Bacterial Genetics
Stanley Maloy, San Diego State University, San Diego, CA, USA
r 2013 Elsevier Inc. All rights reserved.
This article is a revision of the previous edition article by Michael
Travisano, volume 1, pp 339–350, r 2001, Elsevier Inc.
Glossary
Alleles Alternative forms of a gene. For example, the
mutants putA601 and putA736 each have a different
mutation in the putA gene.
Ames test A genetic test for the identification of
carcinogens based on their mutagenic activity initially
developed by Bruce Ames. The test relies on the ability of a
chemical or physical agent to promote the reversion of
different classes of mutations that cause histidine
auxotrophy.
Auxotroph A mutant that will only grow when a
particular nutritional requirement (e.g., amino acid,
nucleotide, or vitamin) is provided.
Genotype A specific description of the genetic
constitution of an organism. The genotype is defined by the
allelic form of each gene in an organism, but for simplicity
usually only differences from the wild type are described.
Mutations and Mutagenesis
A powerful feature of bacterial genetics is the ability to
examine very large populations of cells (typically 41010) for
extremely rare types of mutants. Mutations can arise in a
variety of ways, including rare errors in DNA synthesis or DNA
repair. The probability that a spontaneous mutation will affect
a particular base pair varies from about 107 to 1011 per
generation. Thus, in a population of bacteria, about 1 in 109
cells may have a mutation in a particular gene. The frequency
of mutations can be increased by certain chemical or physical
agents called mutagens. Mutagens may act by increasing the
frequency of errors during DNA synthesis or repair or by dir-
ectly altering the DNA. Direct exposure of bacteria to a
mutagen may increase the frequency of mutations in a
population of cells from 103- to 106-fold. Thorough genetic
analysis requires many types of mutations, but each particular
method of mutagenesis yields different subsets of mutations.
Effects of Mutations
Bacterial mutants are typically described by comparison to a
standard, well-characterized, reference strain called the ‘wild-
type’ strain. Bacterial mutants have often lost some growth
property (e.g., failure to utilize a particular carbon or nitrogen
source or failure to grow without a particular nutrient), or
acquisition of some new growth property (e.g., ability to grow
in the presence of some toxic substance). Genes can be divided
Encyclopedia of Biodiversity, Volume 1 http://dx.doi.org/10.1016/B978-0-12-384719-5.00431-7
Mutagen A chemical or physical agent that increases the
frequency of mutation, usually by directly damaging
the DNA.
Mutation Any heritable alteration in the base sequence of
the genetic material.
Phenotype The appearance or other observable
characteristics of an organism. The phenotype expressed by
an organism depends on the particular forms of its genes
(e.g., its wild-type or mutant alleles) and the environmental
conditions.
Suppression The restoration (or partial restoration) of a
wild-type phenotype by a second mutation. There are many
different potential mechanisms of suppression.
Transposon A genetic element which, in addition to
encoding the proteins required for its own transposition,
confers one or more new observable phenotypes (often
resistance to one or more specific drugs) on the host cell.
into two categories based on the phenotypes of the corres-
ponding mutants: nonessential gene products are only re-
quired under specific growth conditions, whereas essential
gene products are required under all conditions. The genes of
lactose catabolism are nonessential because they are only re-
quired for growth on medium with lactose as the sole carbon
source. In contrast, the genes encoding RNA polymerase are
essential because they are required for growth on all media.
Null mutations in a nonessential gene will prevent growth on
a medium that requires that gene product but such mutants
will still grow on other media. In contrast, null mutations in
an essential gene are lethal. Consequently, such mutants
cannot be recovered from haploid bacteria. Nevertheless, it is
possible to isolate more subtle mutations in essential genes.
For example, it is possible to isolate mutations that alter a
subunit of RNA polymerase that make the organism resistant
to the antibiotic rifampicin. It is also possible to isolate mu-
tations where some phenotype is observed under certain
‘nonpermissive’ conditions but not under other ‘permissive’
conditions (Table 1).
Because not all mutations have an observable effect, it is
important to distinguish the genotype from the resulting
phenotype. In bacterial genetic nomenclature a three-letter
mnemonic refers to a pathway or discrete cluster of physio-
logically connected systems. A fourth, capitalized letter repre-
sents a particular gene of that set. The genotype is written in
lower case letters and italicized (e.g., purB), with a plus
superscript indicating the wild-type genotype. (The purB gene
encodes one of the enzymes required for purine biosynthesis.)
317
318 Bacterial Genetics
The phenotype is indicated by the same mnemonic but the
