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CHAPTER 5

Why is Relevant to Know the Genetics of Bacteria

and Their Viruses?

Phylogenetic Tree of Life Domains on Planet Earth

Common Ancestor

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

bya

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CHAPTER 5 OUTLINE1. Viruses and Bacteria in Genetics

2. Mechanisms of Genetic Exchange in BacteriaConjugation

Transformation

Transduction3. The Evolutionary Significance

of Genetic Exchange in

Bacteria

Physical maps and linkage

maps compared

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The fruits of DNA technology, made possible by

bacterial genetics and their phages

Insert 8-10 kb

The making of a genomic library

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

SmaI

CCC GGG

GGGCCC

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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.

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The Miracle of Bolsena:

In 1263* the German priest

Peter of Prague was breakingbread for communion at the

church of Saint Christina in

Bolsena, Italy.

Peter was surprised when hebroke the communion wafer

and saw it had blood on it!

*Note: Antonie van

Leeuwenhoek first

observed bacteria

in 1663

In1264 to honor of the miracle of Bolsena, Pope

Urban instituted the feast of Corpus Christi (Body ofChrist). Neither the Pope nor Peter the Priest could

ever have known that a red bacterium, Serratia

marscesens, was the probable cause of this blood-

like substance on the communion bread.

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

Raphaels Vatican Painting

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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

Asexual reproduction by simple binary fission. Wild-type bacteria are prototrophs; they can

synthesize everything they need to grow andreproduce on minimal medium (carbon energysource, some inorganic salts, and water)

Auxotrophic mutant bacteria require additional

metabolites for growth.

Fimbriae

Flagelum

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Escherichia coliis the centerpiece in recombinant

DNA technology

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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

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Bacterial colonies, each derived from a single cell

Figure 5-3

(10)7

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Distinguishing lac+ (prototroph) and lac- (auxotroph) by

using an indicator red dye

Figure 5-4

Auxothroph: A strain that

will grow when a nutrient

(building blocks) is

supplemented in the

medium

Prototroph: A strain that

can grow on minimalmedium, containing only

inorganic salts, carbon

source for energy, and

waterlac+can use lactose sugar

(glucose-galactose)

lac-cannot use lactose;

lacks -galactosidase

function

lac+

WT

lac-

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Table 5-1

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Mixing bacterial genotypes produces rare recombinants

Figure 5-5b

Lederberg and Tatum, 1946 cross-feeding ?

Supplemented

medium

(Minimal medium)

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No recombinants (WT) are produced without cell contact

Figure 5-6

Bernard Davis U-tube experiment

Supplemented medium Auxotrophic

Do not allow passage of bacteria

When auxotrophic strains were plated

out on minimal mediumno prototrophic bacteria recovered,therefore, conjugation was required

K P i

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Key Points

Parasexual recombination mechanisms

produce new combinations of genesin bacteria.

Parasexual mechanisms enhance the

ability of bacteria to adapt to changes

in the environment.

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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.

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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.

Mechanisms of Genetic Exchange in Bacteria

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Key Points

Transformation involves the uptake of free DNA by

bacteria. This DNA can recombine with the hostgenome.

Conjugation (Lederberg and Tatum, 1945) occurs

when a donor cell makes contact with arecipient cell and then transfers DNA (F plasmid-sex like) to the recipient cell and recombining;conjugation is not reciprocal.

Transduction occurs when a virus carries bacterialgenes from a donor cell to a recipient cellcreating recombination on host chromosome.

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Bacteria exchange DNA by several processes

Figure 5-2

phage T4

Structure and

function of

phage T4

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Bacteria conjugate by using pili (singular, pilus)

Figure 5-7

Pilus

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F (fertility factor) plasmids transfer during conjugation

OriT

F plasmid

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Conjugation: In Hfr (high frequency of recombination) strains the F factor

integrates into the bacterial chromosome behaving as F+ cells

Figure 5-10

Fluorescent antibodies

Hfr

F-, GFP + Mutant

Wollman and Jacob, 1957

Exconjugantes

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Conjugation (Hfr = F+): Crossovers integrate parts of the

transferred donor fragment

Figure 5-11

(Crossovers)

Luca Cavalli-Sforza

High frequency of recombination Fertility factor

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Tracking time of marker entry generates a chromosome map

Figure 5-12a

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Tracking time of marker entry generates a chromosome map

Figure 5-12b

f

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A single crossover inserts F at a specific locus, which then

determines the order of gene transfer

Figure 5-13

Th F i t ti it d t i th d f t f

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The F integration site determines the order of gene transferin HFRs

Figure 5-14

T t f DNA t f t k l d i j ti

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Two types of DNA transfer can take place during conjugation

Figure 5-15

A i l t d i bl bi t

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A single crossover cannot produce a viable recombinant

Figure 5-16

The generation of various recombinants by crossing over in

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Figure 5-17

The generation of various recombinants by crossing over in

different regions

F lt tl i d F F l id th t t i

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Figure 5-18

Faulty outlooping produces F, an F plasmid that contains

chromosomal DNA

Pl id

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Plasmids

A plasmid is a genetic element that can

replicate independently of the mainchromosome in an extrachromosomal state.

