Glycocalyx

Many prokaryotes secrete on their surface a substance called Glycocalyx. Glycocalyx (meaning sugar coat) is the general term used for substances that surround cells. The bacterial glycocalyx is a viscous (sticky), gelatinous polymer that is external to the cell wall and composed of polysaccharide, polypeptide, or both. Its chemical composition varies widely with the species. For the most part, it is made inside the cell and secreted to the cell surface.

If the substance is organized and is firmly attached to the cell wall, the glycocalyx is described as a capsule. The presence of a capsule can be determined by using negative stainingIf the substance is unorganized and only loosely attached to the cell wall, the glycocalyx is described as a slime layer.e.g. Bacillus anthracis produces a capsule of d-glutamic acid. (Recall

The glycocalyx is a very important component of biofilms (see page 160). A glycocalyx that helps cells in a biofilm attach to their target environment and to each other is called an extracellular polymeric substance (EPS). The EPS protects the cells within it, facilitates communication among them, and enables the cells to survive by attaching to various surfaces in their natural environment.

A glycocalyx also can protect a cell against dehydration, and its viscosity may inhibit the movement of nutrients out of the cell.

Some bacterial cells are surrounded by a viscous substance forming a covering layer or envelope around the cell wall called capsule. Capsule consist of a mesh or network of fine strands. This capsule only helpsin disease causing ability of a few types of bacteria. This capsule is divided into twogroups:(a) Macrocapsule: It is about O.21lm thick and can be seen under light microscope.(b) Microcapsule: It can't be seen under light microscope but can be demonstrated immunologically.

Chemically the capsules are made up of di or polysaccharide or polypeptide. Thepolysaccharide may be homo polysaccharide (composed of single kind of sugar) e.g.Streptococcus mutans ot it may be heteropolysaccharide (composed of several kind ofsugars) e.g. Klebsiella pneumoniae.

The a hemolytic Streptococcus mutans,the primary organism found in dental plaque is able to synthesis a large extracellularmucoid glucans from sucrose. Not all bacterial species produce capsules; however, thecapsules of encapsulated pathogens are often important determinants of virulence.Encapsulated species are found among both Gram-positive and Gram-negative bacteria.In both groups, most capsules are composed of highmolecular-weight viscouspolysaccharides that are retained as a thick gel outside the cell wall or envelope. Thecapsule of Bacillus anthracis is unusual in that it is composed of a g-glutamyl polypeptide.A plasma membrane stage is involved in the biosynthesis and assembly of the capsularsubstances, which are extruded or secreted through the outer wall or envelope structures.Mutational loss of enzymes involved in the biosynthesis of the capsular polysaccharidescan result in the smooth-to-rough variation seen in the pneumococci.

Fimbriae and PiliMany gram-negative bacteria contain hair-like appendages that are shorter, straighter, and thinner than flagella Used for attachment and transfer of DNA rather than for motility. These structures, which consist of a protein called pilin arranged helically around a central core, are divided into two types, fimbriae and pili, having very different functions.

Fimbriae (singular: fimbria) can occur at the poles of the bacterial cell or can be evenly distributed over the entire surface of the cell. They can number anywhere from a few to several hundred per cell Fimbriae have a tendency to adhere to each other and to surfaces. As a result, they are involved in forming biofilms and other aggregations on the surfaces of liquids, glass, and rocks. Fimbriae can also help bacteria adhere to epithelial surfaces in the body. For example, fimbriae on the bacterium Neisseria gonorrhoeae (n-se r-a go-nor-r ), the causative agent of gonorrhea, help the microbe colonize mucous membranes. Once colonization occurs, the bacteria can cause disease.

The fimbriae of E. coli O157 enable this bacterium to adhere to the lining of the small intestine, where it causes a severe watery diarrhea. When fimbriae are absent (because of genetic mutation), colonization cannot happen, and no disease ensues.

Pili (singular: pilus) are usually longer than fimbriae and number only one or two per cell. Pili are involved in motility and DNA transfer. In one type of motility, called twitching motility, a pilus extends by the addition of subunits of pilin, makes contact with a surface or another cell, and then retracts (powerstroke) as the pilin subunits are disassembled. This is called the grappling hook model of twitching motility and results in short, jerky, intermittent movements. Twitching motility has been observed in Pseudomonas aeruginosa, Neisseria gonorrhoeae, and some strains of E. coli. The other type of motility associated with pili is gliding motility, the smooth gliding movement of myxobacteria. Although the exact mechanism is unknown for most myxobacteria, some utilize pilus retraction. Gliding motility provides a means for microbes to travel in environments with a low water content, such as biofilms and soil.