first letter is upper case and it not italicized (e.g., PurB), with a
plus superscript indicating the functional phenotype and a
minus indicating a mutant phenotype. The genotype of a cell
is usually inferred from its phenotype but may also be deter-
mined indirectly by recombination experiments or directly by
DNA sequencing
Isolation and Characterization of Bacterial Mutants
Genetic analysis begins with the isolation of mutants that af-
fect some property of the bacteria. Since mutations are very
rare, mutants must be isolated from large populations of wild-
type cells. Thus, some tactic is needed to find the rare mutants
within a vast excess of parental bacteria. It is possible to
identify mutations by physical methods such as DNA se-
quencing, but because mutations are so rare this approach is
not usually practical. Instead, mutants are usually identified
on the basis of an observable effect on the physiology of the
cell. Detection of mutants requires a genetic selection or
screen (Table 2).
A selection is an experimental arrangement that allows
specific mutants, but not the parental cells, to grow. Genetic
selections are very powerful because they allow the direct
isolation of rare mutations from a very large population of
cells. Some useful selections include resistance to antibiotics,
resistance to phage, or the ability to grow on a medium where
the parental cells cannot grow. For example, selection for
streptomycin-resistant (StrR) mutants simply requires ex-
posing a large number of bacteria to medium containing
Table 1 Some types of conditional mutations used in bacterial
genetics
Conditional Permissive conditions Nonpermissive
mutation conditions
Temperature 30 1C 42 1C
sensitive (Ts)
Cold sensitive (Cs) 42 1C 30 1C
Osmoremedial High osmotic strength Low osmotic strength
Suppressor Host with suppressor Host lacking suppressor
sensitive mutation mutation
Table 2 Isolation of bacterial mutants
Approach Features
Selection Condition where only mutant cells can grow
Screen Condition where both mutants and parental cells can 102–103
grow, but the mutants have a phenotype that is
distinguishable from the parent
Enrichment Condition where survival of the mutant is favored
over the parental cells; usually employs an
antibiotic that selectively kills growing cells or a
condition that kills cells able to incorporate a
particular substrate; a genetic screen is needed to
identify the resulting mutants
streptomycin-sensitive bacteria are killed by the antibiotic and
resistant mutants grow.
The ability to grow bacteria on solid media (i.e., agar
plates) under conditions where each cell forms a single col-
ony allows one to carry out screens that distinguish a par-
ticular mutant from other bacteria in the population. If the
mutation is relatively common and no direct selection for the
mutant phenotype is available, it is possible to screen for
mutants on media where both the mutant and parental cells
grow, but where the mutant has a readily scorable phenotype.
For example, mutants of Escherichia coli unable to degrade
lactose can be identified on indicator media, such as Mac-
Conkey-lactose color indicator plates. MacConkey-lactose
plates contain a carbon source that can be used by Lac þ cells,
and peptides that can be used as a carbon source by both
Lac þ and Lac cells. Both the Lac mutant and the Lac þ
parental cells can therefore grow on MacConkey-lactose me-
dium. However, MacConkey-lactose medium also contains a
pH indicator that is colorless at high pH but red when the pH
decreases owing to fermentation of the lactose, so Lac þ
bacteria form red colonies and Lac bacteria form white
colonies. Hence, it is possible visually to screen for Lac
mutants by looking for rare white colonies on MacConkey-
lactose plates.
Screens for other types of mutants are not this simple. For
example, when mutations disrupt a biosynthetic pathway, the
bacteria cannot grow unless the endproduct of the pathway is
provided in the medium (permissive medium). It is not pos-
sible to isolate such ‘auxotrophic mutants’ by directly screen-
ing for growth on media lacking the endproduct because the
desired mutants will not grow (nonpermissive medium).
However, auxotrophs are readily identified by replica plating.
The bacteria are grown on permissive medium at a density of
several hundred colonies per plate. Each of these ‘master
plates’ is then replicated onto two other plates, containing
either the permissive medium or the nonpermissive medium.
The bacteria are transferred to an identical position on each of
the replica plates. Once colonies are identified that grow only
on the permissive medium, the mutants can be isolated from
the master plate (Figure 1).
The difference between a selection and a screen has im-
portant practical consequences. Compare the selection for a
StrR mutant with a screen for a His mutant. Isolation of a StrR
colony is a direct selection because as many as 1010 cells can be
Sensitivity Examples
9
410 Resistance to antibiotics or toxic substrate analogs;
resistance to phage
Indicator medium to identify mutants unable to
ferment a carbon source; replica plating to identify
auxotrophs
102 per cycle Penicillin enrichment; D-cycloserine enrichment;
radioisotope suicide