Most plasmids are not required for the survivalof the host cell.

Plasmids in E. coli

F Factor (Fertility Factor) R Plasmids (Resistance Plasmids)

Col Plasmids (synthesize compounds that killsensitive cells)

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Table 5-2

A plasmid with segments from many former bacterial hosts

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A plasmid with segments from many former bacterial hosts

Figure 5-19

An R plasmid with resistance genes carried in a transposon

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An R plasmid with resistance genes carried in a transposon

Figure 5-20

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Key Points

Plasmids are self-replicating extrachromosomal geneticelements.

Episomes can replicate autonomously or as integratedcomponents of bacterial chromosomes.

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

Closely linked genes can be mapped in bacteria by three-

factor crosses.

Transformation mechanism of DNA uptake by bacteria

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Transformation mechanism of DNA uptake by bacteria

Figure 5-21

Th G ti f Vi

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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 discoveredthrough studies of bacteriophages.

TheNobel Prize-winning biologist

David Baltimore

devised the

Baltimoreclassification

system

B t i h T4

http://www.ask.com/wiki/Nobel_Prize?qsrc=3044http://www.ask.com/wiki/David_Baltimore?qsrc=3044http://www.ask.com/wiki/Virus_classification?qsrc=3044%23Baltimore_classificationhttp://www.ask.com/wiki/Virus_classification?qsrc=3044%23Baltimore_classificationhttp://www.ask.com/wiki/Virus_classification?qsrc=3044%23Baltimore_classificationhttp://www.ask.com/wiki/Virus_classification?qsrc=3044%23Baltimore_classificationhttp://www.ask.com/wiki/David_Baltimore?qsrc=3044http://www.ask.com/wiki/Nobel_Prize?qsrc=3044

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Bacteriophage T4

Double-stranded

DNA genome

Protein headGenome contains

168,800 base

pairs and 150

characterizedgenes

Lytic phage

Transduction: Structure and function of phage T4

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Transduction: Structure and function of phage T4

Figure 5-22

Electron micrograph of phage

infection

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

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

Bacteriophage

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BacteriophageDouble-stranded DNA genome

Genome contains, 48,502 base pairs and about 50 genes

May be lytic or lysogenic

Cos Site*

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Bacteriophage

CG

GCCCCGCCGCTGGA

GGGCGGCGACCTCG

GC

CGGGGCGGCGACCTCG

GCCCCGCCGCTGGAGCcleavage

(during

packaging)

ligation

(after

infection)

cos coslong (left) arm short (right) armnonessential region

48.5 kb

*cos = cos site or cohesive end

19 kb 9 kb20 kb

bacteriophage

protein coat

DNA

Needed for integration

Cos Site

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K P i t

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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 lyticpathway, like T4, or it can enter a lysogenicpathway, during which its chromosome isinserted into the chromosome of the bacterium.

In its integrated state, the chromosome is calleda prophage, and its lytic genes are kept turnedoff.

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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

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Figure 5-27

p g y y g

parental phages

Generalized transduction by random incorporation of

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Figure 5-29

y pbacterial DNA into phage heads

Mapping Genes in Bacteriophage

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Mapping Genes in Bacteriophage

Genes may be mapped based on recombination

frequencies.

Host bacteria are infected with two types ofphage; progeny phage are screened forrecombination.

Map distances are calculated as the averagenumber of crossovers between genetic markers.

From high cotransduction frequencies, close linkage is

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From high cotransduction frequencies, close linkage is

inferred

Figure 5-30

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Table 5-3

Transfer of prophage during conjugation can trigger lysis

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p p g g j g gg y

Figure 5-31

Transduction

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Transduction

In transduction, a bacteriophage transfers DNA

from a donor cell to a recipient cell.

In generalized transduction, a randomfragment of bacterial DNA is packaged in the

phage head in place of the phage DNA.

In specialized transduction, recombinationbetween the phage chromosome and the hostchromosome produces a phage chromosomecontaining a piece of bacterial DNA.

phage inserts by a crossover at a specific site

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p g y p

Figure 5-32

Faulty outlooping produces phage containing bacterial

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y p g p p g g

DNA

Figure 5-33a

Faulty outlooping produces phage containing bacterial

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y p g p p g g

DNA

Figure 5-33b

Faulty outlooping produces phage containing bacterial

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y p g p p g g

DNA

Figure 5-33c

A map of the E. coli genome obtained genetically

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p g g y

Figure 5-34

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

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sequencing

Figure 5-35

Physical map of the E. coli genome

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Figure 5-36

Proportions of the genetic and physical maps are similar but

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not identical

Figure 5-37

Transposon mutagenesis can be used to map a mutation in

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Figure 5-38

the genome sequence