PILI OR FIMBRAEThese are hair like appendages present on the surface of most of the gram negativebacteria (Enterobacteriaceae, Pseudomondaceae and Caulobacter). They are smaller thanflagella, have no role in the motility of bacteria.

They measure 0.2-20J.! in length and 30-140Ao in width.

A single bacterial cells bears about 100-500 pili which are arrangedperitrichously.

There origin is from cytoplasm and penetrate through the peptidoglycanlayers of the cell wall.

Chemically they are composed of 100% protein named fimbrilinwith a molecular weight of about 16,000. Fimbrilin consist of about 163 amino acids.Following two types of pili are found in bacteria viz.

(a) Somatic pili (b) Sex pili or conjugate pili

(a) Somatic Pili : Each bacterial cell bears about 100 somatic pili whose main function is to help the bacterium for attachment to a substratum.

(b) Sex Pili or Conjugate Pili : They are also known as F pili and are controlled by sex factors. These pili are comparatively long (20 ll) and broad in width (65-135AO).There number ranges from 1-10 in male or donor bacterium, but in some bacterium it is found in both viz. Male donor (+ factors) or female receptor/ receiver (- factor).

At the time of conjugation the sex pili of male donor recognize the receptor protein on the surface of female or recipient and get attached with the help of conjugation tube.The DNA from the donor to recipient is transferred through this conjugation tube. There are two types of sex pili in E.coli. They are F. pili and I.pili.In certain pathogenic bacteria these pili help the bacteria in the attachment of pathogenic bacterial cell to the host cells. There are generally four types of pili classified on the basis of their attachment ability to the host cell :(a) Type I : There diameter is about 90A and found in E.coli, Serratia & Salmonella.(b) Type II : They lack the attachment ability e.g. some species of Salmonella.(c) Type III : Their attachment ability is effected by mannose sugar. They are about50 A in diameter e.g. Klebsiella and some spp. of Salmonella.(d) Type IV : They are found in Proteus bacteria.

Functions of Pili(i) They help the bacteria to attach themselves to the natural substrate or to other organism due to its adhesive properties.(ii) They bear antigenic properties. (iii) Sex pili are helpful in chromosome transfer during conjugation by acting as conjugation tube.(iv) They act as bacteriophage receptor.

PiliThe terms pili and fimbriae are usually used interchangeably to describe the thin, hairlikeappendages on the surface of many Gram-negative bacteria and proteins of pili arereferred to as pilins.

Pili are more rigid in appearance than flagella. In some organisms, such as Shigella species and E coli, pili are distributed profusely over the cell surface, with as many as 200 per cell. As is easily recognised in strains of E coli, pili can come in two types: short, abundant common pili, and a small number of very long pili known as sex pili.

Sex pili can be distinguished by their ability to bind male-specific bacteriophages.The sex pili attach male to female bacteria during conjugation. Pili in many enteric bacteria confer adhesive properties on the bacterial cells, enabling them to adhere to various epithelial surfaces, to red blood cells, and to surfaces of yeast and fungal cells.

These adhesive properties of piliated cells play an important role in bacterial colonisation of epithelial surfaces and are therefore referred to as ~colonisation factors. The common pili found on E coli exhibit a sugar specificity analogous to that of phytohemagglutinins and lectins, in that adhesion and hemagglutinating capacities of the organism are inhibited specifically by mannose. Organisms possessing this type of hemagglutination are called mannose-sensitive organisms. Other piliated organisms, such as gonococci, are adhesive and hemagglutinating, but are insensitive to the inhibitory effects of mannose. Extensive antigenic variations in pilins of gonococci are well known

Functions(i) They provide protection against temporary drying by binding water molecules.(ii) They may be antiphagocytic i.e. they inhibit the engulfment of pathogenic bacteria by W.B.C. and contribute to invasive ability.

Axial FilamentsSpirochetes are a group of bacteria that have unique structure and motility. One of the best-known spirochetes is Treponema pallidum (tre-p-n ma pal li-dum), the causative agent ofsyphilis. Another spirochete is Borrelia burgdorferi (bor-rel--a burg-dor fer-), the causative agent of Lyme disease. Spirochetes move by means of axial filaments, or endoflagella, bundles of fibrils that arise at the ends of the cell beneath an outer sheath andspiral around the cell (Figure 4.10).Axial filaments, which are anchored at one end of the spirochete, have a structure similar to that of flagella. The rotation of the filaments produces a movement of the outer sheath that propels the spirochetes in a spiral motion. This type of movement is similar to the way a corkscrew moves through a cork.This corkscrew motion probably enables a bacterium such as T. pallidum to move effectively through body fluids.

SheathsSome species of bacteria of freshwater and marine environment form chains ortrichomes which are enclosed by a hollow tube called sheath. Sheaths may be sometimesimpregnated with ferric or manganese hydroxides which strengthen them.