Place sterile Hold outside of
velvet on block ring and press
‘Fuzzy-side-up’ down over velvet
Press surface of
Petrie dish with
bacterial colonies
Figure 1 Replica plating. This simple technique makes it possible to test many individual mutants for their ability to grow on a variety of
different types of media. After pressing the sterile plates to the velvet replica of an original (‘master’) plate, the newly inoculated plates are
incubated to allow the colonies to grow.
spread on a single plate containing streptomycin and only StrR
mutants will form colonies. Thus, StrR mutants as rare as 1 in
1010 can be isolated easily. In contrast, finding an auxotrophic
mutant involves screening through a large number of colonies
for one that fails to grow in the absence of the required nu-
trient. To score the growth behavior of individual colonies,
only a few hundred colonies can be examined on any given
plate. Thus, if 1 in 105 cells in the population were a particular
auxotrophic mutant, thousands of plates would be needed to
find a single mutant. Treating populations of cells with a
mutagen may increase the fraction of mutants to 1 in 103,
allowing screening for the desired mutant on a reasonable
number of replica plates. Nevertheless, random mutagenesis
may not increase the mutant fraction sufficiently to screen
easily for the desired mutants, and mutagenesis may lead to
other, undesirable mutations as well.
Isolating rare mutants is generally achieved by using some
sort of enrichment, a method that favors the growth of the
desired mutants relative to nonmutant bacteria. Penicillin
enrichment is a classical example of this approach. This anti-
biotic disrupts the synthesis of the bacterial cell wall. Non-
growing bacteria do not engage in cell wall synthesis, so
penicillin only kills actively growing bacteria. The differential
survival of growing versus nongrowing bacteria can be used to
enrich for a desired mutant. For example, penicillin enrich-
ment can be used to isolate a rare auxotrophic mutant from a
population of bacteria. If a mixture of wild-type and auxo-
trophic bacteria are suspended in the nonpermissive medium
containing penicillin, the auxotrophic mutants do not grow
and thus will survive, whereas 99% of the wild-type bacteria
will grow and be killed by the penicillin. When the surviving
bacteria are washed free of the penicillin, and resuspended in
permissive medium, both the mutant and wild-type bacteria
will grow, but the ratio of mutant to wild-type bacteria will be
enriched 100-fold. Repeating this procedure multiple times
eventually increases the proportion of mutants in the popu-
lation (Figure 2).
Bacterial Genetics 319
Pressing sterile Petrie
dishes to surface of
Cells from colonies
velvet produces
attach to velvet in
replica of colonies
same pattern
on each Petrie dish
Medium #1
onto velvet
Medium #2
Medium #3
Genetic Exchange
Exchange of DNA between bacteria plays an important role in
evolution. Gene transfer in nature can result in the acquisition
of antibiotic resistance and new virulence traits. Gene transfer
is also a useful tool in the laboratory, allowing genetic map-
ping and complementation tests, and the construction of
bacterial strains with multiple mutations. The three most
common methods of gene transfer between bacteria are
transformation, conjugation, and transduction (Table 3). A
suitable selectable marker is required to identify recipients
that have inherited the desired region of donor DNA.
Transformation
The uptake of naked DNA is called transformation. Some
species of bacteria are naturally transformable. At some stage
during growth they express gene products that facilitate the
uptake of exogenous DNA, a condition called ‘competence.’
The physiological conditions required to induce competence
differs for different species of bacteria. However, most natural
transformation seems to involve the degradation of one strand
of the exogenous DNA during the transfer of the other strand
of DNA into the cell. Stable inheritance of the donor DNA
requires either that it be replicated (e.g., some plasmids and
phage) or that it integrate into the recipient chromosome via
homologous recombination.
Many types of bacteria are not naturally transformable but
can be induced to take up DNA by treatment with specific
chemicals or by electric shock, processes that are mech-
anistically different from natural transformation. A common
method of chemical transformation in E. coli uses hypotonic
Ca2 þ shock. To prepare competent bacteria by this method,
an early-exponential phase culture of cells is suspended in a
cold hypotonic CaCl2 solution. When DNA is added to these
bacteria it forms a calcium–DNA complex that adsorbs to the
320 Bacterial Genetics

