Chapter 6

Genetic Analysis and Mapping in Bacteria and Bacteriophages

Genetic Phenomena in Prokaryotes and Bacteriophages

• Simple model systems– Very short life cycles– Easily studied in pure cultures– Inexpensive to cultivate in large numbers– Very few “rights”….– Spontaneous mutation

Terminology

• Minimal medium

• Prototroph/auxotroph

• Lag phase/log phase/stationary phase

Mechanisms of Genetic Exchange

• Bacterial Conjugation – Mechanism of transferring genetic information from

one bacterium to another, followed by recombination with the recipient bacterium’s genetic material

• Transformation– Uptake of DNA from the surrounding medium and

recombination into the recipient bacterium’s genetic material

• Transduction– Transfer of genetic material from one bacterium to

another via bacteriophage

Bacterial Conjugation

• Discovered by Lederberg and Tatum (1946)– Two auxotroph strains (one was met bio and the

other thr leu thi)– Culture together on complete medium– Subculture on minimal medium

• Some cells were prototrophs (10-7)… and 2-3 independent gene mutations unlikely to create revertants

• One strain had provided genetic material to replace defective genes in the other

Lederberg and Tatum: More Work

• Transfer is unidirectional– Some strains are always donors, some always

recipients in an exchange

• Strains designated F+ (fertility, donor) or F-

(recipient)

Bernard Davis

• Demonstrated using a U-tube culture that contact between donor and recipient cells was necessary for the transfer of genetic material

• Now know transfer through F pilus

Bacterial Conjugation

F factor

• Fertility conferred by a factor that could be lost and regained by a strain (from another F+ strain)– Mobile element

• now known to be a plasmid (autonomous genetic element)

• 100 kbp in size

• Encodes 20 genes for genetic transfer (plus others)

– Nearly always transferred to recipient cell during conjugation

• Converting recipient to F+

Hfr Strains and Chromosome Mapping

• Nitrogen mustard treatment of F+ strain to induce mutations (1950, 1953))– Mutant had recombination rate of 10-4 (vs. 10-7)– Strains called Hfr for high-frequency

recombination– Unlike “normal” F+ strains, Hfr strains do not

convert recipient cell to F+

– Genes transferred at different rates• Some very commonly, some not at all… something had

changed…

Hfr Strains and Chromosome Mapping

• Wollman and Jacob’s Interrupted Mating Technique– Allow mating (conjugation) to proceed for specified

time and then transfer to blender • Sheer forces terminate transfer through pilus

– Used antibiotic sensitive donor and resistant recipient– Some genes always transferred sooner than others

• Seemed to be a specific order

• Chromosome transferred linearly from a specific start point

Time Map• Times when

individual genes first observed to have been transferred

• Time could vary depending upon Hfr strain– Order same

– Start point varied

Order of Transfer Same, First Gene and Direction Varies

First Prokaryotic Genetic Maps

• Map units in minutes, not recombination frequency

• E. coli K12 map approximately 100 minutes total

• Original map had about 1000 genes noted – Modern sequence map identified over 4000 putative

genes (ORFs)

Conversion of F+ to Hfr

• F plasmid integrates into host chromosome

• Transfer always begins from one end of integrated F

• One strand of duplex “peeled off” and transferred through pilus

• Second strand synthesis and recombination occurs in recipieint

Hfr to F’ Conversion

• Integrated F plasmid can excise– Often includes portion of

host chromosome

– New plasmid called F’– Cell with F’ is partially

diploid and called a merozygote (very useful for studying genetic regulation in bacterial systems)

Discovery of rec Genes

• Mutants isolated with diminished recombination ability– recA, recB, recC, and recD genes (at first)– RecA protein involved in strand transfer

reaction, integrating donor strand into recipient duplex (strand displacement)

– RecBCD complex cuts and unwinds strand from donor duplex

Plasmids

• F factors are plasmids (F plasmids)– Small, generally circular, independent replicons

• R plasmids encode resistance transfer factor (RTF, essential for genetic transfer) and one or more antibiotic resistance genes (r-determinants)