Prostheceae and StalksThese are semirigid extensions of cell wall and cytoplasmic membrane. They arecharacteristic of a number of aerobic bacteria from freshwater and marine environment.These prosthecae may be single (ex. Caulobacter) or several (Ancalomicrobrillm)The main function of prostheceae is that they increase the surface area of the cellsfor nutrient absorption in the dilute environment.Stalk are non living ribbon like or tubular appendages that are excreted by thecell. The~ stalk aid in attachment of the cells to surfaces e.g. Plallctomyces.

The Cell Wall:

General characteristics:

The cell wall of the bacterial cell is a complex, semi-rigid structure responsible for the shape of the cell. The cell wall surrounds the underlying, fragile plasma (cytoplasmic) membrane and protects it and the interior of the cell from adverse changes in the outside environment. Almost all prokaryotes have cell walls.

The major function of the cell wall is to prevent bacterial cells from rupturing when the water pressure inside the cell is greater than that outside the cell. It also helps maintain the shape of a bacterium and serves as a point of anchorage for flagella. As the volume of a bacterial cell increases, its plasma membrane and cell wall extend as needed.

The cell wall is resistant to extremely high pressure. The cell wall constitutes a significant portion of the dry weight of the cell, it may account for as such 10-40% of the dry weight of bacterial cellClinically, the cell wall is important because it contributes to the ability of some species to cause disease and is the site of action of some antibiotics. In addition, the chemical composition of the cell wall is used to differentiate major types of bacteria.

Although the cells of some eukaryotes, including plants, algae, and fungi, have cell walls, their walls differ chemically from those of prokaryotes, are simpler in structure, and are less rigid.

The thickness of these different layers varies both in gram +ve and gram -ve bacteria. The walls of gram -ve species are generally thinner (10-15 nm) than those of gram +ve species (20-25 nm).

Composition and CharacteristicsThe bacterial cell wall is composed of a macromolecular network called peptidoglycan (also known as murein), which is present either alone or in combination with other substances. which is an insoluble, porous, cross-linked polymer of enormous strength and rigidity. This peptidoglycan is only found in prokaryotes.

Peptidoglycan consists of a repeating disaccharide attached by polypeptides to form a lattice that surrounds and protects the entire cell. The disaccharide portion is made up of monosaccharides called N acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) (from murus, meaning wall), which are related to glucose.

It is basically a polymer of N-acetyl glucosamine (NAG), N acetylmuramic acid (NAM), and 4 amino acids (L-alanine, D-alanine, D-glutamate and a diamino acids).

The structural formulas for NAG and NAM Figure 4.12.The various components of peptidoglycan are assembled in the cell wall (Figure 4.13a).

Alternating NAM and NAG molecules are linked in rows of 10 to 65 sugars to form a carbohydrate backbone (the glycan portion of peptidoglycan). Adjacent rows are linked by polypeptides (the peptide portion of peptidoglycan).

Although the structure of the polypeptide link varies, it always includes tetrapeptide side chains, which consist of four amino acids attached to NAMs in the backbone. The amino acids occur in an alternating pattern of d and l forms (see Figure 2.13, page 43).

The tetrapeptide of one peptidoglycan layer is cross linked with the other peptidoglycan layer and as a result a strong framework is formed around the cell and impart great rigidity to the total structure. Some antibiotics viz. penicillin inhibit the synthesis of this framework thus the cell wall synthesis is stopped.

This is unique because the amino acids found in other proteins are l forms. Parallel tetrapeptide side chains may be directly bonded to each other or linked by a peptide cross-bridge, consisting of a short chain of amino acids.

NOTE: Penicillin interferes with the final linking of the peptidoglycan rows by peptide cross-bridges (see Figure 4.13a). As a result, the cell wall is greatly weakened and the cell undergoes lysis, destruction caused by rupture of the plasma membrane and the loss of cytoplasm.

Gram-Positive Cell WallsIn most gram-positive bacteria, the cell wall consists of many layers of peptidoglycan, forming a thick, rigid structure (Figure 4.13b). The cell wall of these bacteria consist of about 40- 80% of peptidoglycan of the dry weight of cell wall.

By contrast, gram-negative cell walls contain only a thin layer of peptidoglycan

In addition, the cell walls of gram-positive bacteria contain teichoic acids, which consist primarily of an alcohol (such as glycerol or ribitol) and phosphate. There are two classes of teichoic acids: lipoteichoicacid, which spans the peptidoglycan layer and is linked to the plasma membrane, and wall teichoic acid, which is linked to the peptidoglycan layer.

Because of their negative charge (from the phosphate groups), teichoic acids may bind and regulate the movement of cations (positive ions) into and out of the cell. They may also assume a role in cell growth, preventing extensive wall breakdown and possible cell lysis.