cell surface. The bacteria are then briefly warmed (heat
shocked), which allows the DNA to enter the cell. In electro-
poration, cells are exposed to an electric field, so that a voltage
potential develops across the membrane, transiently forming
small pores that allow entry of exogenous DNA. In contrast to
natural transformation and chemical transformation, which
Population of 108 cells
Grow in minimal medium + supplement
Outgrowth
Both aux+ and aux− cells grow
Ratio of mutant to wild-type unchanged
Grow in minimal medium − supplement + Penicillin
Enrichment
aux+ cells grow and 99% die
aux− cells cannot grow so they survive
Ratio of mutant to increases 100 ×
Population of cells with auxotrophs enriched by 102
Grow in minimal medium + supplement
Outgrowth
Both aux+ and aux− cells grow
Ratio of mutant to wild-type unchanged
Grow in minimal medium − supplement + Penicillin
Enrichment aux+ cells grow and 99% die
aux− cells cannot grow so they survive
Ratio of mutant to increases 100 ×
only seem to work in certain bacteria, a wide range of bacteria
can be transformed by electroporation.
Conjugation
DNA can also be transferred between bacteria by a process
called conjugation. Conjugation occurs via cell–cell contact
and the formation of a mating channel. This structure is
formed by specific proteins that form a pore between the
juxtaposed membranes through which single-stranded DNA
and some associated proteins are transferred into the recipient
cell. Conjugation requires four events: (1) contact between
donor and recipient cells and formation of a mating ap-
pendage; (2) nicking of the donor DNA at specific sites; (3)
translocation of the nicked strand of donor DNA to the mat-
ing bridge and into the recipient cell; and (4) replication of
the transferred DNA in the recipient cell. The proteins required
for conjugation are usually encoded on specific plasmids or
transposons. These conjugal plasmids or transposons can
transfer themselves or, if integrated into another region of the
genome, any adjacent genomic sequence. Thus, conjugation
can result in the transfer of very large DNA fragments – even
chromosome length DNA fragments. Furthermore, some
conjugal plasmids are quite promiscuous. In addition to
transferring DNA into a wide variety of bacteria, some con-
jugal plasmids can transfer DNA into Archaea and eukaryotes
as well (Figure 3).

