with T and G can pair with C. Because these strands are mirrors of each other, the amount of A is equal to the amount of T and the amount of C is equal to the amount of G in any double stranded DNA molecule. In solution, the two strands will usually find each other and form a double helix. This reaction is favorable because of the numerous hydrogen bonds that can

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be formed between the complementary bases. The DNA molecule can stretch for millions of base pairs and the DNA sizes of organisms can vary greatly. Note however that the size of the DNA genome is not always a measure of how advanced an organism is; otherwise one may think that news (genome size 19,000,000 kb) are more advanced than humans (genome size 3,500,000 kb). DNA is double stranded- two strands line up antiparellal to each other and the bases are linked together with hydrogen bonds. Due to the nature of these bonds the strands form left handed double helix.

Figure 43: The DNA Double Helix

Figure 44: Wire frame model of DNA (Side and Top View) The entire DNA molecule is a linear sequence of bases. The typical E. coli cell has 4.6 x 106 bases, this would make a strand of DNA 1400 m long, but E. coli is only 2-3 m long. The size of bacterial DNA is halved by the circular structure of the DNA, but much more is needed of

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course. A series of strategies are employed by microbes to compact the DNA, yet leave it accessible for its necessary functions. The relaxed circular chromosome, with a diameter of approximately 430 m, is first segregated into about 50 chromosomal domains by DNA-protein interactions with nucleotide binding proteins such as HU, IHF and H-NS. There is sufficient HU protein in the cell to bind the DNA every 300 to 400 bp and these proteins are thought to act in a fashion similar to the histones in eukaryotic organisms. An intact chromosome with its full complement of DNA binding proteins will result in a particle with a maximum diameter of 17 m. The DNA is further compacted by twisting the DNA in each domain around itself, called supercoiling.

Figure 45: Supercoiling of DNA A collection of enzymes (one of the more notable being DNA gyrase) is responsible for winding the DNA. Imagine the DNA as a rubber band held at one end. DNA gyrase twists the DNA about itself causing it to fold over. By repeating this process many times the DNA is organized into a series of supercoiled regions. A fully supercoiled chromosome will be about 1 m in diameter and is small enough to fit inside a bacterium. Functions of DNA 1) Protein production-The single most important purpose of the genetic material of any cell is

that it holds all the information necessary for a cell to carry out its many functions. The sequence of bases in the DNA contains this information or genetic code, which is transcribed into RNA and then translated into protein, the phenomena called Central Dogma.

Figure 46: DNA is transcribed into RNA that is then translated into protein by the

ribosome

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2) Replication - DNA must be able to replicate itself to pass on this genetic information. Since DNA is double stranded, the two strands separate and each one serves as a template to make another complementary strand.

This process is known as semi conservative replication. At each cell division, each cell gets one old strand and one new strand. Replication is accomplished by the coordinated efforts of many cellular enzymes (about 20). One of the best-understood enzymes is polymerase, which forms the phosphodiester bond between the phosphate residue on the sugar of the incoming nucleotide and OH residue on the sugar of the growing DNA chain. Synthesis is always 5' ->3'. This enzyme can also proof read and correct any mistakes made along the way. Because bacterial chromosomal DNA is supercoiled it makes replication a little trickier. For most bacterial chromosomes, replication of the circular DNA is bi-directional. This leads to a characteristic structure known as a theta structure (looks like the Greek letter theta)

Replication Fork

Figure 47: A sketch of Replicating DNA Plasmids Although bacterial cells have only one main chromosome, they may have other pieces of genetic material. These smaller pieces of DNA are known as plasmids and are defined as extrachromosomal pieces of DNA which are capable of autonomous (or regulated) replication. Structure It is similar to chromosome i.e. covalently closed circular DNA although considerably smaller. In a few species linear plasmids have been found. Size The size of a piece of DNA is referred by the number of 1000s of base pairs {kilobases (kb)}. Chromosomal DNA is typically about 4000 kb, plasmid DNA ranges from 1-200 kb. There may also be anywhere from 1-700 copies of a plasmid in a cell.

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Function The function of plasmids is not always known, but they are not normally essential to the host, although their presence generally gives the host some advantage. Advantages plasmids bestow on the host

• Antibiotic resistance - Some plasmids code for proteins that degrade antibiotics- a big advantage for pathogens.

• Some encode for proteins, which confer virulence factors on the host. For example- E. coli plasmid Ent P307 codes for an enterotoxin, which makes E. coli pathogenic.

• Conjugative plasmids - These allow exchange of DNA between bacterial cells B: Ribosomes The distinct granular appearance of prokaryotic cytoplasm is due to the presence and distribution of ribosomes. Ribosomes are a combination of protein and RNA. The ribosomes of prokaryotes are smaller than cytoplasmic ribosomes of eukaryotes. Prokaryotic ribosomes are 70S in size, being composed of 30S and 50S subunits. Ribosomes are involved in the process of translation (protein synthesis), but some details of their activities differ in eukaryotes, Bacteria and Archea. RNA RNA is similar in structure to DNA, except that uracil (U) takes the place of thymine in the molecule and the ribose unit on each sugar contains a hydroxyl group. RNA serves 3 functions in ribosomes. These functions center on translating the genetic information in DNA into protein. The three different RNA are as follows: Ribosomal RNA (rRNA) rRNA is part of the ribosome structure and assists in the catalytic role of the ribosome. During rapid growth the cell has to synthesize large amounts of rRNA and there are several copies of the rRNA genes on the chromosome.

Figure 48: A wire frame model of the 5S ribosomal RNA of E. coli

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Transfer RNA (tRNA) These are adapter molecules in protein synthesis that convert the genetic code from the language of nucleic acid to that of amino acids - the building blocks of proteins.

Figure 49: A diagrammatic representation of transfer RNA Messenger RNA (mRNA) Messenger RNA directs the incorporation of amino acids into proteins. It can be said as a "photocopy" of DNA that the ribosome works from. Protein There are 52 different proteins in ribosomes that perform all sorts of functions. These polypeptides, along with the ribosomal RNA will self-assemble into a functional unit if they are added together in the proper order. Functions of Ribosomes Ribosomes are the protein synthesizing factories of the cell. They translate the information in mRNA into protein sequences. Ribosomes also give the cytoplasm its granular look in the Electron Microscope. Often they aggregate to form structures known as "polysomes". Ribosomes sit down on mRNA at two sites. The A site, where the new amino acid is accepted and the P site, where the growing polypeptide is held.

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mRNA strand

Small subunit

Large subunit Growing polypeptide

Ribosome mRNA moves through the ribosomes in this direction

Completed polypeptide

end

Start

Figure 50: mRNA (Yellow strand) at work in the cell. The ribosomes (red and green) in this

picture are in a polysome structure Inclusions The cytoplasm of bacterial cells often contains, one or another of some type of inclusion granule. These Inclusions or Internal structures are microscopically visible bodies in the cell that are distinguishable from the general cytoplasm. They are aggregates of various compounds that are normally involved in storing energy reserves or building blocks for the cell. Inclusions accumulate when a cell is grown in the presence of excess nutrients and they are often observed under laboratory conditions. In strict sense Inclusions are distinct granules that may occupy a substantial part of the cytoplasm and are usually reserve materials of some sort. For example, carbon and energy reserves may be stored as glycogen (a polymer of glucose) or as polybetahydroxybutyric acid (a type of fat) granules. Polyphosphate inclusions are reserves of PO4 and possibly energy; elemental sulfur (sulfur globules) are stored by some phototrophic and some lithotrophic bacteria as reserves of energy or electrons. Some inclusion bodies are actually membranous vesicles or intrusions into the cytoplasm, which contain photosynthetic pigments or enzymes. Poly-beta-hydroxyalkanoate (PHA) One of the more common storage inclusions is PHA. It is a long polymer of repeating hydrophobic units that can have various carbon chains attached to them. The most common form of this class of polymers is poly-beta-hydroxybutyrate (PHB) that has a methyl group as the side chain to the molecule. Some PHA polymers have plastic like qualities and there is some interest

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in exploiting them as a form of biodegradable plastic. The function of PHA in bacteria is as a carbon and energy storage product.

