Complementation Test – rII Locus

• Infection by the bacteriophage T1 leads to reproduction of the virus at the expense

of the bacterial host, from which new phages are released as the host cell is
disrupted, or lysed.

• If a plate of E. coli is uniformly sprayed with T1, almost all cells are lysed.

• Rare E. coli cells, however, survive infection and are not lysed.

• If these cells are isolated and established in pure culture, all their descendants are
resistant to T1 infection.

• The adaptation hypothesis, - the interaction of the phage and bacterium is

essential to the acquisition of immunity. In other words, exposure to the phage
“induces” resistance in the bacteria.
 Spontaneous mutations in Bacteria

• Occur regardless of the presence or absence of bacteriophage T1.

• suggested an alternative model to explain the origin of resistance in E. coli.

• 1943, Salvador Luria and Max Delbrück presented the first convincing evidence that
bacteria, like eukaryotic organisms, are capable of spontaneous mutation.

• Mutant cells that arise spontaneously in otherwise pure cultures can be isolated and
established independently from the parent strain by the use of selection techniques.

• Selection refers to culturing the organism under conditions where only the desired
mutant grows well, while the wild type does not grow.
 Prototroph and Auxotroph
• growth medium for Bacteria consist only of an organic carbon source (such as glucose
or lactose) and various inorganic ions, including Na+, K+, Mg 2+, Ca2+, and NH4 +
present as inorganic salts, it is called minimal medium.

• To grow on such a medium, a bacterium must be able to synthesize all essential

organic compounds (e.g., amino acids, purines, pyrimidines, sugars, vitamins, and fatty
acids) – prototroph (wild type for all growth requirements).

• if a bacterium loses, through mutation, the ability to synthesize one or more organic
components, it is an auxotroph.

• For example, a bacterium that loses the ability to make histidine is designated as a his-
auxotroph, in contrast to its prototrophic his+ counterpart. For the his- bacterium to
grow, this amino acid must be added as a supplement to the minimal medium.

• Medium that has been extensively supplemented is called complete medium.

Growth of Bacteria in the Liquid Medium
Growth of Bacteria in the Semi Solid Medium
 transfer of genetic information from one bacterium to another
• conjugation,
• transformation, and
• transduction.
• vertical gene transfer - When transfer of genetic information occurs between members of
the same species.

• horizontal gene transfer - When transfer occurs between members of related but distinct
bacterial species.

• The horizontal gene transfer process has played a significant role in the evolution of
bacteria.
• Example - horizontal transfer of genes that confer survival advantages to the recipient
species. For example, one species may transfer antibiotic resistance genes to another
species. Or genes conferring enhanced pathogenicity may be transferred.
Conjugation in Bacteria: The Discovery of F1 and F2 Strains
• 1946, Joshua Lederberg and Edward Tatum showed that bacteria
undergo conjugation, a process by which genetic information
from one bacterium is transferred to and recombined with that of
another bacterium.
• genetic recombination had occurred.
• unidirectional transfer of genetic material.

• F+ cells (F for “fertility”) - cells serve as donors of parts of their

chromosomes.

• Recipient bacteria, designated as F− cells - receive the donor

chromosome material (now known to be DNA) and recombine it
with part of their own chromosome.
Davis U-tube Experiments (Bernard Davis, who designed the Davis U-tube )

• At the base of the tube is a sintered glass filter with a pore size that
allows passage of the liquid medium but is too small to allow
passage of bacteria.

• When Davis plated samples from both sides of the tube on minimal
medium, no prototrophs were found,

• Conclusion - that physical contact between cells of the two strains

is essential to genetic recombination.

• Physical interaction is mediated by a structure called the F pilus (or

sex pilus; pl. pili), a 6- to 9-nm tubular extension of the cell.

