CHAPTER 5

Why is Relevant to Know the Genetics of Bacteria and Their Viruses?

Phylogenetic Tree of Life Domains on Planet Earth

bya

Common Ancestor

Like it or not, we truly live in a bacterial world (Pierce, 2012)

CHAPTER 5 OUTLINE
1. Viruses and Bacteria in Genetics 2. Mechanisms of Genetic Exchange in Bacteria

Conjugation Transformation Transduction

3. The Evolutionary Significance of Genetic Exchange in Bacteria
Physical maps and linkage maps compared

The fruits of DNA technology, made possible by bacterial genetics and their phages
The making of a genomic library

Insert 8-10 kb

Serratia marscescens: frequent contaminant of petri plates in the lab. SmaI CCC GGG GGG CCC

Serratia marcescens. A gram negative producing mucoid colonies. Red colonies (red pigment prodigiosin) produced at 30 oC and white colonies do not produce prodigiosin at 37 oC. This is an example of temperature-regulated phenotypic expression.

The Miracle of Bolsena: In 1263* the German priest Peter of Prague was breaking bread for communion at the church of Saint Christina in Bolsena, Italy. Peter was surprised when he broke the communion wafer and saw it had blood on it! *Note: Antonie van Leeuwenhoek first observed bacteria in 1663

Raphaels Vatican Painting

In1264 to honor of the miracle of Bolsena, Pope Urban instituted the feast of Corpus Christi (Body of Christ). Neither the Pope nor Peter the Priest could ever have known that a red bacterium, Serratia marscesens, was the probable cause of this bloodlike substance on the communion bread.

http://microbezoo.commtechlab.msu.edu/zoo/microbes/serratia.html

Model Organism Escherichia coli

First described by Theodore Escherich (1885) Lederberg and Tatum demonstrated sexual recombination Workhorse: rapid reproduction, small size, easy to grow One chromosome with 4.64 x 106 bp and 4300 genes (~35% UF) Haploid genome, therefore no dominance (masking) Human homology 8% Contain plasmids and episomes, some adapted as vectors, and now genetic engineered constructs Platform for genetic transformation Discoveries: elucidation of the genetic code, replication, and regulation Flagelum Asexual reproduction by simple binary fission. Wild-type bacteria are prototrophs; they can synthesize everything they need to grow and reproduce on minimal medium (carbon energy source, some inorganic salts, and water) Auxotrophic mutant bacteria require additional metabolites for growth.

Fimbriae

Escherichia coli is the centerpiece in recombinant DNA technology

Phenotypes in Bacteria Genetics

Colony color and morphology Nutritional mutants (auxotrophic) for energy sources Prototrophs and auxotrophs Antibiotic resistance Colony size and type Mutated genotypes by transgenes that are at the center of modern genetic engineering

Bacterial colonies, each derived from a single cell

Figure 5-3

(10)7

Distinguishing lac+ (prototroph) and lac- (auxotroph) by using an indicator red dye Auxothroph: A strain that will grow when a nutrient (building blocks) is lacsupplemented in the medium Prototroph: A strain that Figure 5-4 can grow on minimal medium, containing only inorganic salts, carbon source for energy, and water lac+ can use lactose sugar (glucose-galactose) lac+ lac- cannot use lactose; WT lacks -galactosidase function

Table 5-1

Mixing bacterial genotypes produces rare recombinants

Supplemented medium

Figure 5-5b

(Minimal medium)

Lederberg and Tatum, 1946

cross-feeding ?

No recombinants (WT) are produced without cell contact

Figure 5-6

Supplemented medium

Auxotrophic
When auxotrophic strains were plated out on minimal medium no prototrophic bacteria recovered, therefore, conjugation was required

Do not allow passage of bacteria

Bernard Davis U-tube experiment

Key Points Parasexual recombination mechanisms produce new combinations of genes in bacteria.

Parasexual mechanisms enhance the ability of bacteria to adapt to changes in the environment.

Genetic Exchange in Bacteria

Mutation is the source of new genetic variation.

Recombination produces new combinations of allele.

Transformation, conjugation, and transduction generate new combinations of genes in bacteria to allow bacteria to adapt to new environments.

Mechanisms of Genetic Exchange in Bacteria

Three parasexual processestransformation, conjugation, and transductionoccur in bacteria. These processes can be distinguished by two criteria: Gene transfer is inhibited by deoxyribonuclease Whether it requires cell contact.

Key Points
Transformation involves the uptake of free DNA by bacteria. This DNA can recombine with the host genome. Conjugation (Lederberg and Tatum, 1945) occurs when a donor cell makes contact with a recipient cell and then transfers DNA (F plasmidsex like) to the recipient cell and recombining; conjugation is not reciprocal. Transduction occurs when a virus carries bacterial genes from a donor cell to a recipient cell creating recombination on host chromosome.