Table 4: Some important cytoplasmic inclusions

Cytoplasmic inclusions Where found Composition Function

Glycogen many bacteria e.g. E. coli

polyglucose reserve carbon and energy source

Polybetahydroxybutyric acid (PHB)

many bacteria e.g. Pseudomonas

polymerized hydroxy butyrate

reserve carbon and energy source

Polyphosphate (volutin granules)

many bacteria e.g. Corynebacterium

linear or cyclical polymers of PO4

reserve phosphate; possibly a reserve of high energy phosphate

Sulfur globules phototrophic purple and green sulfur bacteria and lithotrophic colorless sulfur bacteria

elemental sulfur reserve of electrons (reducing source) in phototrophs; reserve energy source in lithotrophs

Gas vesicles aquatic bacteria especially cyanobacteria

protein hulls or shells inflated with gases

buoyancy (floatation) in the vertical water column

Parasporal crystals endospore-forming bacilli (genus Bacillus)

protein unknown but toxic to certain insects

Magnetosomes certain aquatic bacteria

magnetite (iron oxide) Fe3O4

orienting and migrating along geo- magnetic field lines

Carboxysomes many autotrophic bacteria

enzymes for autotrophic CO2 fixation

site of CO2 fixation

Phycobilisomes cyanobacteria phycobiliproteins light-harvesting pigments

Chlorosomes Green bacteria lipid and protein and bacteriochlorophyll

light-harvesting pigments and antennae

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Figure 51: Poly-beta-hydroxybutyrate (PHB) in a Rhodospirillum species

PHB is one type of PHA.

Figure 52: The general structure of a PHA monomer

Glycogen Glycogen is another common carbon and energy storage product. Glycogen is a polymer of repeating glucose units. Phosphate and sulfur globules Many organisms accumulate granules of polyphosphate, since this is a limiting nutrient in the environment. The globules are long chains of phosphate. Photosynthetic bacteria that do not evolve oxygen often use sulfides as their source of electrons; some of them accumulate sulfur globules. These globules may later be further oxidized and disappear if the sulfide pool dries up. Gas vesicles Gas vesicles are the exception to the rule that all bacterial cells have one contiguous membrane. Gas vesicles are found in Cyanobacteria, which are photosynthetic and live in aquatic systems.

Figure 53: A Gas vesicle

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Structure Gas vesicles are aggregates of hollow cylindrical structures composed of rigid proteins. They are impermeable to water, but permeable to gas. The amount of gas in the vacuole is under the control of the microorganism. Function Gas vesicles regulate the buoyancy of the microbes by changing the amount of gas contained within them. Release of gas from the vesicle causes the bacterium to fall in the water column, while filling the vesicle with gas increases their height in the water. Endospores Endospores are highly resistant resting structures produced within cells. They are common to organisms which live in soil and may need to wait out some rough times such as >100 0C heat, radiation, or drying. Spores are resistant to heat, radiation, chemicals, and desiccation. The mechanism that accounts for this, include the dehydration of the protoplast and the production of special proteins that protect the spores DNA. Spores are capable of detecting their environment and under favorable nutrient conditions germinating and returning to the vegetative state.

Figure 54: Endospore-forming bacilli (phase contrast illumination) The two important genera that form endospores are Bacillus, which are aerobic sporeformers in the soils, and Clostridium, whose species are anaerobic sporeformers of soils, sediments and the intestinal tracts of animals. Characteristics Endospores are produced within cells and are refractile - light cannot penetrate them so that they are very easy to see in the phase microscope. They are resting structures, meaning that there is little or no metabolism inside the spore and it is a real form of suspended animation. Spores can survive for a very long time, and then regerminate. Spores that were dormant for thousands of years in the great tomes of the Egyptian Pharaohs were able to germinate and grow when placed in appropriate medium. There are even claims of spores that are over 250 million years old being able to germinate when placed in appropriate medium. These results have yet to be validated. Endospores differ from the vegetative cells that form them in a variety of ways. Several new surface layers develop outside the core (cell) wall, including the cortex and spore coat. The cytoplasm is dehydrated and contains only the cell genome and a few ribosomes and enzymes. The endospore is cryptobiotic (exhibits no signs of life) and is remarkably resistant to

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environmental stress such as heat (boiling), acid, irradiation, chemicals and disinfectants. Some endospores have remained dormant for 25 million years preserved in amber, only to be shaken back into life when extricated and introduced into a favorable environment.

Table 5: Differences between endospores and vegetative cells Property Vegetative cells Endospores

Surface coats Typical Gram-positive murein cell wall polymer

Thick spore coat, cortex, and peptidoglycan core wall

Microscopic appearance Nonrefractile Refractile

Calcium dipicolinic acid Absent Present in core

Cytoplasmic water activity High Very low

Enzymatic activity Present Absent

Macromolecular synthesis Present Absent

Heat resistance Low High

Resistance to chemicals and acids Low High

Radiation resistance Low High

Sensitivity to lysozyme Sensitive Resistant

Sensitivity to dyes and staining Sensitive Resistant

Parts of the Spore

• Core - The core is dehydrated cytoplasm containing DNA, ribosomes, enzymes etc. It is the central part of the spore and is made up of dipicolinic acid (DPA). DPA occurs in combination with calcium; calcium- DPA complex provides resistance to endospores.

• Cortex - The cortex is a modified cell wall/peptidoglycan layer that is not as cross-linked as in a vegetative cell. It is largely composed of a unique peptidoglycan, containing three repeating N-acetyl-glucosamine-muramic acid dimers differing with respect to substitutions on the lactic acid moiety of muramic acid: a muramic lactam subunit, without any attached amino acids; an alanine subunit, bearing only an L-alanyl residue; and a tetrapeptide subunit, bearing the sequence L-ala-D-glu-meso-DAP-D-ala. These subunits represent 55, 15 and 30 percent respectively.

• Coats - Outside of the cortex are several protein layers that are impermeable to most chemicals. The coat is responsible for the spores’ resistance to chemicals. Outer spore coat represents 30 to 60 percent of the dry weight of the spore and is composed of protein. Spore coat proteins have an unusually high content of cysteine and of hydrophobic amino acids, and are highly resistant to treatments that solubilize most proteins.

• Exosporium – An outer protective layer

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DNA

Core wall

Spore CoatCortex

Exosporium

Figure 55: Bacterial endospores Spore life cycle The formation of a spore is an expensive and complex process for the bacterial cell. Spores are only made under conditions where cell survival is threatened such as starvation for certain nutrients or accumulation of toxic wastes. Regulation of sporulation is tight and the first few steps are reversible. This helps the cell conserve energy and only sporulate when necessary. Sporulation is a seven stage process. The first step is the formation of an axial chromatin thread and formation of a separate compartment for the spore in the mother cell by a transverse septum. The membrane of the larger cell rapidly grows around the smaller cell, which thus becomes completely engulfed within cytoplasm of the larger cell, to produce a so-called forespore. Once this occurs, sporulation is irreversible. The next stages involve laying down the various layers of the spore. The first to appear is the cortex, which developes between the two membranes surrounding the fore spore. Afterward, a more electron dense layer, the spore coat begins to form exterior to both membranes surrounding the cortex. Once the sporecoat is synthesized, the maturing spore becomes refractile. The heat resistance of the spore is due to the presence of Calium- dipicolinic acid. In the final stages, the spore dehydrates its cytoplasm and is released from the cell.

