The Genetics of Bacteria and Their Viruses ! Problems assigned the week of Jan 27, for presentation during the week of Feb 3, as model for quiz during the week of Feb 10 !Chapter 5: 2, 6, 9, 14, 15, 20, 24, 26, 34, 40

Gene linkage – Gene mapping in Eukaryotes !Physically linked alleles are inherited together except when there is a cross-over in the region of the chromosomes between the genes.

A B

a B

a b

A b

All the events that we have been observing are reciprocal; information can be exchanged, but the number of copies of a given

allele remains the same during the process

Meiosis is a carefully controlled process to insure the proper segregation of the genetic information

Chromosomes segregate independently, and even genes on the same chromosome get segregated by recombination

A B

a B

A b

a b

A B

a B

a b

A b

Bacteria are single-celled, prokaryotic organisms Their cells do not have a nucleus or other organelles. They typically have circular chromosomes. They are typically haploid. They have genes; they have phenotypes. They have cell division (binary fission), but not meiosis. !

Escherichia coli or E. coli is the most commonly

used bacterial organism for genetic studies.

Bacteria and other single celled organisms offer the advantage of large populations. !Rare mutations can be selected from large populations when there is a way to select for or identify the mutant. !Bacteria have fast generation times. Cell division can be as short as 20 minutes. !In haploid organisms the genotype and phenotype are the same since recessive alleles are not masked by a second copy of the gene.

Bacteria used in experiments are most commonly seen as liquid cultures or as colonies on agar plates. Bacteria can be

streaked, spread, or arrayed on semi-solid agar with growth media.

Dr. Axel Oberbremer

E. coli can be grown in liquid media. !It grows best (fastest) at 37ºC and with aeration – that comes with shaking.

Bacteria can easily be counted on agar plates after serial dilution.

Fresh media

1 ml

1/10 1/10 1/10 1/10 1/10 th ml

6 colonies, inoculum was 60 bacteria/ml

The original culture was 6 x 10 5 bacteria /ml

The phenotype of a bacteria is usually seen by whether it grows or not on a selective media.

There are three major classes of genes that are often used for bacterial genetics

!I. Resistance to antibiotics

!II. Requirement for a particular nutrient in order to grow.

III. Ability to grow on a particular compound

as a sole carbon source.

I. Resistance to antibiotics ! ampicillin (ampr) kanamycin (kanr) streptomycin (strr) tetracycline (tetr) neomycin (neor)

Rich media, Rich media + ampicillin no antibiotics

I. Resistance to antibiotics ! ampicillin (ampr) kanamycin (kanr) streptomycin (strr) tetracycline (tetr) neomycin (neor)

Rich media, Rich media + ampicillin no antibiotics

I. Resistance to antibiotics ! ampicillin (ampr) kanamycin (kanr) streptomycin (strr) tetracycline (tetr) neomycin (neor)

Rich media, Rich media + ampicillin no antibiotics

I. Resistance to antibiotics ! ampicillin (ampr) kanamycin (kanr) streptomycin (strr) tetracycline (tetr) neomycin (neor)

Rich media, Rich media + ampicillin no antibiotics

ampramps

Gene symbol systems - E. coli

Escherichia coli and other bacterial genes !In class we will use three letter symbols with + for wild type, normal and – for the mutant type. !leu+ leu– , dcm+ dcm–

!Bacterial geneticists usually shorten leu– to leu

II. Requirement for a particular nutrient in order to grow. Many of these gene mutations are related to a requirement for particular amino acids; eg. arginine arg- cysteine cys- glycine gly- histidine his- leucine leu- lysine lys- methionine met- proline pro-

III. Cannot grow on a particular compound as a sole carbon source. Many of these gene mutations prevent E. coli from growing with a particular sugar as a sole carbon source. ! lactose lac- galactose gal- Rich media Minimal MM+ arginine

media

Wild type (wt) arg-

arg- does not grow on Minimal Media (MM) It does grow on MM + arginine It cannot synthesize arginine. The wild type strain that can grow on minimal media is called a prototroph, the mutants that need supplements are called auxotrophs

Rich media (LB)

Minimal media (MM)

MM + arginine MM + glycine

arg- gly-

arg – mutants cannot synthesize the amino acid arginine. Arginine must be in the media for them to grow

gly- mutants cannot synthesize the amino acid glycine. It must be in the media for them to grow

Rich media (LB)

III. Mutants that cannot grow on a particular compound as a sole carbon source.

SM + lactose SM+ glucose

lac

lac- mutants cannot grow on lactose as a sole source of carbon, i.e energy. They require some other energy source.

Rich media (LB)

III. Mutants that cannot grow on a particular compound as a sole carbon source

SM + lactose SM+ glucose

lac-

lac- lac-

SM + glycerol SM+ fructose

lac- mutants cannot grow on lactose as a sole source of carbon, i.e energy. They require some other energy source.

lac-lac-

II. Requirement for a particular nutrient in order to grow. Many of these gene mutations are related to a requirement for particular amino acids; eg. arginine arg- cysteine cys- glycine gly- histidine his- leucine leu- lysine lys-

III. Cannot grow on a particular compound as a sole carbon source. Many of these gene mutations prevent E. coli from growing with a particular sugar as a sole carbon source. ! lactose lac- galactose gal-

I. Resistance to antibiotics ampicillin (ampr) kanamycin (kanr) streptomycin (strr) tetracycline (tetr)

These mutants provide the tools to look for genetic exchange among bacteria. !If we think about the complexity of the system for genetic exchange in eukaryotes (the whole system of meiosis) the idea that bacteria would undergo genetic exchange seemed, to most researchers, very unlikely