Finally, teichoic acids provide much of the walls antigenic specificity and thus make it possible to identify gram-positive bacteria by certain laboratory tests (see Chapter 10).

Similarly, the cell walls of gram-positive streptococci are covered with various polysaccharides that allow them to be grouped into medically significant types.

Gram + ve bacteria have a much greater amount of peptidoglycan in their cell walls than do gram - ve bacteria. This peptidoglycan is of about 40 or more layers in gram + ve bacteria. The cell wall measures about 30-80 nm in thickness. Teichoic acid or acidic polysaccharide are mainly present in gram positive bacteria and are found associated with peptidoglycan by a single terminal covalent bond.

Teichoic acid is a negatively charged substituted polysaccharide polymer made up of ribitol and glycerol residues joined through diphosphoester linkages. It constitute a major surface antigen. It is hydrophilic and its main function is to transport positively charged substances to the bacterial cell in the storage of phosphorous.

Gram-Negative Cell WallsThe cell walls of gram-negative bacteria consist of one or a very few layers of peptidoglycan and an outer membrane (see Figure 4.13c).The peptidoglycan is bonded to lipoproteins (lipids covalently linked to proteins) in the outer membrane and is in the periplasm-a gel-like fluid between the outer membrane and the plasma membrane.The periplasm contains a high concentration of degradative enzymes and transport proteins. Gram-negative cell walls do not contain teichoic acids. Because the cell walls of gram-negative bacteria contain only a small amount of peptidoglycan, they are more susceptible to mechanical breakage.

The outer membrane of the gram-negative cell consists of lipopolysaccharides (LPS), lipoproteins, and phospholipids (see Figure 4.13c). The outer membrane has several specialized functions.

Its strong negative charge is an important factor in evading phagocytosis and the actions of complement (lyses cells and promotes phagocytosis), two components of the defenses of the host(discussed in detail in Chapter 16).

The outer membrane also provides a barrier to certain antibiotics (for example, penicillin), digestive enzymes such as lysozyme, detergents, heavy metals, bile salts, and certain dyes.

However, the outer membrane does not provide a barrier to all substances in the environment because nutrients must pass through to sustain the metabolism of the cell. Part of the permeability of the outer membrane is due to proteins in the membrane, called porins that form channels. Porins permit the passage of molecules such as nucleotides, disaccharides, peptides, amino acids, vitamin B12, and iron.

The lipopolysaccharide (LPS) of the outer membrane is a large complex molecule that contains lipids and carbohydrates and consists of three components: (1) lipid A, (2) a core polysaccharide, and (3) an O polysaccharide. Lipid A is the lipid portion of the LPS and is embedded in the top layer of the outer membrane. When gram-negative bacteria die, they release lipid A, which functions as an endotoxin (Chapter 15). Lipid A is responsible for the symptoms associated with infections by gram-negative bacteria such as fever, dilation of blood vessels, shock, and blood clotting. The core polysaccharide is attached to lipid A and contains unusual sugars. Its role is structuralto provide stability. The O polysaccharide extends outward from the core polysaccharide and is composed of sugar molecules. The O polysaccharide functions as an antigen and is useful for distinguishing species of gram-negative bacteria.For example, the foodborne pathogen E. coli O157:H7 is distinguished from other serovars by certain laboratory tests that test for these specific antigens. This role is comparable to that of teichoic acids in gram-positive cells.

The wall of gram - ve bacteria are more complex than those of gram + ve bacteria. The envelop of this kind of bacteria is made up of two unit membranes and are separated by 100Ao space known as periplasmic region and contains a peptidoglycan layer.

The outermost membrane is known as cell wall while the inner one is referred as cytoplasmic membrane. Peptidoglycan is only about 5-10% of the dry weight of cell wall. The outer membrane serve as selective barrier for various external chemicals and enzymes that could damage the cells. Its structure is similar to plasma membrane or cell membrane.

The outer membrane is anchored to the underlying peptidoglycan by means of Braun's lipoprotein. The membrane is bilayered structure consisting mainly of phospholipids protein and lipopolysaccharide (L.P.S.). The phospholipids are bilayered consisting of both hydrophilic and hydrophobic ends. The wall contains 4 types of protein components along with other major types of protein called lipoprotein.The Lipopolysaccharide (LPS) has toxic properties and is also known as endotoxin.It occurs only in the outer layer of the membrane and is composed of three covalently linked parts :(i) Lipid A = firmly embedded in the membrane.(ii) Core polysaccharide = located at the membrane surface.(iii) O-antigens = which extend like whiskers from the membrane surface into the surrounding medium. Many antigenic properties of gram - ve bacteria are attributable to O-antigens.