• Col plasmids (e.g. ColE1) encode proteins that are highly toxic to other bacteria (colicins), and complementary immunity proteins for protection

• Plasmids are a key to rDNA technology (Ch. 19)

R Plasmids

• Common reason as to why antibiotics “quit working” after a number of years of use

• Spread rapidly through bacterial populations under selective pressure

Transformation

• Foreign DNA enters the cell from the surrounding medium (Griffith’s experiment)

• Two steps– Entry of foreign DNA into cell– Replacement by donor DNA of resident DNA

(but sometimes the donor DNA remains independent)

Transformation Process

• Competence– A physiological state which allows the cell to take up

foreign DNA into the cell• Natural competence requires specific receptors on the cell

surface, energy and transport molecules

• dsDNA is taken up, one strand is degraded

• Surviving strand integrates into recipient chromosome, forming heteroduplex

• Mismatch repair leads to gene conversion (or replication leads to nonidentical daughter cells)

• Cotransformation identifies “linked” genes

Transformation Process

Bacteriophage

• Viruses with bacterial hosts, phage for short• Valuable models for genetic research• T4 life cycle

– Phage binds to host cell– DNA injected into cell– All host DNA replication, transcription stops– Host chromosome degraded, phage DNA

transcribed/replicated, phage proteins synthesized– Phage particles assembled, host cell lysed to release

progeny– See figure 6-15

Bacteriophage T4

T4 Life

Cycle

Plaque Assays

• Lytic action of phage leads to the formation of a plaque on a plate covered by a lawn of bacteria– When properly spread one obtains one plaque per infected cell

– Serial dilution allows one to count phage particle concentration in a solution

– Allows one to perform elegant analyses of phage genes

Plaque Assays

Lysogeny• Lysogeny

– Lysogenic or temperate phage– Occurs when instead of replicating and lysing host

cell, phage integrates its DNA into host chromosome• prophage

– No new phage produced– Integrated phage passed on to cell progeny– Cell and progeny immune to further infection by

similar phage

• Episome– Genetic element that can either replicate

independently or as part of the bacterial chromosome

Transduction

• Zinder and Lederberg, 1952– Studying Salmonella typhimurium– Recovered prototrophs from culture of two

auxotrophs, but no F plasmid present– U-tube experiment still allowed prototroph production

when two auxotrophs remain separated• Filterable agent involved

– DNA transfer by bacteriophage P22• Portion of bacterial chromosome packaged into phage head

• Transduction

Transduction Experiment

Transduction Process

Transduction

• Specialized transduction– Only few and specific bacterial genes transferred

• Generalized transduction– No phage DNA included– Any bacterial gene transferred, up to 1% of total– Abortive (vs. complete) transduction occurs when

transferred DNA is not integrated into the host chromosome and is passed on to only one daughter cell (until eventually degraded)

• Transduction mapping uses gene cotransfer frequency

Bacteriophage Genetics

• Bacteriophage undergo genetic recombination• Genetic maps can be constructed by mixed

infection experiments– Simultaneous infection with two different phage

mutants/strains (Seymour Benzer, 1950s)– h+r x hr+ gives some hr and h+r+ progeny– Recombinants over total gives frequency and distance

of separation (map distance) proportional to % recombination

– Detection of recombinants at 1 per 106

– Could map multiple positions within single genes..

Complementation• Also discovered by Benzer studying rII locus

of bacteriophage T4– rII mutants can lyse E. coli B but not E. coli

K12()– Simultaneous infection of K12 with certain pairs of

rII mutants did produce plaques• Individual mutants fell into one of 2 groups

– Pairs of mutations that produced plaques were said to complement each other (different complementation groups)

– Smallest unit of complementation called a cistron (equivalent to a gene today)

•

Mapping Within a Cistron

Deletion Mapping

• Mutants created with segments of the chromosome deleted

• Mutants that failed to complement a deletion mutant possessed a mutated locus within the deletion– Preliminary mapping of mutants to a general

locatioin

Deletion Mapping

Benzer’s Significance

• Combining the results from his studies, Benzer had defined an abstract unit (the gene) as a mutational and recombinational unit that was arranged in a specific order (not an indivisible particle such as a marble)