Figure 56: The sequential steps in the process of endospore formation in Bacillus subtilis.

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Figure 57: The developmental cycle of the Endospore

Some sporeformers are pathogens of animals, usually due to the production of powerful toxins. Bacillus anthracis causes anthrax, a disease of domestic animals (cattle, sheep, etc.) which may be transmitted to humans. Bacillus cereus is becoming increasingly recognized as an agent of food poisoning. Clostridium botulinum causes botulism a form of food-poisoning, and Clostridium tetani causes tetanus. In association with the process of sporulation, some Bacillus species form a crystalline protein inclusion called parasporal crystals. The protein crystal and the spore (actually the spore coat) are toxic to lepidopteran insects (certain moths and caterpillars) if ingested. The crystals and spores of Bacillus thuringiensis are marketed as "Bt" a natural insecticide for use on garden or crop plants. Another species of Bacillus, B. cereus, produces an antibiotic that inhibits growth of Phytophthora, a fungus that attacks alfalfa seedling roots causing a "damping off" disease. The bacteria, growing in association with the roots of the seedlings, can protect the plant from disease. Also, apparently in association with the sporulation process, some Bacillus species produce clinically-useful antibiotics. Bacillus antibiotics such as polymyxin and bacitracin are usually polypeptide molecules that contain unusual amino acids.

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Cysts Cysts are dormant, thick-walled, desiccation-resistant forms that develop by differentiation of a vegetative cell and which can later germinate under suitable conditions. Cysts in some way resemble endospores, but their structure and chemical composition are different and they do not have the high heat resistance of endospores. An example of a cyst is the structurally complex type produced by the genus Azotobacter. Several other bacteria can differentiate into cyst like forms, but these lacks the degree of structural complexity characteristic of Azotobacter cysts. Eukaryotic Microbial Cell: General Structure and Function

The structures that make up a eukaryotic cell are determined by the specific functions carried out by the cell. Nevertheless, eukaryotic cells generally have three main components: a cell membrane, a nucleus, and other organelles.

Figure 58: Structural details of typical eukaryotic cell

The Cell Membrane

The cell membrane is a complex barrier separating the cell from it's external environment. The membrane is "selectively permeable" and regulates passage of substances into and out of the cell. All the cells of all organisms are surrounded by a cell membrane. The cell membrane controls the ease with which substances pass into and out of the cell; some substances easily cross the membrane, while others cannot cross at all and thus it is said to be selectively permeable. Structure Electron microscopic examinations of cell membranes have led to the development of the lipid bilayer model (also referred to as the fluid-mosaic model). FLUID MOSAIC MODEL proposed

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by Singer and Nicolson is based on the fact that lipids and proteins can readily move laterally and also undergo rotation. The degree of membrane fluidity is determined by temperature and lipid composition.

Figure 59: Structure of cell membrane

Cell membranes are chiefly composed of phospholipid molecules (Phosphate + Lipid). Phospholipids are a kind of lipids that consist of two fatty acids (tails), and a phosphate group (head). Phospholipids are amphipathic meaning that they possess both a hydrophobic and hydrophilic end. A phospholipid molecule has a polar "head" and two nonpolar "tails". The phosphate head is hydrophilic meaning "water loving". Because of its hydrophilic nature, the head of a phospholipid orients itself such that it is as close as possible to water molecules. The lipid tails are hydrophobic meaning "water-fearing", and tend to orient themselves away from water. Cell membranes consist of two phospholipid layers called a lipid bilayer. The lipid bilayer behaves as a fluid. Heads face the aqueous environment inside and outside the cell whereas the lipid tails are sandwiched within the bilayer. A variety of protein molecules are embedded in the lipid bilayer. Some proteins are attached to the surface of the cell membrane; these are called peripheral or extrinsic proteins, and are located on both the internal and external surface. The proteins embedded in the lipid bilayer are called integral or intrinsic proteins. Some integral proteins extend across the entire cell membrane and are exposed to both the inside of the cell and the exterior environment. Others extend only to the inside or only to the exterior surface. There occur many kinds of proteins in membranes; they help to transport substances into and out of the cell. Some integral proteins form channels or pores through which selective substances can pass. Other proteins called carrier proteins bind to a substance on one side of the membrane and carry it to the other side of the membrane. Integral proteins exposed to the cell's external environment often have carbohydrates attached to them serving as identification badges that allow cells to recognize each other and may act as sites where viruses or chemical messengers such as hormones can attach. Lipids with shorter fatty acid chains are less rigid and remain fluid at lower temperatures because interactions between shorter chains are weaker than for longer chains. Lipids containing unsaturated fatty acids increase membrane fluidity. The double bonds introduce kinks, preventing tight packing of the fatty acids. Cholesterol with its hydrocarbon ring structure plays a distinct role in determining membrane fluidity. Polar hydroxyl group positions close to the

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phosphate head group. Rigid rings interact with regions of fatty acid chain adjacent to phospholipid head groups. This interaction decreases the mobility of the outer portions of the fatty acid chains, making this region of the membrane more rigid, even at higher temperatures. On the other hand insertion of cholesterol interferes with interactions between fatty acids, thereby maintaining fluidity at lower temperatures. Cholesterol is not present in bacteria or plant cells. Plant cell membranes do contain sterols that function in a manner similar to cholesterol. In the fluid mosaic model of the membrane, there are membrane proteins inserted into the lipid bilayer. The lipids provide the basic structure, but proteins carry out the specific functions of the different types of membranes. The four major phospholipids found in cell membranes are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin - non-glycerol phospholipids. Various glycolipids are also found in the outer margin of the cell membrane. Cholesterol is another important constituent of the animal cell membrane. Lipid composition differs in the various types of cells and in different types of organisms. The average eukaryotic plasma membrane comprises 50% by mass of lipid and 50% protein.

Figure 60: Basic principle of fluid mosaic model The integral membrane proteins are also referred to as transmembrane proteins. Commonly, integral membrane proteins have membrane spanning domains, which are alpha helical. There may be 1, 2, 7 or more membrane spanning alpha helical domains. The alpha helix neutralizes the polar character of the peptide bonds. The hydrophobic side chains associated with these amino acids interact with the fatty acid chains of membrane lipids. Most transmembrane proteins are also glycosylated (have carbohydrate groups attached). The peripheral membrane proteins are not embedded in the bilayer but remain indirectly associated with the membrane through interactions with integral membrane proteins or by weak electrostatic interactions with the hydrophilic head groups of membrane lipids. They are located extracellular or associated with the cytoplasmic surface of the bilayer. There occur lipid-anchored proteins located outside the lipid bilayer, but covalently linked to a lipid molecule that is situated within the bilayer. An increasingly large number of proteins are found to be linked by a short oligosaccharide to a molecule of glycophosphatidylinositol (GPI) that is embedded in the outer portion of the lipid bilayer. Another group of proteins are actually present on the cytoplasmic side of the membrane and are anchored by long hydrocarbon chains embedded in the inner leaflet of the lipid bilayer.

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The extracellular portion of the plasma membrane proteins is generally glycosylated. LIkewise, the carbohydrate portions of glycolipids are exposed on the outer face of the plasma membrane. Consequently, the glycocalyx, is formed by the oligosaccharides of glycolipids and transmembrane glycoproteins. Functions

• Maintains cell shape. • Serves as a barrier between the external and internal environment of the cell. • Renders flexibility to the cell. • Functions as a mode of various forms of cellular transport. • Markers for cell-cell interactions.