The outer membrane is although impermeable to large molecules but can allow smaller molecules such as nucleosides, oligosaccharide, monosaccharides, pep tides and amino acids. This is accomplished by means of channels in special proteins called porins.

Cell Wall and Gram-Negative Cell Envelope

The Gram stain broadly differentiates bacteria into Gram-positive and Gram-negative groups; a few organisms are consistently Gram-variable.

Gram-positive and Gramnegative organisms differ drastically in the organisation of the structures outside the plasma membrane but below the capsule: in Gram-negative organisms these structures constitute the cell envelope, whereas in Gram-positive organisms they are called a cell walLMost Gram-positive bacteria have a relatively thick (about 20 to 80 nm), continuouscell wall, which is composed largely of peptidoglycan. In thick cell walls, other cell wallpolymers are covalently attached to the peptidoglycan. In contrast, the peptidoglycanlayer in Gram-negative bacteria is thin; in E coli, the peptidoglycan is probably only amonolayer thick. Outside the peptidoglycan layer in the Gram-negative envelope is anouter membrane structure. In most Gram-negative bacteria, this membrane structure isanchored noncovalently to lipoprotein molecules, which, in turn, are covalently linkedto the peptidoglycan.

The lipopolysaccharides of the Gram-negative cell envelope form part of the outer leaflet of the outer membrane structure. The organisation and overall dimensions of the outer membrane of the

Gram-negative cell envelope are similar to those of the plasma membrane. Moreover, in Gram-negative bacteria such as E coli, theouter and inner membranes adhere to each other at several hundred sites; these sitescan break up the continuity of the peptidoglycan layer.The basic differences in surface structures of Gram-positive and Gram-negativebacteria explain the results of Gram staining.

Both Gram-positive and Gram-negative bacteria take up the same amounts of crystal violet (CV) and iodine (I). The CV-I complex, however, is trapped inside the Gram-positive cell by the dehydration and reducedporosity of the thick cell wall as a result of the differential washing step with 95 percentethanol or other solvent mixture. In contrast, the thin peptidoglycan layer and probablediscontinuities at the membrane adhesion sites do not impede solvent extraction of theCV-I complex from the Gram-negative celL The above mechanism of the Gram stainbased on the structural differer.ces between the two groups has been confirmed bysophisticated methods of electron miorost'oPY. Moreover, mechanical disruption of theBacteria: Structure and Functions 43cell wall of Gram-positive organisms or its enzymatic removal with lysozyme results incomplete extraction of the CV-I complex and conversion to a Gram-negative reaction.Therefore, autolytic wall-degrading enzymes that cause cell wall breakage may accountfor Gram-negative or variable reactions in cultures of Gram-positive organisms.PeptidoglycanUnique features of almost all prokaryotic cells are cell wall peptidoglycan and the specificenzymes involved in its biosynthesis. These enzymes are target sites for inhibition ofpeptidoglycan synthesis by specific antibiotics.