• F+ cells contain a fertility factor (F factor) that confers the ability to

donate part of their chromosome during conjugation.
 fertility factor (F factor)
• Joshua and Esther Lederberg and by William Hayes and Luca Cavalli-Sforza showed that certain environmental
conditions eliminate the F factor from otherwise fertile cells. However, if these “infertile” cells are then grown with
fertile donor cells, the F factor is regained.

• Conclusion - F factor is a mobile element.

• F factor has been shown to consist of a circular, double-stranded DNA molecule;

• it is equivalent in size to about 2 percent of the bacterial chromosome (about 100,000 nucleotide pairs) and contains as
many as 40 genes.

• Many are tra genes, whose products are involved in the transfer of genetic information, including the genes essential to
the formation of the sex pilus

• E. coli cell may or may not contain the F factor.

• When it is present, the cell is able to form a sex pilus and potentially serve as a donor of genetic information.

• During conjugation, a copy of the F factor is almost always transferred from the F + cell to the F- recipient, converting the
recipient to the F+ state.
 Hfr Bacteria
• 1950, Cavalli-Sforza treated an F+ strain of E. coli K12 with nitrogen mustard,

• genetically altered strain of donor bacteria that underwent recombination at a rate of

1/104 (or 10-4 ), 1000 times more frequently than the original F+ strains.

• strains were designated Hfr, for high-frequency recombination.

• Hfr cells constitute a special class of F+ cells.

 Hfr Bacteria and Chromosome Mapping – Interrupted Mating Technique
1950s, Ellie Wollman and François Jacob showed how Hfr strains would allow genetic mapping of the E. coli chromosome.

• culture containing a mixture of an Hfr and an F- strain was

incubated

• samples were removed at intervals and placed in a blender.

• The shear forces created in the blender separated conjugating

bacteria so that the transfer of the chromosome was terminated.
The order of gene transfer in four Hfr strains, suggesting that the E. coli chromosome is circular
 Conversion of F1 to an Hfr state occurs by
integration of the F factor into the bacterial
chromosome

Pg 176
 Role of the Bacterial Rec Proteins in Recombination
• mutations that impaired the process of recombination and led to the discovery of rec (for recombination)
genes.

• series of mutant genes labeled recA, recB, recC, and recD.

• rec mutations reduced recombination by about 100 times.

• Rec A - plays an important role in recombination involving either a single-stranded DNA molecule or the
linear end of a double-stranded DNA molecule that has unwound.

• single-strand displacement is a common form of recombination

• When double-stranded DNA enters a recipient cell, one strand is often degraded, leaving the complementary
strand as the only source of recombination. This strand must find its homologous region along the host
chromosome, and once it does, RecA facilitates recombination.

• RecBCD protein - important when double-stranded DNA serves as the source of genetic recombination.
RecBCD unwinds the helix, facilitating recombination that involves RecA
 F Factor - Plasmids
• F factor is a plasmid - extrachromosomal heredity unit, exists autonomously in the bacterial cytoplasm, composed of a
double-stranded closed circle of DNA.

• plasmids that can exist autonomously or can integrate into the chromosome are further designated as episomes

• The F factor plasmid confers fertility and contains genes essential for sex pilus formation, on which conjugation and
subsequent genetic recombination depend.

• Other examples of plasmids include the R and the Col plasmids.

• R plasmids consist of two components: the resistance transfer factor (RTF) and one or more r-determinants.

• RTF encodes genetic information essential to transferring the plasmid between bacteria. r-determinants - genes
conferring resistance to antibiotics or heavy metals such as mercury.

• Bacterium Shigella (1950) - bacteria resistant to as many as five of the above antibiotics. major health threat. Fortunately,
a bacterial cell sometimes contains r-determinant plasmids but no RTF. Although such a cell is resistant, it cannot transfer
the genetic information for resistance to recipient cells.