These mutants provide the tools to look for genetic exchange among bacteria. !If we think about the complexity of the system for genetic exchange in eukaryotes (the whole system of meiosis) the idea that bacteria would undergo genetic exchange seemed, to most researchers, very unlikely !However, with collections of auxotrophic, drug resistant and sugar-requiring mutants, researchers looked for genetic exchange !They mixed together strains of E. coli with different combinations of multiple auxotrophic mutants, and looked for the appearance of prototrophs

Bacterial Mating

The F+ fertility factor is about 100 kb in length, about 1/50 the size of the 4600 kb bacterial chromosome

www.bio.utexas.edu/faculty/sjasper/bio212/microbial.html

In high frequency recombination (Hfr) bacterial strains the F factor is integrated into the chromosome.

Hfr strains can transfer a copy of their chromosome to a recipient (F - ) strain.

New DNA introduced into the bacterial cell is incorporated into the circular chromosome by a double crossover.

The linear fragments are lost during cell replication.

Individual strains have a fixed starting point for the transfer a copy of their chromosome.

It takes about 60 minutes to transfer the whole chromosome. Gene order can be determined by the time it takes to

transfer different genes to the recipient strain.

Bacterial mating - can be interrupted by vigorous agitation of the culture.

Donor Recipient

Media: no arginine no leucine no histidine lactose

after 30 min

disrupt

Repeat at 10, 20, 30 and 40 minutes

arg+ leu+ his+ lac+

Bacterial mating !Strs, arg+, leu+, his+, lac+ (Hfr) X (F-) Strr, arg-, leu-, his-, lac-

!Selective media to determine the genotype. !Media Phenotype selected MM + Str (control) Strr, arg+,leu+his+ !MM + Str + leu, his, glucose (no arg) Strr, arg+

!MM + Str + arg, his, glucose (no leu) Strr, leu+

!MM + Str + arg, leu, glucose (no his) Strr his+ !MM + Str + arg, leu, his, lactose Strr lac+

Strs, arg+,leu+,his+,lac+ (Hfr) X Strr,arg-,leu-,his-,lac- !phenotype 10 min. 20 min. 30 min. 40 min. arg+ 0 0 3 20 leu+ 6 30 60 61 his+ 0 8 41 56 lac+ 0 0 0 10 !

What is the gene order?

Strs, arg+,leu+,his+,lac+ (Hfr) x Strr,arg-,leu-,his-,lac- !phenotype 10 min. 20 min. 30 min. 40 min. arg+ 0 0 3 20 leu+ 6 30 60 61 his+ 0 8 41 56 lac+ 0 0 0 10 !

The gene order is: leu, his, arg, lac

Different High Frequency Recombination (Hfr) strains have different starting points and different orientations

for the transfer of a copy of their chromosome. !

Mapping data from different Hfr strains can be combined to produce the complete circular genetic

map of a bacteria.

Different Hfr strains have the fertility factor in different positions and orientations in their genome

Genes are in the same order in different strains, but the first genes

to be transferred will be different in different strains.

Bacterial genetic maps are circular. !Bacterial chromosomes are circular

There are three E. coli strain types to remember in reference to bacterial conjugation. !1. F+ strains have the fertility factor as a plasmid. F+ can donate the fertility factor but cannot receive DNA. They donate the F+ factor readily. A very small proportion of the population have the F+ factor integrated into the chromosome and can donate other genes. !

2. F- strains can receive DNA but cannot donate DNA. If they receive the F+ factor, they become F+. !3. Hfr strains have the F+ integrated into a specific place in their chromosome. They transfer their chromosome into a F- strain, starting at the place adjacent to the F factor, with the F factor itself being the last DNA to transfer.

Masamichi Kohiyama, Sota Hiraga, Ivan Matic, Miroslav Radman Science (2003) 301:802-803

2003 - 50th Anniversary of discovery of Bacterial Mating !Donor: C (spots are hemi- methylated DNA) !Recipient: D !Mated cells are green with yellow spots - which mark the presence of hemi-methylated DNA !A shows Hfr mating, B an F+ transfer

Some background information !

Bacteria have simple “immune” systems that result from enzymes called restriction enzymes that cut foreign DNA

that enters the cell Bacteria protect their own DNA from these systems by modifying the DNA, typically by methylation of specific

bases – these combined systems are restriction-modification systems.

We will talk more about restrictions enzymes when we talk about genetic engineering

The Dam methylase methylates the A of the sequence GATC

Recipient F- bacteria are expressing GFP (green fluorescent protein) and thus are green, and are dam- and thus cannot methylate their DNA !Donor F+ or Hfr bacteria have red labeled DNA because a fluorescent antibody is binding the SeqA protein, which in turn is binding to hemi-methylated DNA (this is DNA that has a methyl group added to specific bases, but only on one strand of the double helix). Hemi-methylated regions occur transiently during replication before the newly synthesized strand is itself methylated !The F- strains do not have visible DNA because they are methylation defective dam- mutants, and fail to methylate the dam sites, so SeqA does not bind

Because only one strand of the F+ or Hfr donated DNA enters the recipient cell, it is always hemi-methylated. The incoming strand is methylated because the donor is Dam+, the recipient duplicates the DNA but does not add any methyl groups, so the GATC sites of the transferred DNA are completely hemi-methylated