Population of cells with auxotrophs enriched by 104

Dilute and plate on minimal medium + supplement
Outgrowth to obtain about 200 colonies per plate
Both aux+ and aux− cells grow
Ratio of mutant to wild-type unchanged
Replica plate onto:
Screen
(i) Minimal medium without supplement
(ii) Minimal medium with supplement
Compare the replica plates: the desired auxotrophs will not
grow on minimal medium without supplement but grow on
minimal medium with supplement
Figure 2 An example of the use of penicillin enrichment to isolate
auxotrophic (aux  ) mutants from a population of prototrophic
(aux þ ) bacterial cells. Each cycle results in about 100  enrichment
of the desired mutants.
Transduction
Transduction is the transfer of bacterial DNA from one cell to
another by a phage particle. Phage particles that contain bac-
terial DNA are called transducing particles. There are two types
of transduction: generalized and specialized.
Generalized transduction requires an error during the
packaging phase of phage maturation, such that random,
phage-size fragments of chromosomal DNA get inserted into
the phage head in place of phage DNA. Thus, in a lysate of a
generalized transducing phagea some particles contain DNA
obtained from the host bacterium rather than phage DNA.
The bacterial DNA fragment can be derived from any part of
the bacterial chromosome. When these phage particles adsorb
to a recipient, the double-stranded chromosomal DNA is in-
jected. Stable inheritance of the donor DNA requires either

Table 3 Common mechanisms of natural gene transfer in bacteria

Transfer Vehicle Properties

mechanism

Transformation – Uptake of naked DNA from environment; recipient cell must be competent
Conjugation Conjugal plasmid or Requires cell-to-cell contact; very long fragments of DNA can be transferred to recipient cell
transposon
Generalized Phage Phage-length fragments of chromosomal DNA transferred to recipient cell; phage head only carries
transduction host DNA
Specialized Lysogenic phage Short fragments of chromosomal DNA transferred to recipient cell; phage head carries host DNA
transduction covalently attached to phage DNA
 Bacterial Genetics 321

Donor Recipient that it be replicated (e.g., following transfer of plasmids) or

that it integrate into the recipient chromosome via homolo-
gous recombination. Generalized transducing particles arise
during lytic growth of both virulent and lysogenic phages
(Figure 4).
F-Plasmid Specialized transduction results from the aberrant excision
of an integrated lysogenic phage. In contrast to generalized
transduction, the phage head packages contain a contiguous
molecule having both host and phage DNA. Only regions of
the host DNA that flank the prophage are packaged.
By using genetic tricks to force lysogenic phages to integrate
Chromosome at many sites in the genome, it is possible to isolate specialized
transducing phage from many different regions of a bacterial
genome. Stable inheritance of specialized transducing frag-
ments can occur either by phage-specific processes or by homo-
logous recombination mediated by bacterial recombinases.

Genetic Analysis of Bacterial Mutants

Three of the most important tools for the analysis of bacterial

mutations are suppressor analysis, genetic recombination, and
complementation. Suppressor analysis facilitates the dis-
section of the roles of gene products and how they interact
with other gene products. Recombination allows the con-
struction of new combinations of genes and the elucidation of
the positions of genes on a chromosome relative to other
genes. Complementation can reveal how many genes are re-
sponsible for a particular phenotype and can distinguish
regulatory genes from regulatory sites.

Reversion and Suppressor Analysis

Mutant organisms can regain their wild-type characteristics by

means of a mutation that restores function, by a process called
reversion. Reversion can either be owing to ‘true reversion,’ a
back mutation that restores the original genotype, or ‘sup-
pression,’ a second mutation that produced a change that
partially or fully compensated for the first mutation. The
ability to analyze very large populations of bacteria facilitates
the isolation of rare classes of such revertants.
The reversion frequency is a useful criterion for classifying
mutants. The reversion frequency can distinguish a single point
mutant from a double mutant or a deletion mutant: single
Donor Donor point mutants (which can be repaired by a single back mu-
tation) revert at a much higher frequency than double mutants
(which require two changes and thus revert very rarely) or
deletion mutants (which cannot directly revert). The effect of