Cell Wall

Certain eukaryotic microbial cells possess definite cell walls such as Fungi and Algae. Their cell walls exhibit important differences based on their chemical composition. Structure

Cell walls of fungi are composed of chitin. Algae cells are surrounded by a rigid cell wall enclosing the cell membrane. The rigidity of cell walls helps to support and protect the cell. Cell walls of algal cells contain polysaccharide, cellulose a complex carbohydrate. Cell walls are of two types:

A. Primary cell wall - While a algal cell is being formed, a primary cell wall develops just outside the cell membrane. As the cell expands in length, cellulose and other molecules are added, enlarging the cell wall. When the cell reaches full size, a secondary cell wall may form.

B. Secondary cell wall - The secondary cell wall forms between the primary cell wall and the cell membrane. The secondary cell wall is tough and rigid. Once a secondary cell wall forms, an algal cell can grow no further.

Functions

• Provide a rigid shape to the cells. • Protects the cell from adverse external factors.

Cytoplasm

The space between the cell membrane and the nucleus is occupied by the cell's cytoplasm. Cytoplasm consists of two main components: cytosol and organelles. Cytosol is a gel-like matrix that consists mostly of water, along with proteins, carbohydrates, salts, minerals and organic molecules (pH usually around 6.8 – 7.0). Suspended in the cytosol are tiny organelles (organs). Organelles are structures that function like miniature organs, carrying out specific functions in the cell. The organelles alongwith the cytosol make up the cytoplasm. In eukaryotic microbial cells, a membrane surrounds most organelles. The cytoplasm was defined earlier as the material between the plasma membrane (cell membrane) and the nuclear envelope. Fibrous proteins that occur in the cytoplasm, referred to as the cytoskeleton maintain the shape of the cell as well as anchoring organelles, moving the cell and controlling internal movement of structures. Microtubules function in cell division and serve as a "temporary scaffolding" for other

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organelles. Actin filaments are thin threads that function in cell division and cell motility. Intermediate filaments are between the size of the microtubules and the actin filaments. Contains many ribosomes, which are particles on which proteins are synthesized, and many enzymes for general cell metabolism. Functions

• Serves as the ground substance for life. • It contains all the cell organelles. • It maintains the chemical composition of the cell. • It maintains the cell pressure. • Provides medium for all major cell reactions.

Nucleus

The nucleus is the most prominent structure within a eukaryotic cell. It maintains its shape with the help of a protein skeleton known as the nuclear matrix. The nucleus is the control center of the cell. Most eukaryotic microbial cells have a single nucleus; some cells have more than one. Structure

A double layer membrane called the nuclear envelope surrounds the nucleus. Nuclear membranes act as barriers that prevent the free passage of molecules between the nucleus and the cytoplasm. Two concentric membranes (phospholipid bilayers) are called the inner and outer nuclear membranes. The outer nuclear membrane is continuous with the Endoplasmic Reticulum (ER) so the space between the inner and outer membrane is directly connected with the lumen of the ER. This space is termed the perinuclear space. The nuclear envelope possesses many small pores through which proteins and chemical messages from the nucleus can pass. Channels through membrane form nuclear pores through which selective traffic of proteins and RNAs occur. The nucleus contains DNA, the hereditary material of cells. The DNA is in the form of a long strand called chromatin. During cell division, chromatin strands coil and condenses into thick structures called chromosomes. The chromosomes in the nucleus contain coded "blueprints" that control all cellular activities. Most nuclei contain at least one nucleolus. The nucleolus synthesizes ribosomes, which in turn, build proteins. When a cell prepares to reproduce, the nucleolus disappears. The nucleus occurs only in eukaryotic cells, and is the location of the majority of different types of nucleic acids. Ribonucleic acid, RNA, is formed in the nucleus by coding off of the DNA bases. RNA moves out into the cytoplasm. The nucleolus is an area of the nucleus (usually 2 nucleoli per nucleus) where ribosomes are constructed. Functions

The nucleus serves as the repository of genetic information and the cell's control center. DNA replication, transcription, RNA processing all take place within the nucleus. Site of RNA synthesis.

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Figure 61: Structure of the nucleus

Mitochondria

Found in most eukaryotic cells called the powerhouse of the cell. It is the site where tricarboxylic acid cycle activity and the generation of ATP by the electron transport and oxidative phosphorylation occurs. Mitochondria are found dispersed throughout the cytosol, and are relatively large organelles. A few cells have a singular giant tubular mitochondrion twisted into a continuous network permeating the cytoplasm (some yeasts, unicellular, algae, and trypanosome protozoa). Mitochondria are the sites of chemical reactions releasing energy from organic compounds to ATP during cellular respiration. ATP is the molecule that the cells use as their main energy currency. Therefore, mitochondria are termed as the "powerhouse" of the cell. Mitochondria are usually more numerous in cells that have a high energy requirement. Structure

Mitochondria is surrounded by two membranes, the outer and inner membrane possessing different lipids. The smooth outer membrane serves as a boundary between the mitochondria and the cytosol. The inner membrane has many long folds; known as cristae. The cristae greatly increase the surface area of the inner membrane, providing more space for the chemical reactions to occur. The inner membrane encloses the dense mitochondrial matrix containing ribosomes, DNA, enzymes and electron carriers involved in electron transport system and large calcium phosphate granules. F1 particles are spheres attached by stalk to the inner surface of the inner membrane and synthesize ATP during cellular respiration. Mitochondrial ribosomes are smaller than cytoplasmic ribosomes and resemble bacterial ribosomes in size, subunit composition and closed circular DNA. Mitochondria have their own DNA, and new mitochondria arise by the division of the existing ones.

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Functions

• Mitochondria function as the sites of energy release (following glycolysis in the cytoplasm) and ATP formation (by chemiosmosis).

• They possess a compendium of enzymes in the mitochondrial matrix and membrane. • They function in cellular respiration.

Figure 62: Structure of a mitochondrion Endoplasmic Reticulum (ER)

Endoplasmic reticulum is a mesh of interconnected membranes that serve a function involving protein synthesis and transport. Structure

The endoplasmic reticulum is a system of membranous tubules and sacs. The amount of ER inside a cell fluctuates, depending on the cell's activity. ER may be either rough or smooth.

Figure 63: The endoplasmic reticulum

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• Rough ER is studded with ribosomes and processes proteins to be exported from the cell. Rough ER is an extensive network of membranes that connect the nuclear envelope to the cell membrane.

• Smooth ER lacks the ribosomes and processes lipids and carbohydrates. The smooth ER is involved in the synthesis of steroids in gland cells, the regulation of calcium levels in muscle cells, and the breakdown of toxic substances by liver cells.

Functions

• ER functions primarily as an intracellular highway, a path along which molecules move from one part of the cell to another.

• Poisons, waste, and other toxic chemicals are made harmless. • They transport proteins, lipids and other materials through the cell and serve as the major

site of cell membrane synthesis. Ribosomes

Unlike most other organelles, a membrane does not surround ribosomes. They are not membrane-bound and thus occur in both prokaryotes and eukaryotes. They are the most numerous organelles in almost all the cells. Some occur free in the cytoplasm; others line the membranes of rough endoplasmic reticulum. Ribosomes are the sites of protein synthesis. Structure

The ribosome consists of a small and larger subunit. Eukaryotic ribosomes (80S) are larger than the prokaryotic (70S) ribosomes and are composed of 60S and 40S subunits. Biochemically the ribosome consists of ribosomal RNA (rRNA) and some 50 structural proteins. Often ribosomes cluster on the endoplasmic reticulum, in which case they resemble a series of factories adjoining a railroad line. Ribosome size measured in Svedberg (S) units; derived from sedimentation in ultracentrifuge.