The primary chemical structures of peptidoglycans of both Gram-positive and Gram-negative bacteria have been established; they consist of a "glycan backbone of repeating groups of bt 4-linked disaccharides of b1,4-N-acetylmuramyl-N-acetylglucosamine.Tetrapeptides of L-alanine-D-isoglutamic acid-L-Iysine -n-alanine are linked throughthe carboxyl group by amide linkage of muramic acid residues of the glycan chains; theD-alanine residues are directly cross-linked to the e-amino group of lysin"e ordiaminopimelic acid on a neighboring tetrapeptide, or they are linked by a peptidebridge. In S aureus peptidoglycan, a glycine pentapeptide bridge links the two adjacentpeptide structures. The extent of direct or peptide-bridge cross-linking varies from onepeptidoglycan to another.The staphylococcal peptidoglycan is highly cross-linked, whereas that of E coli ismuch less so, and has a more open peptidoglycan mesh. The diamino acid providingthe e-amino group for cross-linking is lysine or diaminopimelic acid, the latter beinguniformly present in Gram-negative peptidoglycans. A peptidoglycan with a chemicalstructure substantially different from that of all eubacteria has been discovered in certainarchaebacteria. Instead of muramic acid, this peptidoglycan contains talosaminuronicacid and lacks the D-amino acids found in the eubacterial peptidoglycans. Interestingly,organisms containing this wall polymer are insensitive to penicillin, an inhibitor of thetranspeptidases involved in peptidoglycan biosynthesis in eubacteria.The 15-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamineis specifically cleaved by the bacteriolytic enzyme lysozyme. Widely distributed innature, this enzyme is present in human tissues and secretions and can cause completedigestion of the peptidoglycan walls of sensitive organisms. When lysozyme is allowedto digest the cell wall of Gram-positive bacteria suspended in an osmotic stabilizer,protoplasts are formed. These protoplasts are able to survive and continue to grow onsuitable media in the wall-less state. Gram-negative bacteria treated similarly producespheroplasts, which retain much of the outer membrane structure. The dependence ofbacterial shape on the peptidoglycan is shown by the transformation of rod-shapedbacteria to spherical protoplasts (spheroplasts) after enzymatic breakdown of the44 Introductory Microbiologypeptidoglycan. The mechanical protection afforded by the wall peptidoglycan layer isevident in the osmotic fragility of both protoplasts and spheroplasts.There are two groups of bacteria that lack the protective cell wall peptidoglycanstructure, the Mycoplasma species, one of which causes atypical pneumonia and somesenitourinary tract infections and the L-forms, which originate from Gram-positive or. Gram-negative bacteria and are so designated because of their discovery and descriptionat the Lister Institute, London. The mycoplasmas and L-forms are all Gram-negative andinsensitive to penicillin and are bounded by a surface membrane structure. L-formsarising "spontaneously" in cultures or isolated from infections are structurally relatedto protoplasts and spheroplasts; all three forms revert infrequently and only underspecial conditions.Teichoic AcidsWall teichoic acids are found only in certain Gram-positive bacteria; so far, they havenot been found in gram- negative organisms. Substituent groups on the polyol chainscan include D-alanine, N-acetylglucosamine, N-acetylgalactosamine, and glucose; thesubstituent is characteristic for the teichoic acid from a particular bacterial species andcan act as a specific antigenic determinant. Teichoic acids are covalently linked to thepeptidoglycan. These highly negatively charged polymers of the bacterial wall can serveas a cation-sequestering mechanism.Accessory Wall PolymersIn addition to the principal cell wall polymers, the walls of certain Gram-positive bacteriapossess polysaccharide molecules linked to the peptidoglycan. For example, the Cpolysaccharide of streptococci confers group specificity. Acidic polysaccharides attachedto the peptidoglycan are called teichuronic acids. Mycobacteria have peptidoglycolipids,glycolipids, and waxes associated with the cell wall.LipopolysaccharidesA characteristic feature of Gram-negative bacteria is possession of various types ofcomplex macromolecular lipopolysaccharide (LPS). So far, only one Gram-positiveorganism, Listeria monocytogenes, has been found to contain an authentic LPS. The LPSof this bacterium and those of all Gram-negative species are also called endotoxins,thereby distinguishing these cell-bound, heat-stable toxins from heat-labile, proteinexotoxins secreted into culture media. Endotoxins possess an array of powerful biologicactivities and play an important role in the pathogenesis of many Gram-negativebacterial infections. In addition to causing endotoxic shock, LPS is pyrogenic, can activatemacrophages and complement, is mitogenic for B lymphocytes, induces interferonBacteria: Structure and Functions 45production, causes tissue necrosis and tumor regression, and has adjuvant properties.The endotoxic properties of LPS reside largely in the lipid A components. Usually, theLPS molecules have three regions: the lipid A structure required for insertion in the outerleaflet of the outer membrane bilayer; a covalently attached core ~omposed of 2-keto-3deoxyoctonic acid (KDO), heptose, ethanolamine, N-acetylglucosamine, glucose, andgalactose; and polysaccharide chains linked to the core.The polysaccharide chains constitute the O-antigens of the Gram-negative bacteria,and the individual monosaccharide constituents confer serologic specificity on thesecomponents. The demonstration of the structure of lipid A of LPS of a heptoseless mutantof Salmonella typhimurium has established that amide-linked hydroxymyristoyl andlauroxymyristoyl groups are attached to the nitrogen of the 2- and 2'-carbons,respectively, and that hydroxymyristoyl and myristoxymyristoyl groups are attached tothe oxygen of the 3- and 3'-carbons of the disaccharide, respectively. Therefore, onlyposition 6' is left for attachment of KDO units.LPS and phospholipids help confer asymmetry to the outer membrane of the Gramnegativebacteria, with the hydrophilic polysaccharide chains outermost. Each LPS isheld in the outer membrane by relatively weak cohesive forces and can be dissociatedfrom the cell surface with surface-active agents. As in peptidoglycan b~osynthesis, LPS 'molecules are assembled at the plasma or inner membrane. These newly formedmolecules are initially inserted into the outer-inner membrane adhesion sites.The outer membranes of Gram-negative bacteria appear broadly similar to the plasmaor inner membranes; however, they differ from the inner membranes and walls of Grampositivebacteria in numerous respects. The lipid A of LPS is inserted with phospholipidsto create the outer leaflet of the bilayer structure; the lipid portion of the lipoprotein andphospholipid form the inner leaflet of the outer membrane bilayer of most Gramnegativebacteria. In addition to these components, the outer membrane possessesseveral major outer membrane proteins; the most abundant is called porin. Theassembled subunits of porin form a channel that limits the passage of hydrophilicmolecules across the outer membrane barrier to those having molecular weights that areusually less than 600 to 700. Evidence also suggests that hydrophobic pathways existacross the outer membrane and are partly responsible for the differential penetration andeffectiveness of certain b-Iactam antibiotics that are active against various Gram-negativebacteria.Although the outer membranes act as a permeability barrier or molecular sieve, theydo not appear to possess energy-transducing systems to drive active transport. Severalouter membrane proteins, however, are involved in the specific uptake of metabolitesand,iron from the medium. Thus, outer membranes of the Gram-negative bacteriaprovide a selective barrier to external molecules and thereby prevent the loss of46 Introductory Microbiologymetabolite-binding proteins and hydrolytic enzymes found in the periplasmic space. Theperiplasmic space is the region between the outer surface of the inner membrane andthe inner surface of the outer membrane. Thus, Gram-negative bacteria have a cellularcompartment that has no equivalent in Gram-positive organisms. In addition to thehydrolytic enzymes, the periplasmic space holds binding proteins involved in membranetransport and chemotactic receptor activities. Moreover, plasmid-encoded b-Iactamasesand aminoglycoside-modifying enzymes in the periplasmic space produce antibioticresistance by degrading or modifying an antibiotic in transit to its target sites on themembr ane or on the ribosomes.