F-Plasmid F-Plasmid Figure 3 Conjugation is a process of DNA transfer via direct contact
between a donor cell that carries a conjugal plasmid such as the
F-plasmid, and a recipient cell that lacks the conjugal plasmid. A
pilus protruding from the surface of the donor cell recognizes the
recipient cell and promotes close contact between the two cells,
forming a bridge through which DNA is transferred. On cell–cell
contact the conjugal plasmid is nicked at a specific site called oriT
(indicated by the arrowhead), and the DNA is transferred from this
site into the recipient cell. After conjugation is complete, both cells
have a complete copy of the conjugal plasmid.
322 Bacterial Genetics
arg+
Grow phage on donor cell
arg+
Phage morphogenesis and
packaging of DNA
Lysis and release of phage
arg+
arg−
Infect recipient cell
arg+
Figure 4 Transduction is the process of packaging of a portion of
the genome from a donor cell into a virus particle (e.g., a
bacteriophage) and the subsequent delivery of the genetic material to
a recipient cell. Once in the recipient cell the fragment of donor DNA
can recombine with the genome of the recipient yielding a new
phenotype (in the case shown, via conversion of the recipient from
an arginine auxotroph into an arginine prototroph). The altered
recipient cell is called a transductant.
mutagens on the reversion frequency can also be used to dis-
tinguish different types of mutations – reversion of frameshift
mutations is stimulated by frameshift mutagens, and so on. For
most purposes this approach has been superceded by direct
DNA sequence analysis; however, the reversion of known bac-
terial mutations remains an important assay to detect potential
human carcinogens (for example, using the Ames test).
Second site mutations that result in suppression can occur
within the same gene as the original mutation (intragenic
suppression), or within a different gene (intergenic sup-
pression). Intragenic suppressors can provide information
about the role of particular amino acids in a protein, protein
folding, and interactions between different amino acids within
a protein. Intergenic suppressors can occur in a variety of ways,
and each provides different insights into the structure or
function of the gene products involved. Suppressor mutations
often result from ‘gain-of-function’ mutations that are dom-
inant over the wild-type gene. Thus, characterization of sup-
pressors requires genetic recombination to construct strains
with different combinations of mutant alleles, and com-
plementation analysis to determine dominance.
Homologous Recombination
Genetic recombination is the physical breakage, exchange, and
rejoining of two DNA molecules. Homologous or general re-
combination can be mediated by several different pathways in
bacteria. Each of these pathways requires the RecA protein to
align the DNA molecules between regions of substantial DNA
sequence identity. The DNA molecules are broken between
random but matching nucleotides, and then the DNA frag-
ments are exchanged and rejoined to form two new combin-
ations of genes. For example, recombination between two
DNA molecules with the genotypes a þ b and ab þ can yield
two recombinant DNA molecules with the genotypes a þ b þ
and ab. Owing to the efficiency of gene transfer and the ability
to work with large populations of bacteria, recombination
analysis can be sufficiently sensitive to detect recombination
events between adjacent base pairs.
Recombination can be used not only to construct strains
with different genotypes, but can also reveal the relative map
location of genes. The probability of recombination between
any two adjacent base pairs is very low, but the probability
of recombination between base pairs within a homologous
DNA sequence is essentially random. Hence, the physical
distance between two genes located on the same DNA mol-
ecule determines the frequency of recombination between
the genes: the probability of recombination is less if the genes
are close to each other than if the genes are farther apart
(Figure 5).
Genetic mapping exploits the recombination frequency
between genes to measure the relative distance between genes.
In bacterial genetics, the probability that recombination did
not occur between genes is usually determined. If recombin-
ation does not occur between two genes, the genes will be
coinherited. For two genetic markers on the same DNA mol-
ecule, the closer two genetic markers are to each other, the
more often they will be coinherited. The frequency that two
genes are coinherited is defined as their linkage. Determining
the linkage of two genetic markers is called a two-factor cross.
It is also possible to determine the relative location of genetic
mutations by using three genetic markers (three-factor crosses)
or by genetic crosses involving a set of defined deletion mu-
tations (deletion mapping). Although it is possible to deter-
mine the relative location of genes by hybridization or DNA
sequencing, genetic mapping often provides a simple and
 Bacterial Genetics 323

Given the following pathway for the synthesis of Z from A.