Figure 64: Structure of the ribosome

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Functions

• Ribosomes provide sites for protein synthesis in a cell. Golgi Apparatus

The golgi apparatus is the processing, packaging and secreting organelle of the cell. Structure

The golgi apparatus is a system of membranes composed of flattened sac like structures called cisternae and spherical vacuoles.

Figure 65: Structure of the Golgi apparatus Functions

It works in association with the endoplasmic reticulum and the golgi apparatus modifying proteins for export by the cell.

Figure 66: Functioning of Golgi apparatus

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Lysosomes

Lysosomes are small spherical organelles that enclose hydrolytic enzymes within a single membrane. Lysosomes are common in the cells of fungi, and protists. Lysosomes are the site of food digestion in the cell. Structure

Lysosomes are formed from pieces of the golgi apparatus that break off. Structurally, they are small membrane-bound vesicles that contain digestive (hydrolytic enzymes). The lysosome membrane itself is able to resist the digestive action of its own enzymes. The vesicles are released from the golgi complex and are dispersed throughout the cytoplasm. Over 50 different enzymes present. Functions

• Lysosomes play an important role in the digestion of internalized particles (phagocytosis) and macromolecules (pinocytosis/receptor-mediated endocytosis).

• Lysosomes function for the digestion and break down of old cellular components, which otherwise tend to accumulate and interfere with proper cell function. In addition, when a cell dies, the lysosome membrane breaks down, releasing digestive enzymes into the cytoplasm, where they break down the cell itself.

• Some forms of tissue damage as well as the aging process may be related to "leaky lysosomes".

Vacuoles

A prominent structure in algal cells is the large vacuole. Cells of other eukaryotic microorganisms may also contain vacuoles, but they are much smaller and are usually involved in food digestion. Structure

The vacuole is a large membrane-bound sac that occupies a large amount of space in most algal cells. The vacuole serves as a storage area, and may contain stored proteins, ions, waste, or other cell products. Vacuoles of some algae contain toxic substances that protect the organism from enemies. Vacuoles are single-membrane organelles that are essentially part of the outside that is located within the cell. The single membrane is known as a tonoplast. Contains acid hydrolases and the pH of the vacuole is maintained at a low value.

Functions

1. The vacuoles play an important function as a storage compartment. 2. Vacuoles are also involved in plant cell enlargement and in maintaining cell rigidity. 3. Protozoa often have food or digestion vacuoles, which fuse with lysosomes so that the food

can be digested. 4. Many also have contractile vacuoles which function to remove excess water from the cell. 5. Carries out many of the functions of the lysosome in the algal cell.

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6. Compounds noxious to predators or various compounds used in the algal defense against pathogens are often stored in vacuoles.

Plastids

Plastids are cytoplasmic organelles of algae that often possess pigments such as chlorophylls and carotenoids and are the site of synthesis and storage of food reserves. A common kind of plastid is the chloroplast, an organelle that converts carbon dioxide, and water in the presence of sunlight into sugars by photosynthesis. Structure

Plastids are encompassed by two membranes. Stroma is the matrix lying within the inner membrane containing DNA, ribosomes, lipid droplets, starch granules, and thylakoids. Chloroplasts are variable in size and shape usually oval. Each chloroplast encloses a system of flattened, membranous sacs called thylakoids. The thylakoids are stacked on each other like coins to form grana (granum). Photosynthesis occurs in the thylakoids. Chloroplasts are green because they contain chlorophyll, a pigment that absorbs energy of sunlight. Chloroplasts contain many different types of accessory pigments (carotenes and xanthophylls), depending on the taxonomic group of the organism being observed. They are found in algae, such as seaweed, and in green plants. Other plastids store reddish-orange pigments that color fruiting bodies and certain other parts. Chloroplasts of many algae contain a pyrenoid. Pyrenoid is a dense region of protein surrounded by starch or another polysaccharide and participates in polysaccharide synthesis. Leukoplasts store starch, sometimes protein or oils. Chromoplasts store pigments associated with the bright colors of fruiting bodies.

Figure 67: Structure of the chloroplast

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Functions

• Plastids serve as sites of synthesis and storage of food reserves. • Chloroplasts are the chief organelles for photosynthesis. • Chromoplasts store varied coloured pigments imparting colouration to the parts of the

organisms. • Leukoplasts store starch, protein or oils.

Microbodies

Microbodies are membrane-bound organelles containing enzymes that regulate various metabolic reactions. One type of microbody, the peroxisome, regulates the conversion of fats to carbohydrates. During the breakdown of fats, hydrogen peroxide is produced. Peroxisomes contain enzymes (including Catalase) that split hydrogen peroxide into water and oxygen, making it harmless. Proteins found in the peroxisomes are synthesized in the cytoplasm of the cell and then transported to the peroxisome. Peroxisomes often contain a dense crystalline core consisting of one of the oxidative enzymes. Peroxisomes in certain cells may be important in detoxifying certain compounds such as ethanol in alchoholic beverages. Glyoxysomes are abundant in the seeds of certain plants. They contain enzymes, which convert stored fats to sugars. These sugars are used as an energy source and as a component for making essential compounds. They also contain catalase and the enzymes for fatty acid oxidation like peroxisomes. Functions

• They contain various enzymes regulating several metabolic processes. • The enzymes catalyse the conversion of fats to carbohydrates.

Cytoskeleton

Cytoskeleton is an internal framework serving to maintain the shape and size of the cells. The cytoskeleton maintains the three-dimensional structure of the cell, participates in the movement of organelles within the cytosol, and helps the cell to move. Structure

The cytoskeleton is a network of long protein strands located in the cytosol, that are not surrounded by a membrane. The cytoskeleton consists of two types of structures: microfilaments and microtubules. Another filamentous component found in the cytoplasm alongwith the microtubules and microfilaments are intermediate filaments making up the cytoskelton. 1. Microfilaments

• Microfilaments have a structure resembling rope made of two twisted chains of protein called actin. Microfilaments, which can contract, causing movement. Found in cytoplasm of most eukaryotes.

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• Involved in cell support, pinching off of daughter cells after mitosis (in animals), cytoplasmic streaming (in plants).

Figure 68: The cytoskeleton

2. Microtubules • Microtubules are hollow tubes found in cytoplasm of all eukaryotes. They are the largest

strands of the cytoskeleton. Microtubules are made of a protein called tubulin (alpha tubulin and beta tubulin). Microtubules serve three functions: maintain the shape of the cell, serve as tracks for organelles to move along within the cell, and when the cell is about to divide, bundles of microtubules known as spindle fibers come together and extend across the cell to assist in the movement of chromosomes during cell division.

• Involved in many structures: cilia, flagella (9+2 arrangement); spindle fibers that polymerize from centrioles during mitosis/meiosis.

• Two motor proteins allow motion along microtubules – dynein and kinesin. 3. Intermediate filaments

• Made from keratin subunits. • Not so quickly assembled and disassembled as microtubules or microfilaments. • May be involved in resisting tension, reinforcing cell shape, fixing location of nucleus

Cilia and Flagella

Cilia and flagella are hair like organelles extending from the surface of the cell, where they assist in movement.

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Structure

Microtubules are bundled into structures called cilia and flagella. Cilia are short hair like projections. Flagella are long whip like projections. They both have the characteristic 9 + 2 arrangement of microtubules. The cilia and flagella of all eukaryotes consist of one central pair of microtubules surrounded by nine peripheral pairs. Cilia are often numerous, flagella are often single. Unicellular organisms such as Paramecium and Euglena use cilia and flagella to move through water. Cell movement; is internal, referred to as cytoplasmic streaming and external, referred to as motility. Internal movements of organelles are governed by actin filaments.