The NucleoidThe nucleoid of a bacterial cell (see Figure 4.6) usually contains asingle long, continuous, and frequently circularly arranged threadof double-stranded DNA called the bacterial chromosome. Thisis the cells genetic information, which carries all the informationrequired for the cells structures and functions. Unlike thechromosomes of eukaryotic cells, bacterial chromosomes are notsurrounded by a nuclear envelope (membrane) and do not includehistones. The nucleoid can be spherical, elongated, or dumbbellshaped.In actively growing bacteria, as much as 20% of the cellvolume is occupied by DNA because such cells presynthesize nuclearmaterial for future cells. The chromosome is attached to theplasma membrane. Proteins in the plasma membrane are believedto be responsible for replication of the DNA and segregation ofthe new chromosomes to daughter cells during cell division.In addition to the bacterial chromosome, bacteria often containsmall usually circular, double-stranded DNA moleculescalled plasmids (see the F factor in Figure 8.26a, page 234).These molecules are extrachromosomal genetic elements; that is,they are not connected to the main bacterial chromosome, andthey replicate independently of chromosomal DNA. Researchindicates that plasmids are associated with plasma membraneproteins. Plasmids usually contain from 5 to 100 genes that aregenerally not crucial for the survival of the bacterium under normalenvironmental conditions; plasmids may be gained or lostwithout harming the cell. Under certain conditions, however,plasmids are an advantage to cells. Plasmids may carry genes forsuch activities as antibiotic resistance, tolerance to toxic metals,the production of toxins, and the synthesis of enzymes. Plasmidscan be transferred from one bacterium to another. In fact, plasmidDNA is used for gene manipulation in biotechnology.RibosomesAll eukaryotic and prokaryotic cells contain ribosomes, whichfunction as the sites of protein synthesis. Cells that have highrates of protein synthesis, such as those that are actively growing,have a large number of ribosomes. The cytoplasm of a prokaryoticcell contains tens of thousands of these very small structures,which give the cytoplasm a granular appearance (see Figure 4.6).Ribosomes are composed of two subunits, each of which consistsof protein and a type of RNA called ribosomal RNA (rRNA).Prokaryotic ribosomes differ from eukaryotic ribosomes in thenumber of proteins and rRNA molecules they contain; theyare also somewhat smaller and less dense than ribosomes ofeukaryotic cells. Accordingly, prokaryotic ribosomes are called70S ribosomes (Figure 4.19), and those of eukaryotic cells areknown as 80S ribosomes. The letter S refers to Svedberg units, whichindicate the relative rate of sedimentation during ultra-high-speedcentrifugation. Sedimentation rate is a function of the size,weight, and shape of a particle. The subunits of a 70S ribosomeare a small 30S subunit containing one molecule of rRNA and alarger 50S subunit containing two molecules of rRNA.Several antibiotics work by inhibiting protein synthesis onprokaryotic ribosomes. Antibiotics such as streptomycin andgentamicin attach to the 30S subunit and interfere with proteinsynthesis. Other antibiotics, such as erythromycin andchloramphenicol, interfere with protein synthesis by attachingto the 50S subunit. Because of differences in prokaryotic andeukaryotic ribosomes, the microbial cell can be killed by the antibioticwhile the eukaryotic host cell remains unaffected.