• The Col plasmid, ColE1 (derived from E. coli) - encodes one or more proteins that are highly toxic to bacterial strains
that do not harbor the same plasmid. These proteins, called colicins, can kill neighboring bacteria, and bacteria that carry
the plasmid are said to be colicinogenic
Transformation can also lead to genetic recombination
Life cycle of bacteriophage T4
The Viral Plaque assay
 Lysogeny
• Upon entry, the viral DNA is integrated into the bacterial chromosome instead of replicating in the bacterial
cytoplasm.

• each time the bacterial chromosome is replicated, the viral DNA is also replicated and passed to daughter
bacterial cells following division.

• No new viruses are produced, and no lysis of the bacterial cell occurs.

• Under certain stimuli, such as chemical or ultraviolet-light treatment, the viral DNA loses its integrated status
and initiates replication, phage reproduction, and lysis of the bacterium.

• The viral DNA integrated into the bacterial chromosome is called a prophage.

• Viruses that can either lyse the cell or behave as a prophage are called temperate phages.

• that can only lyse the cell are referred to as virulent phages.

• A bacterium harboring a prophage has been lysogenized and is said to be lysogenic.

 Transduction Is Virus-Mediated Bacterial DNA Transfer

• 1952, Norton Zinder and Joshua Lederberg were investigating possible recombination in
the bacterium Salmonella typhimurium.

• Trying to recover prototrophs from mixed cultures of two different auxotrophic strains,

• Recombination was not due to the presence of an F factor and conjugation, as in E. coli.

• A process of bacterial recombination mediated by bacteriophages and now called

transduction.
Transduction Is Virus-Mediated Bacterial DNA Transfer
Zinder and Lederberg Experiment
• P22, present initially as a prophage in the
chromosome of the LA-22 Salmonella cells.
• P22 prophages sometimes enter the vegetative, or
lytic, phase, reproduce, and are released by the LA-22
cells.

• being much smaller than a bacterium, then cross the

filter of the U-tube and subsequently infect and lyse
some of the LA-2 cells.

• In the process P22 phages occasionally package a

region of the LA-2 chromosome in their heads -
contains the phe+ and trp+ genes,

• the phages subsequently pass back across the filter

and infect LA-22 cells

• This is a process of transduction, where bacterial

recombination is mediated by bacteriophage P2.
Generalized Transduction
 Bacteriophage Mutations
• rapid lysis (r) - Hershey named the mutant rapid lysis (r) , plaques’ are of
larger size due to a more rapid or more efficient life cycle of the phage.

• We now know that, in wild-type phages, reproduction is inhibited once a

particular-sized plaque has been formed. The r mutant T2 phages
overcome this inhibition, producing larger plaques.

• Wildtype T2 phages can infect E. coli B (a unique strain), they normally

cannot attach or be adsorbed to the surface of E. coli B-2 (a different
strain).

• Salvador Luria - The h mutation, however, confers the ability to adsorb to

and subsequently infect E. coli B-2 in addition to B strain.
 mixed infection experiments, in which two distinct mutant
strains were allowed to simultaneously infect the same
bacterial culture.

 These studies were designed so that the number of viral

particles sufficiently exceeded the number of bacterial
cells to ensure simultaneous infection of most cells by both
viral strains.

 the parental viruses were of either the h+r (wild-type host

range, rapid lysis) or the hr + (extended host range, normal
lysis) genotype.

 recombinational frequency = (h+r + + hr)/total plaques *

100
 rII Locus of T4 Phage

• Mutants at the rII locus produce distinctive plaques when plated on E. coli strain B.

• Benzer isolated 20,000 rII mutants.

• Wanted to produce a genetic map of this locus through recombination.