Figure 69: The 9+2 arrangement of microtubules in a flagellum or cilium

Figure 70: Detailed cross-sectional view of eukaryotic flagella

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Functions

• Flagella work as whip pulling (as in Chlamydomonas or Halosphaera) or pushing (dinoflagellates, a group of single-celled Protista) the organism through the water.

• Cilia work like oars on a viking longship (Paramecium has 17,000 such oars covering its outer surface).

• Pseudopodia are used by many cells, such as Amoeba.

Figure 71: Movement of cilia and flagella

Intracellular Junctions

In multicellular organisms, several types of specialized junctions hold adajcent cells together. Tight junctions (found in animals): specialized "belts" that bind two cells tightly to each other, and prevent fluid from leaking into intracellular space. Desmosomes (found in animals): intercellular "rivets" that create tight bonds between cells, but allow fluids to pass through intracellular spaces. Gap junctions (found in animals): formed by two connecting protein rings embedded in cell membrane of adjacent cells. Allows passage of water, small solutes, but not macromolecules (proteins, and nucleic acids). Plasmodesmata (found in plants): channels connecting cells; allow free passage of water and small solutes, but not macromolecules (proteins, and nucleic acids).

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Specialized structures

Algae

The algae (singular alga) consist of several different groups of living organisms that capture light energy through photosynthesis, converting inorganic substances into simple sugars using the captured energy. Forms of algae

Most of the simpler algae are unicellular flagellates or amoeboids, but colonial and non-motile forms have developed independently among several of the groups. Some of the more common organizational levels, more than one of which may occur in the life cycle of a species, are:

• Colonial - small, regular groups of motile cells • Capsoid - individual non-motile cells embedded in mucilage • Coccoid - individual non-motile cells with cell walls • Palmelloid - non-motile cells embedded in mucilage • Filamentous - a string of non-motile cells connected together, sometimes branching • Parenchymatous - cells forming a thallus with partial differentiation of tissues

In three lines even higher levels of organization have been reached, leading to organisms with full tissue differentiation. These are the brown algae—some of which may reach 70 m in length (kelps)—the red algae, and the green algae. The most complex forms are found among the green algae (Charales), in a lineage that eventually led to the higher land plants. Representative algae

• Red algae (Division Rhodophyta) Evolution: Red algae are some of the oldest eukaryotic organisms on the planet. Fossils of red algae have been found that are over 2 billion years old. Habitat: There are 4000 different species of red algae. They are very abundant in tropical and warm waters, although many are found in cooler waters. Red algae are typically found in marine waters attached to rocks or other plants in the calmer, deeper waters beyond the tidal zone. Some red algae are reef builders in tropical seas. Structure: Their size and complexity vary from thin films growing on rocks to complex filaments or membranes growing to heights approaching one meter. Their accessory pigments called phycobilins mask the chlorophyll a and give them their red color. Due to these specialized pigments, red algae are often able to photosynthesize in deeper water than other algae. Red algae do not have flagella at any stage of their life cycle.

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Figure 72: Morphology of Laurencia, a marine algae

• Diatoms (Golden-brown algae; Division Bacillariophyta) Largest group of algae but many of its species still undescribed. Evolution- A relatively recent group; diatoms did not exist in the age of the dinosaurs. Habitat: cool marine oceans. Structure: mostly unicellular with silica in cell walls.

• Kelps (brown algae; Division Phaeophyta) Evolution- Closely related to diatoms and also a young group, but very different in appearance. Habitat: rocky coasts in temperate zones or open seas (cold water algae). Structure: multicellular. Some attain great size- 180 feet and grow 2 feet per day. Examples: shoreline: Laminaria; open ocean: Sargassum.

• Dinoflagellates (Division Pyrrhophyta or Dinophyta) Habitat: Especially important in food chains in warm, tropical oceans Structure: Mainly unicellular.

o Green and colorless forms o Biflagellate o Nucleus unusual- chromosomes always visible o Some bioluminescent forms- light up when water is disturbed

• Green algae (Division Chlorophyta)

The green algae are the second largest group of algae. They are also the most diverse of the algae, with at least 7000 species. Evolution- almost as old as red algae. Habitat: They are found mostly in fresh waters and on land. Most species float in rivers, lakes, reservoirs, and creeks. They can also live on rocks, soil, and tree bark.

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A few species, such as sea lettuce (Ulva), live in the salt water along the coast. Large; thin sheets of sea lettuce often totally obscure the muddy bottom in sheltered bay and estuary habitats. Structure: Green algae are organisms with a variety of body forms including single cells, filaments, colonies, and thalli (singular - thallus, multicellular forms that have a leaf-like shape). The higher terrestrial plants arose from a green algal ancestor. They possess the same photosynthetic pigments (chlorophyll a and b) and some green algae have stiff cell walls composed of cellulose, as do plants. Examples - fresh water: Volvox, Spirogyra; marine: sea lettuce (Ulva). Cell Walls of Algae

Algae are the plants with the simplest organization. Many of them are single-celled, some have no cell wall, others though possess cell wall, its composition and structure differ significantly from that of higher plants. In many classes of algae cellulose is the main structural element of the wall, though remarkable variations of the fibrillary structure exist. Reliable X-ray analytical data prove the fact that cellulose could aggregate in many more or less uniform crystalline structures. This may differ considerably from species to species. In some classes of algae exist only disperse textures, while others (specially many Chlorophyta-species) have a higher degree of organization (layers of parallel microfibrils). Such layers do usually alternate with layers of an amorphous material. No clear difference between primary and secondary cell wall exists in most algae. Although the evolution of plants from early eucaryotic cells is not known in detail, it is commonly agreed on that primitive algae are flagellates closely related to the non-green flagellates. Many, though not all species of this stage of evolution, among which the Euglenophyta are typical green representatives, have no cell wall. They are separated from the surrounding by a pellicle of quite complex organization. It consists mainly of glycoproteins organized in regular patterns as two-dimensional crystals. Helical ribs wind round the cell's surface.

Figure 73: Structure of a typical algal cell

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Most single-celled algae like the Volvocales possess real cell walls. The most-studied species is Chlamydomonas reinhardii. Its wall lacks long, fibrillary carbohydrates. Most of it is made up of glycoproteins, and an extension-like protein rich in hydroxyproline can occur. Among the identified sugar residues are arabinosyl-, galactosyl- and mannosyl residues. In the electron microscope the wall appears consisting of seven layers. The middle layer contains an extensive grid-shaped framework of polygonal plates consisting mainly of the glycoproteins, while the layers above and below display fibre-like structures. The thickness of the outer layer varies since it includes components that the cell takes up from its surrounding. Certain common cell wall components of algae are described below. Mannans occur in a number of marine green algae (Codium, Dasycladus, Acetabularia, etc.) as well as in the walls of some red algae (Porphyra, Bangia) as the main structural elements. They are linear and the mannosyl residues are 1 > 4 glycosidically linked. Hydrogen bonds that are (just like in cellulose) the cause of the partially crystalline organization of microfibrils may develop. In Codium the carbohydrates are tightly associated with protein. Xylanes are polymers where the beta-D-xylosyl residues are linked via 1 > 3 and 1 > 4 glycosidic bonds. In species with xylan-containing walls exists a layered structure with an orientation of the microfilaments. They contain mostly linear polymers. Alginic acid and its salts, the alginates are important components of the walls of Phaeophyta (brown algae). They are singular in many respects. They consist exclusively of uronic acids such as mannuronic acid and beta-L-glucuronic acid in different ratios and of small amounts of beta-D-glucuronic acid. The alginates of brown algae exist both within the cell wall and in the intercellular substance. Their part in the cell wall may be as high as 40 per cent of the dry matter. They have a high affinity for divalent cations (calcium, strontium, barium, magnesium) and the tendency to gel. The main portion of the magnesium ions isolated from brown algae stem from the alginic acid fraction.

