Fundamentals of Microbiology (Biology 240)

Course notes
Dr. Josh D. Neufeld
Topic 9: Bacterial Genetics
Learning Objectives
By the end of this module you should be able to

 list reasons why bacteria are ideal genetic research tools;

 correctly use terminology and conventions for microbial genotypes and
phenotypes;
 discuss the differences between selection and screening of mutants, providing
examples of each;
 describe the mechanisms of restriction and modification enzymes;
 explain how plasmids can be used to clone DNA for screening or selection;
 contrast transformation, conjugation, and transduction;
 explain the following conjugation terminology: F+, F-, F′, Hfr, tra, oriT;
 explain how mobile genetic elements can mobilize DNA through replicative and
non-replicative tranposition; and
 contrast generalized and specialized transduction.

For several reasons, microbial genetics is of fundamental importance for

biology. First, cell function is determined by gene function and gene regulation.
Second, because genetics of prokaryotic microorganisms is generally much more
accessible than genetics of eukaryotic organisms, with the possible exception of
yeast, microbial systems are very useful as experimental systems. Microbial
genetics gave rise to molecular biology and most of the tools used for molecular
cloning are of prokaryotic origin.
Terminology
 Mutant: a strain carrying a mutation
 Genotype: genetic description of a strain (e.g., hisC)
 Phenotype: observable properties of a strain (e.g., His-)
 Wild-type strain: original isolate from which mutant strains are derived

Depending on their phenotype, mutations may be selectable or nonselectable.

Selectable mutations confer the ability to grow under conditions that do not allow
growth of a strain that does not carry the mutation. For example, an antibiotic
resistance phenotype would be selectable, whereas an antibiotic sensitivity
phenotype would be nonselectable. It is possible to select directly for the
antibiotic resistance phenotype on antibiotic-containing medium. In contrast, the
antibiotic sensitivity phenotype cannot be selected for, but it could be screened.
Replica plating (Figure 10.6 - 3rd edition or Figure 9.6 - 2nd edition) is a
technique for screening many colonies for a nonselectable phenotype. This
technique is especially useful for the isolation of auxotrophs, which are mutants
 Fundamentals of Microbiology (Biology 240)
Course notes
Dr. Josh D. Neufeld
that have a specific nutritional requirement (e.g., amino acid, vitamin). Note that
the non-mutant parental strain is referred to as a prototroph.

Homologous recombination refers to the exchange, or crossing over, between

identical or highly similar (=homologous) DNA sequences. The process of
recombination (Figure 10.19 - 3rd edition or Figure 9.19 - 2nd edition) is complex
and involves many different proteins. The most important protein for
recombination in bacteria is RecA, encoded by the recA gene. You might
remember RecA as having also been involved in the SOS response system
regulon from Topic 8. Genetic crosses involving homologous recombination
between genetically distinct sequences form the basis of classical genetics.

How are genetic crosses involving recombination carried out in bacteria? First,
DNA from a donor strain must enter the recipient cell. This can occur by
transformation, in which naked DNA is taken up by the recipient cell,
transduction, in which a phage injects DNA from the donor cell into the recipient
cell, or conjugation, in which plasmid or chromosomal DNA is transferred from
the donor cell to the recipient cell as a single strand. After the DNA from the
donor strain enters the recipient cell, homologous recombination might occur. In
order to detect a recombination event, it is necessary that the presence of the
genetically distinct but homologous sequences can be differentiated by
phenotype of the recombinant organism. Use of selectable markers allows the
detection of recombination events that occur at even very low frequencies.

Some types of bacterial cells are naturally transformable by DNA (i.e.,

competent). In these naturally competent bacteria, specific competence proteins
aid in the transformation event (Figure 10.22 - 3rd edition or Figure 9.22 - 2nd
edition). Some other types of bacterial cells can be made competent by physical
or chemical treatment. For example, Escherichia coli cells can be made
competent by treatment with calcium ions and this has made possible the
development of E. coli as the preferred host for molecular biology and genetic
engineering. Electroporation, a relatively new method in which electric fields are
used to create small pores in the cell membranes through which DNA molecules
can enter, is also popular as a method of transformation.

Plasmid replication is usually carried out by host cell DNA polymerases.

Nonetheless, plasmids encode genes involved in regulating the rate of plasmid
replication and thus determine plasmid copy number within the cell. If the
replication of two plasmids is controlled by the same genes, then those plasmids
cannot be maintained together in the same cell; they are said to be
incompatible. Plasmids that are incompatible with one another are closely
related and belong to the same Inc group (for “Incompatible”). One of the best-
studied plasmids is the F plasmid (Figure 10.23 - 3rd edition or Figure 9.23 - 2nd
edition).
 Fundamentals of Microbiology (Biology 240)
Course notes
Dr. Josh D. Neufeld
Many plasmids are able to transfer from donor cell to recipient cell in a process
called conjugation (Figure 10.24 - 3rd edition or Figure 9.24 - 2nd edition). This
transfer process is mediated by the products of tra genes, some of which interact
with the oriT region of the plasmid to initiate the transfer of a single strand of
DNA, whereas others form structures such as pili, which aid in the transfer of the
DNA to the recipient cell.