Bacteria exchange DNA by several processes

Figure 5-2

Structure and function of phage T4

phage T4

Bacteria conjugate by using pili (singular, pilus)

Pilus

Figure 5-7

F (fertility factor) plasmids transfer during conjugation

F plasmid

OriT

Conjugation: In Hfr (high frequency of recombination) strains the F factor integrates into the bacterial chromosome behaving as F+ cells

Hfr
Fluorescent antibodies

Exconjugantes

Figure 5-10

F-, GFP + Mutant

Wollman and Jacob, 1957

Conjugation (Hfr = F+): Crossovers integrate parts of the transferred donor fragment
High frequency of recombination Fertility factor

Figure 5-11

(Crossovers)

Luca Cavalli-Sforza

Tracking time of marker entry generates a chromosome map

Figure 5-12a

Tracking time of marker entry generates a chromosome map

Figure 5-12b

A single crossover inserts F at a specific locus, which then determines the order of gene transfer

Figure 5-13

The F integration site determines the order of gene transfer in HFRs

Figure 5-14

Two types of DNA transfer can take place during conjugation

Figure 5-15

A single crossover cannot produce a viable recombinant

Figure 5-16

The generation of various recombinants by crossing over in different regions

Figure 5-17

Faulty outlooping produces F, an F plasmid that contains chromosomal DNA

Figure 5-18

Plasmids
A plasmid is a genetic element that can replicate independently of the main chromosome in an extrachromosomal state. Most plasmids are not required for the survival of the host cell. Plasmids in E. coli
F Factor (Fertility Factor) R Plasmids (Resistance Plasmids) Col Plasmids (synthesize compounds that kill sensitive cells)

Table 5-2

A plasmid with segments from many former bacterial hosts

Figure 5-19

An R plasmid with resistance genes carried in a transposon

Figure 5-20

Key Points
Plasmids are self-replicating extrachromosomal genetic elements.

Episomes can replicate autonomously or as integrated components of bacterial chromosomes.

F factors that contain chromosomal genes (F factors) are trasnferred to F- cells by sexduction.

Closely linked genes can be mapped in bacteria by threefactor crosses.

Transformation mechanism of DNA uptake by bacteria

Figure 5-21

The Genetics of Viruses

Viruses can only reproduce by infecting living host cells. Bacteriophages are viruses that infect bacteria. Several important genetic concepts have been discovered through studies of bacteriophages.

The Nobel Prizewinning biologist David Baltimore devised the Baltimore classification system

Bacteriophage T4

Double-stranded DNA genome Protein head Genome contains 168,800 base pairs and 150 characterized genes Lytic phage

Transduction: Structure and function of phage T4 Electron micrograph of phage infection

Figure 5-22

Cycle of a phage (T4) that lyses the host cells

Bacteriophage
Double-stranded DNA genome Genome contains, 48,502 base pairs and about 50 genes May be lytic or lysogenic

Cos Site*

protein coat

cleavage (during packaging)

CGGGGCGGCGACCTCG Bacteriophage GCCCCGCCGCTGGAGC

ligation (after infection)

DNA

GGGCGGCGACCTCG CG GC GCCCCGCCGCTGGA

bacteriophage
Needed for integration

cos

long (left) arm 19 kb

cos nonessential region short (right) arm 9 kb 20 kb 48.5 kb

*cos = cos site or cohesive end

Key Points
Viruses are nonliving obligate parasites that can reproduce only by infecting living host cells. Bacteriophages are viruses that infect bacteria. Bacteriophage T4 is a lytic phage that infects E. coli, reproduces, and lyses the host cell. Bacteriophage lambda () can enter a lytic pathway, like T4, or it can enter a lysogenic pathway, during which its chromosome is inserted into the chromosome of the bacterium. In its integrated state, the chromosome is called a prophage, and its lytic genes are kept turned off.

A plaque is a clear area in which all bacteria have been lysed by phages

Plaques from recombinant and parental phage progeny

A phage cross made by doubly infecting the host cell with parental phages

Figure 5-27

Generalized transduction by random incorporation of bacterial DNA into phage heads

Figure 5-29

Mapping Genes in Bacteriophage

Genes may be mapped based on recombination frequencies. Host bacteria are infected with two types of phage; progeny phage are screened for recombination. Map distances are calculated as the average number of crossovers between genetic markers.

From high cotransduction frequencies, close linkage is inferred

Figure 5-30

Table 5-3

Transfer of prophage during conjugation can trigger lysis

Figure 5-31

Transduction
In transduction, a bacteriophage transfers DNA from a donor cell to a recipient cell. In generalized transduction, a random fragment of bacterial DNA is packaged in the phage head in place of the phage DNA. In specialized transduction, recombination between the phage chromosome and the host chromosome produces a phage chromosome containing a piece of bacterial DNA.

 phage inserts by a crossover at a specific site

Figure 5-32

Faulty outlooping produces phage containing bacterial DNA

Figure 5-33a

Faulty outlooping produces phage containing bacterial DNA

Figure 5-33b

Faulty outlooping produces phage containing bacterial DNA

Figure 5-33c

A map of the E. coli genome obtained genetically

Figure 5-34

Part of the physical map of the E. coli genome, obtained by sequencing

Figure 5-35

Physical map of the E. coli genome

Figure 5-36

Proportions of the genetic and physical maps are similar but not identical

Figure 5-37

Transposon mutagenesis can be used to map a mutation in the genome sequence

Figure 5-38