Figure 74: Chemical structure of alginic acid Sulfonated polysaccharides are polysaccharides whose monomers are esterified to sulfuric acid residues and are moreover partially methylated occurring in nearly all marine algae. They occur partially in the cell wall itself and partially in the intercellular substance. Sulfonated galactanes are typical for many red algae, depending on their origin are they called agarose, carrageenan, porphyran, furcelleran and funoran. L- and D-galactose, which are linked by beta 1 > 3 or alpha 1 > 4 glycosidic bonds form the basic pattern of agarose and porphyran, in the latter alternate L- and D-galactosyl residues. Carrageenan and furcelleran contain exclusively D-compounds. Agar,

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whose basic unit is agarose, is yielded mainly from Gelidium and Gracillaria, both genera of red algae.

Figure 75: Chemical structure of agarose Other cell wall compounds: A number of algae contain mineral cell wall components. Silicon, for example, is the main component of the diatom shell, though it occurs also in the cell walls of other groups of algae. Silicon-containing scales enclosed the chrysophyt Synura. In some brown algae and in the green algae Hydrodictyon silicon is a cell wall component. Sporopollenin, an isoprene derivative and component of pollen cell walls also found in the walls of some green algae (Chlorella, Scenedesmus, etc.). Calcium encrustations of cell walls are especially common in species of tropical, marine waters. Some species participate in reef formation. Calcium is always deposited as calcium carbonate. Calcium carbonate occurs in two different crystalline states: calcite and argonite. Calcite is produced in the walls of some groups of red algae and in Charophycea, while argonite is produced by some green (Acetabularia, etc.), brown and red algae. Both states do not occur simultaneously in one species. Functions

• Cell wall is the mediator between the cell and its surrounding. • It protects not only the cell but serves to, communicate with cells of the same or other

types. • It is permeable to metabolites and regulators and / or to carry receptor molecules with

which it may contact other cells. The diversity of these functions (and their specificity) caused the evolution of a variety of differently structured cell walls. Plastids in algae

Many of the algal groups include some members that are non-photosynthetic. Some retain plastids, but not chloroplasts, while others have lost them entirely. The remaining algae all have chloroplasts containing chlorophylls a and c. The latter chlorophyll type is not known from any prokaryotes or primary chloroplasts, but genetic similarities with the red algae suggest a relationship there. These groups include:

• Heterokonts (e.g., golden algae, diatoms, brown algae)

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• Haptophytes (e.g., coccolithophores) • Cryptomonads • Dinoflagellates

In the first three of these groups (Chromista), the chloroplast has four membranes, retaining a nucleomorph in cryptomonads, and it now appears that they share a common pigmented ancestor. The typical dinoflagellate chloroplast has three membranes, but there is considerable diversity in chloroplasts among the group, some members presumably having acquired theirs from other sources. Protozoa

Protozoa are unicellular animals found worldwide in most habitats. Most species are free living, but some are parasitic. Acanthamoeba species are free-living amoebae that inhabit soil and water. Structure

Protozoa are a large group of eukaryotic, single celled organisms, which lack a rigid cell wall and usually chloroplasts. They vary widely in size, cell structure and form, ranging from Amoeba with its very fluid shape and simple internal organization and few specialised organelles through to Paramecium with its fixed shape, complex internal organisation and many specialised organelles.

Table 6: Organelles and their function.

Organelle Function Mitochondria Transfers energy from organic compounds to ATP Ribosome Organizes the synthesis of proteins Endoplasmic reticulum (ER)

Prepares proteins for export (rough ER); synthesizes steroids, regulates calcium levels, breaks down toxic substances (smooth ER)

Golgi apparatus Processes and packages substances produced by the cell Lysosome Digests molecules, old organelles and foreign substances Microfilaments and microtubules

Contribute to the support, movement and division of cells

Cilia and Flagella Propel cells through the environment; move materials over the cell surface

Nucleus Stores hereditary information in DNA; synthesizes RNA and ribosomes

Cell wall∗ Supports and protects the cell Vacuole∗ Stores enzymes and waste products

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Most parasitic protozoa in humans are less than 50 µm in size. The smallest (mainly intracellular forms) are 1 to 10 µm long, but Balantidium coli may measure 150 µm. Protozoa are unicellular eukaryotes. The organelles of protozoa have functions similar to the organs of higher animals. Nucleus: The nucleus is enclosed in a membrane. In protozoa other than ciliates, the nucleus is vesicular, with scattered chromatin giving a diffuse appearance to the nucleus, all nuclei in the individual organism appear alike. One type of vesicular nucleus contains a more or less central body, called an endosome or karyosome. The endosome lacks DNA in the parasitic amebas and trypanosomes. In the phylum Apicomplexa, on the other hand, the vesicular nucleus has one or more nucleoli that contain DNA. The ciliates have both a micronucleus and macronucleus, which appear quite homogeneous in composition. Plasma membrane: The plasma membrane enclosing the cytoplasm also covers the projecting locomotory structures such as pseudopodia, cilia, and flagella. The outer surface layer of some protozoa, termed a pellicle, is sufficiently rigid to maintain a distinctive shape, as in the trypanosomes and Giardia. However, these organisms can readily twist and bend when moving through their environment. Cytoplasm: In most protozoa the cytoplasm is differentiated into ectoplasm (the outer, transparent layer) and endoplasm (the inner layer containing organelles); the structure of the cytoplasm is most easily seen in species with projecting pseudopodia, such as the ameobae. Some protozoa have a cytosome or cell "mouth" for ingesting fluids or solid particles. Contractile vacuoles: Contractile vacuoles for osmoregulation occur in some, such as Naegleria and Balantidium. Cytoskeleton and locomotory organelles: Many protozoa have subpellicular microtubules; in the Apicomplexa, which have no external organelles for locomotion, these provide a means for slow movement. The trichomonads and trypanosomes have a distinctive undulating membrane between the body wall and a flagellum. Most protozoa are motile by various means such as: Pseudopodia, where the protoplasm streams forward, changing the shape of the organism as it moves e.g. Amoeba Flagella - long, whip-like appendages Cilia - short appendages which are distributed all over the surface of the organism, which beat together. Other organelles: Many other structures occur in protozoa, including the Golgi apparatus, mitochondria, lysosomes, food vacuoles, conoids in the Apicomplexa, and other specialized structures. Subgroups of protozoans- There are 4 major subgroups of protozoans: the amoeba-like protozoans (called the Sarcodines), the flagella-bearing protozoans (called the Mastigotes), the cilia-bearing protozoans (called Ciliates) and the apicomplexans (which used to be called the Sporozoans).

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Figure 76: Fine structure of a protozoan, Typanosoma evansi Sarcodines have a feature that sets them apart from most other protozoans, a type of extension of the cell, called pseudopods (from the Latin for "phony feet"). The amoeba has fairly thick pseudopods, produced by moving material inside the cell in such a way that part of it is pushed out. The membrane of a pseudopod is capable of sticking to most surfaces, and the rest of the cell sort of "flows" into the new position. The interior, the cytoplasm, of Sarcodines is often set off in two layers - the outer layer, the ectoplasm, full of contracting molecular cables, and an inner layer, the endoplasm, full of the looser cellular machinery, including the nucleus and most cell organelles. In moving, they may be used as tentacles for crawling. A large number of thin pseudopods may radiate out from a Sarcodine and help it float. They are often used for feeding. They can be extended around food objects forming a food vacuole, inside the amoeba or as tentacles for capturing food as well. Mastigotes are the flagellates characterized by the possession of flagella: one or more (but rarely more than a dozen) long, mobile extensions from the cell. Unlike pseudopods, flagella have a rigidly-organized core of cross-connected microtubules that drive them. Flagella move in various ways: they may spin, or whip, or move like tentacles, among other things. They may carry extra structures, like bristles, or combs, or stiff sections, or a flat outgrowth of membrane that acts like a fin.