• most of these mutations, because they were randomly isolated, would represent different
locations within the rII locus and would thus provide an ample basis for mapping studies
 Complementation in the rII Locus
• Benzer - a control study.
• Wild type – can lyse both E. coli B and K12 strain. Mutant – cannot lyse K12.
• K12 bacteria were simultaneously infected with pairs of different rII mutant strains.
• sometimes found that certain pairs of the rII mutant strains lysed the K12 bacteria.
• simultaneous infection, each mutant strain provided something that
the other lacked.
• complementation
• each mutation fell into one of two possible complementation
groups, A or B.
• Those that failed to complement one another were placed in the
same complementation group.
• those that did complement one another were each assigned to a
different complementation group.
• Benzer coined the term cistron.
• Cistron represents a gene.
• Benzer’s A and B cistrons represent two separate genes in what we
originally referred to as the rII locus.
• Complementation occurs when K12 bacteria are infected with two
rII mutants, one with a mutation in the A gene and one with a
mutation in the B gene.
• Wild type – can lyse both E. coli B and K12 strain. Mutant –
cannot lyse K12.
• If phages from any two different mutant strains were
allowed to simultaneously infect E. coli B, exchanges
between the two mutant sites within the locus would
produce rare wild-type recombinants.

• 99.9 percent rII phages and less than 0.1 percent wild-type
phages, were then allowed to infect strain K12, the wild-
type recombinants would successfully reproduce and
produce wild-type plaques

• By using serial dilution techniques, Benzer was able to

determine the total number of mutant rII phages produced
on E. coli B and the total number of recombinant wild-type
phages that would lyse E. coli K12.

• These data provided the basis for calculating the frequency

of recombination, a value proportional to the distance
within the gene between the two mutations being studied.
 Mapping the mutations into the rII locus – info from the deletions

• some of the rII mutations have deletions of small parts of both cistrons.
• When a deletion mutation was tested using simultaneous infection by two phage strains, one having the
deletion mutation and the other having a point mutation located in the deleted part of the same cistron -
never yielded wild-type recombinants.
• Because the deleted area is lacking the area of DNA containing the point mutation, no recombination is
possible.
Mapping the mutations into the rII locus – info from the deletions

• Depending on whether the viral chromosome

bearing a point mutation does or does not
undergo recombination with the chromosome
bearing a deletion, each point mutation can
be assigned to a specific area of the cistron.

• Further deletions within each of the seven

areas can be used to localize, or map, each rII
point mutation more precisely.
The rII Gene Map

• two cistrons composing the rII locus of phage T4.

• From the 20,000 mutations analyzed, 307 distinct

sites within this locus were mapped in relation to
one another.

• hot spots - were apparently more susceptible to

mutation than other areas in which only one or a
few mutations were found.
Split Gene
 Overlapping Genes

• Several different reading frames within the same mRNA, thus specifying more than one polypeptide.
• Concept of the overlapping genes.
• sometimes also refer to overlapping ORFs.
• phage Ø X174
• The circular DNA chromosome consists of 5386 nucleotides, which should encode a maximum of 1795 amino
acids, sufficient for five or six proteins. However, this small virus in fact synthesizes nine proteins consisting of
more than 2300 amino acids. A comparison of the nucleotide sequence of the DNA and the amino acid
sequences of the polypeptides synthesized has clarified the apparent paradox. At least four instances of
multiple initiation have been discovered, creating overlapping genes
Relative positions of the sequences encoding seven polypeptides of the phage Ø X174

• K and B polypeptides are initiated in separate reading frames within the sequence specifying the A
polypeptide.

• K gene sequence overlaps into the adjacent sequence specifying the C polypeptide.

• E sequence is out of frame with, but initiated within, that of the D polypeptide.

• Finally, the A′ sequence, while in frame with the A sequence, is initiated in the middle of the A sequence.
They both terminate at the identical point.

• Seven different polypeptides are created from a DNA sequence that might otherwise have specified only
three (A, C, and D).
 Overlapping Gene – Advantages and Disadvantages

• Advantage – limited DNA/RNA can code for multiple polypeptides.

• Disadvantage - a single mutation may affect more than one protein and thus increase the chances that the
change will be deleterious or lethal.