Q What properties make endospores resistant to processes that normally killvegetative cells?Cell wallPlasmamembraneBacterialchromosome(DNA)Cytoplasm Spore septum begins to isolatenewly replicated DNA and asmall portion of cytoplasm.(a) Sporulation, the process of endospore formation(b) An endospore of Bacillus subtilisPlasma membrane starts to surround DNA,cytoplasm, and membrane isolated in step 1.Peptidoglycan layer forms between membranes.Spore coat forms.123456 Endospore is freed from cell.Spore septum surrounds isolated portion,forming forespore.Two membranesTEM 0.5 _ mEndosporeChapter 4 Functional Anatomy of Prokaryotic and Eukaryotic Cells 97nitrogen source, becomes scarce or unavailable. In the firstobservable stage of sporulation, a newly replicated bacterialchromosome and a small portion of cytoplasm are isolated byan ingrowth of the plasma membrane called a spore septum. Thespore septum becomes a double-layered membrane that surroundsthe chromosome and cytoplasm. This structure, entirelyenclosed within the original cell, is called a forespore. Thicklayers of peptidoglycan are laid down between the two membranelayers. Then a thick spore coat of protein forms aroundthe outside membrane; this coat is responsible for the resistanceof endospores to many harsh chemicals. The original cell is degraded,and the endospore is released.The diameter of the endospore may be the same as, smallerthan, or larger than the diameter of the vegetative cell. Dependingon the species, the endospore might be located terminally (atone end), subterminally (near one end; Figure 4.21b), or centrallyinside the vegetative cell. When the endospore matures, the vegetativecell wall ruptures (lyses), killing the cell, and the endosporeis freed.Most of the water present in the forespore cytoplasm iseliminated by the time sporulation is complete, and endosporesdo not carry out metabolic reactions. The endospore contains alarge amount of an organic acid called dipicolinic acid (DPA),which is accompanied by a large number of calcium ions. Evidenceindicates that DPA protects the endospore DNA againstdamage. The highly dehydrated endospore core contains onlyDNA, small amounts of RNA, ribosomes, enzymes, and a fewimportant small molecules. These cellular components are essentialfor resuming metabolism later.Endospores can remain dormant for thousands of years.An endospore returns to its vegetative state by a process calledgermination. Germination is triggered by physical or chemicaldamage to the endospores coat. The endospores enzymes thenbreak down the extra layers surrounding the endospore, water enters,and metabolism resumes. Because one vegetative cell forms asingle endospore, which, after germination, remains one cell, sporulationin bacteria is not a means of reproduction. This process doesnot increase the number of cells. Bacterial endospores differ fromspores formed by (prokaryotic) actinomycetes and the eukaryoticClinical Case ResolvedIt is the glycocalyx that enables bacteria in water to stickinside a pipe. The bacteria grow slowly in the nutrient-poortap water but do not get washed away by the flowingwater. A slimy layer of bacteria can accumulate in a pipe.Irene discovers that the disinfectant in the hospitals watersupply was inadequate to prevent bacterial growth. Somebacteria can get dislodged by flowing water, and evennormally harmless bacteria can infect a surgical incisionor weakened host.76 86 88 95 97principal differences between prokaryotic and eukaryotic cellsare summarized in Table 4.2 page 100.The following discussion of eukaryotic cells will parallel ourdiscussion of prokaryotic cells by starting with structures thatextend to the outside of the cell.As mentioned earlier, eukaryotic organisms include algae, protozoa,fungi, plants, and animals. The eukaryotic cell is typicallylarger and structurally more complex than the prokaryoticcell (Figure 4.22). When the structure of the prokaryotic cell inFigure 4.6 is compared with that of the eukaryotic cell, the differencesbetween the two types of cells become apparent. TheThe Eukaryotic Cellfungi and algae, which detach from the parent and develop into anotherorganism and, therefore, represent reproduction.Endospores are important from a clinical viewpoint and in thefood industry because they are resistant to processes that normallykill vegetative cells. Such processes include heating, freezing, desiccation,use of chemicals, and radiation. Whereas most vegetativecells are killed by temperatures above 70C, endospores cansurvive in boiling water for several hours or more. Endospores ofthermophilic (heat-loving) bacteria can survive in boiling waterfor 19 hours. Endospore-forming bacteria are a problem in thefood industry because they are likely to survive underprocessing,and, if conditions for growth occur, some species produce toxinsand disease. Special methods for controlling organisms that produceendospores are discussed in Chapter 7.Chec k Your Understanding Where is the DNA located in a prokaryotic cell? 4-10 What is the general function of inclusions? 4-11 Under what conditions do endospores form? 4-12* * *Having examined the functional anatomy of the prokaryotic cell,we will now look at the functional anatomy of the eukaryotic cell.