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Figure 77: An amoeboid cell Ciliates: The characteristic feature of this group is possession of cilia; there are several other unique or highly unusual features as well. The cilia are small cell projections constructed the same basic way as flagella, with a core of cross-connected microtubules that interact to produce their motion. However, there are several differences between these structures. Ciliates often have a depression or furrow on their surface down with which a current drives food; at the bottom of this feature, which is often called an oral groove, food vacuoles form which often follow a set path through the cell (almost like the track of food through a digestive tract), ending at a special spot at which the undigested contents are dumped outside. Freshwater ciliates, like freshwater protozoans in general, have contractile vacuoles for maintaining osmotic balance. Ciliates each have a micronucleus, is usually small. The micronucleus carries the "master copy" of the cell's DNA, which is used for making new nuclei, usually in preparation for reproduction. Ciliates also each have one or more micronuclei, which are used as the "working" genetic codes - when the cell needs to make proteins, needs to perform some function, it accesses the DNA in a macronucleus for it. Apicomplexans used to be known as the sporozoans. They possess an apical complex, at one end of their cells. Fungi

Structure

Fungi lie at the borderline of unicellular/multicellular organisms, and they show a wide degree of division of labour and specialisation. The main body of most fungi is composed of fine, branching, usually colourless threads called hyphae. Each fungus will have vast numbers of these hyphae, all intertwining to make up a tangled web called the mycelium. Fruiting bodies (such as mushrooms) are made up of thick collections of hyphae. They vary in size from small and insignificant, to large eye-catching structures. They are usually produced at the surface of the substrate, to allow the spores to be shed and dispersed by the wind, or by water, or animals.

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Figure 78: The hyphae are magnified 100 times life size

Figure 79: Hyphal fungi (Moulds) Fungi can be distinguished into two basic morphological forms, yeasts and hyphae. Yeasts are unicellular fungi, which reproduce asexually by blastoconidia formation (budding) or fission. Hyphae are multi-cellular fungi, which reproduce asexually and/or sexually. Dimorphism is the condition where by a fungus can exhibit either the yeast form or the hyphal form, depending on growth conditions. Very few fungi exhibit dimorphism. Most fungi occur in the

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hyphae form as branching, threadlike tubular filaments. These filamentous structures either lack cross walls (coenocytic) or have cross walls (septate) depending on the species. In some cases septate hyphae develop clamp connections at the septa, which connect the hyphal elements. A mass of hyphal elements is termed the mycelium. Aerial hyphae often produce asexual reproduction propagules termed conidia. Relatively large and complex conidia are termed macroconidia while the smaller and more simple conidia are termed microconidia. When the conidia are enclosed in a sac (the sporangium), they are called endogenous spores.

Figure 80: Morphological forms, yeasts and hyphae Hyphae may be aseptate (coenocytic) or septate. They may be specialised for nutrient absorption (haustoria), adhesion (appressoria), reproduction (spores) and surviving the adverse environmental factors (rhizomorphs, sclerotia, chlamydospores). Spores in fungi may be specialized for sexual or asexual reproduction. Spores may be one or many celled in the fungi and motile or non-motile. The various membranes bound organelles embedded in the cell cytoplasm (N, Nucleus; ER, endoplasmic reticulum; D, dictysome or Golgi apparatus; V, vacuole; M, mitochondrion.). The other organelles, endoplasmic reticulum, dictysomes, mitochondria and vesicles also are common. The dark bodies are Woronin bodies, which are composed of protein and block the septal pore when a hyphal compartment becomes physically ruptured such that the contents of the undamaged compartments are not lost externally. The structural specificities include chitin in cell wall, incorporation of ergosterol in the plasma membrane and absence of chloroplasts.

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Figure 81: Fungal appressorium on and haustorium within host cell

Figure 82: Rhizomorph

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Table 7: Differences between Archae, Bacteria and Eukaryotes

Characteristic Archea Bacteria Eukaryotes Predominantly multicellular No No Yes Cell contains a nucleus and other membrane Bound organelles

No No Yes

DNA occurs in a circular form∗ Yes No No Ribosome size 70S 70S 80S Membrane lipids ester- linked∗∗ No Yes Yes Photosynthesis with chlorophyll No Yes Yes Capable of growth at temperatures greater than 80 °C

Yes Yes No

Histone proteins present in cell Yes No Yes Methionine used as tRNA initiator∗∗∗ Yes No Yes Operons present in DNA Yes Yes No Intron present in DNA No No Yes Capping and Poly-A tailing of mRNA No No Yes Gas Vesicles present Yes Yes No Capable of Methanogenesis Yes No No Sensitive to chloramphenicol, Kanamycin and Streptomycin

No Yes No

Transcription factors required No Yes Yes Capable of Nitrification No Yes No Capable of Denitrification Yes Yes No Capable of Nitrogen fixation Yes Yes No Capable of Chemolithotrophy Yes Yes No

∗ Eukaryotic DNA is linear ∗∗ Archae membrane lipids are ether linked ∗∗∗ Bacteria use formylmethionine

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Table 8: Differences between Archael, Bacterial and Eukaryotic cytoplasmic membranes

Characteristics Archea Bacteria Eukaryotic Protein content High High Low Lipid composition

Sulfolipids, glycolipids, isoprenoid lipids, phospholipids

Phospholipid Phospholipids

Lipid structure Straight chain Straight chain Branched Lipid linkage Ether linked (di and

tetraethers) Ester linked Ester linked

Sterols Absent Absent Present

Archaea

Eubacteria, Eukarya

Figure 83: Lipids in Archae, Bacteria and Eukarya Suggested Readings 1. Garrett, R.A., P. Redder, B. Greve, K. Brügger, L. Chen and Q. She. 2004. Archaeal plasmids. In Plasmid

Biology. Eds. B.E. Funnell and G.J. Phillips. A.S.M. Press, Washington, DC, pp 377–392. 2. Madigan.T.M., Martinko.J.M. and Parker.J.1997. Brock biology of Microorganisms. 8th edition. Prentice hall

International, New Jersey 3. Metlina. A. L. 2004. Bacterial and Archaeal Flagella as Prokaryotic Motility Organelles. REVIEW.

Biochemistry (Moscow), Vol. 69, No. 11, 2004, pp. 1203-1212. Biochemistry (Moscow) Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1477-1488.

4. Pelczar. M., Chan.E.C and Krieg.N.R. 1993. Microbiology. 5th edition. Tata McGraw-Hill Publishing Company Ltd, New Delhi.

5. Prescott.L.M., Harley.J.P and Klein.D.A.1996. Microbiology. 3rd edition. Wm. C. Brown Publishers, USA.

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6. Rabel, C., A.M. Grahn, R. Lurz and E. Lanka. 2003. The VirB4 family of proposed traffic nucleoside triphosphatases: common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption. J. Bacteriol. 185:1045–1058.

7. Schlegel.H.G.1993. General microbiology. 7th edition. Cambridge University press, New York. 8. Stanier.R.Y., Ingraham.J.L., Wheelis.M.L and Painter.P.R. 1995. General Microbiology. 5th edition.

MacMillan Press Ltd. HongKong. 9. Starr, Cecie and Ralph Taggart. 1995. Biology: The Unity and Diversity of Life. Wadsworth Publishing Co.

Belmont, CA 10. Starr, Cecie. 1994. Biology: Concepts and Applications. Wadsworth Publishing Co. Belmont, CA

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