As well as being able to conjugate themselves between bacterial cells,

conjugative plasmids can sometimes integrate into the bacterial chromosome
and transfer part of the chromosome between bacterial cells. Donor cells are
called "males", and recipient cells are called "females". Only the donor cells have
the sex pilus, which is a structure that is encoded by some of the plasmid-
encoded tra genes and helps to bring the donor and recipient cells together
(Figure 10.24 - 3rd edition or Figure 9.24 - 2nd edition). Once the cells have been
brought together, one strand of the plasmid is nicked at the oriT by a tra-encoded
endonuclease, the resulting single strand is unwound from the helix, and is
transferred into the recipient cell. The strand that is transferred is replaced in the
donor cell by replication, and the complementary strand is also synthesized in the
recipient cell. Under appropriate conditions, plasmids can spread rapidly within
and between bacterial populations by conjugation.

How is it that plasmids can integrate into the chromosome and mediate
conjugation of part of the chromosome? Some plasmids, such as F, have
insertion sequences (IS), which are specific DNA sequences that are also
found on the chromosome, and homologous recombination can therefore occur
between the plasmid and the chromosome at these sequences (Figure 10.23 - 3rd
edition or Figure 9.23 - 2nd edition). Strains that have F integrated in the
chromosome are called Hfr (for "high frequency of recombination"). A given Hfr
strain will be able to transfer a particular gene at a characteristic frequency.
Genes that are nearest the site of insertion in the direction of transfer are
transferred at the highest frequency (Figure 10.25 and 10.26 - 3rd edition or
Figure 9.25 and 9.26 - 2nd edition). This is because the genes nearest the oriT of
the integrated F will be most likely to be transferred into the recipient cell before
the DNA strand is broken. The sequential nature of gene transfer can be
demonstrated using interrupted mating experiments (Figure 10.26 - 3rd edition
or Figure 9.26 - 2nd edition) and this property can be used to map genes.

To detect a chromosome conjugation event between donor and recipient cells, it

is necessary to use appropriate markers and selection. Usually, this entails the
use of a combination of antibiotic resistance and auxotrophic markers (Figure
10.26 - 3rd edition or Figure 9.26 - 2nd edition). The conditions for selection must
be such that the desired transconjugant bacteria are able to grow, but the donor
and recipient are not able to grow.

Sometimes, an integrated F plasmid is able to excise from the chromosome, and

sometimes when it does this, it brings along part of the chromosome as well. This
 Fundamentals of Microbiology (Biology 240)
Course notes
Dr. Josh D. Neufeld
results in a plasmid that carries chromosomal genes. Such a plasmid is called a
F' plasmid.

Some genes can move from one part of the genome to another at low
frequencies. This process is called transposition and is carried out by
transposable elements. Insertions sequences, such as those found on F
(Figure 10.23 - 3rd edition or Figure 9.23 - 2nd edition), are transposable elements,
as are transposons. These elements contain genes that encode transposase
enzymes, which recognize terminal inverted repeat sequences (Figure 10.31 - 3rd
edition or Figure 9.31 - 2nd edition). The transposition process is illustrated in
Figure 10.30 - 3rd edition or Figure 9.30 - 2nd edition. You should note that
although transposition is a recombination process - it does not involve
homologous recombination, but rather site-specific recombination.

Transposition is actually a very useful tool for mutagenesis. If a transposon

inserts within a gene, it will disrupt the function of that gene (Figure 10.32 - 3rd
edition or Figure 9.32 - 2nd edition). It is possible to set up a transposition
experiment where a transposon that confers antibiotic resistance is introduced
into a recipient cell, and all selected antibiotic resistant colonies contain a
transposon insertion at a unique location. The insertion containing colonies can
then be screened for the desired phenotype.

Bacteriophage are sometimes able to transfer genes between bacterial strains by

transduction. Generalized transduction involves the transfer of DNA from any
region of the chromosome. When lytic bacteriophage DNA is packaged into
phage particles, some of the host cell's DNA can be accidentally packaged into a
small proportion of the phage particles. These rare particles thus contain only
host cell DNA and no phage DNA. They are called transducing particles,
because they can inject the packaged DNA from the host cell into an appropriate
recipient cell, where it is free to recombine into the recipient cell's genome.
Figure 10.33 - 3rd edition or Figure 9.33 - 2nd edition shows the formation of
transducing particles arising from generalized transduction.

There is a different mechanism of forming transducing particles associated with

lysogenic phage, called specialized transduction. Specialized transduction is
associated with lysogenic bacteriophage infections. The bacteriophage genome
integrates into the host genome at a specific location. Yet, when it excises again,
it occasionally does so imperfectly, taking with it a little bit of the bacterial
genome. When this happens, very specific genes close to the integration site are
taken with it into the bacteriophage particle, along with lots of virus DNA.

Thus, specialized transduction involves packaging with site-specific genes

contained within transducing particles. When specialized transduction happens
with a lysogenic virus, all particles are transducing particles. Transducing
particles have virus genes in addition to host genome.