Gene a Gene b Gene c

Enzyme a Enzyme b Enzyme c

W X Y Z
(a)

A mutant in gene b that does not make enzyme b cannot

make Y, therefore will not be able to make the product Z.
Gene a Gene b − Gene c

Enzyme a Enzyme c
W X
(b)

If the cell has two mutant copies of any of these genes, it

will be unable to make all the necessary intermediates
and, thus, will be unable to make the product Z.

Gene a Gene b − Gene c

Enzyme a Enzyme c
No complementation
W X (because mutations
affect same function)
Enzyme a Enzyme c

Gene a Gene b − Gene c

(c)

If the cell has at least one good copy of each gene, it will
be able to make all the necessary intermediates and, thus,
will be able to make the product Z.

Gene a Gene b− Gene c

Enzyme a Enzyme c
Complementation
W X Y Z (because mutations
affect different function)

Enzyme b Enzyme c

Gene a− Gene b Gene c

(d)

Figure 5 Complementation analysis provides a genetic test for particular functions encoded by different genes. (a) Assume that in a cell
substrate W is converted in three enzymatic steps to the endproduct Z, and that each of these enzymatic steps is encoded by a different genes
(a, b, and c). (b) If any one of these genes is defective in a haploid bacterial cell, then that cell will be unable to make the endproduct. (c) If two
copies of these genes are placed in a single bacterial cell, and if both copies are defective for the same gene, then the cell will be unable to
make the endproduct. In this case, we say that the second copy did not complement the original mutation. (d) If the two copies of the genes are
defective for different genes, then at least one functional gene is present for each step in the pathway, and the endproduct will be made. If the
second copy compensates for the defective function, then we say that the mutation was complemented.

inexpensive way to determine rapidly the location of mu-
tations in bacteria.
Recombination also provides an invaluable tool for con-
structing strains with multiple mutations. If a mutation involves
a directly selectable phenotype, it can be transferred with ease
from a donor to an appropriate recipient strain. If the mutation
cannot be directly selected, linkage to an adjacent, selectable
marker can be used to move the mutation into a recipient
strain. For example, an auxotrophic mutation may be trans-
ferred by selecting for coinheritance of a nearby gene.
324 Bacterial Genetics
To determine the genetic or biochemical effects of a mu-
tation, it is necessary to compare a mutant with a strain that only
differs by a single mutation. If several mutations are present, it is
not obvious which of the mutations is responsible for an ob-
served phenotypic change. Two organisms that differ by only a
single mutation are said to be isogenic or to have the same
genetic background. The most common way to ensure that two
strains are isogenic is to transfer a small region of DNA carrying
the mutation into the parental strain by recombination.
Complementation Analysis
A particular phenotype frequently reflects the activity of many
genes. To understand any genetic system it is essential to
know the number of genes and regulatory elements involved.
Since multiple genes that affect the same function may map
very close to each other, it is not possible to determine if
two mutations are in the same or different genes simply from
the recombination frequency. For a variety of reasons, it is
also often difficult to prove that two mutations affect the same
gene product by DNA sequence analysis. However, this ques-
tion can be addressed by genetic complementation.
Bacteria are normally haploid, but complementation re-
quires the maintenance of two copies of a particular gene in
the same cell. In bacteria this can be done by constructing a
partial diploid or ‘merodiploid’ that carries two copies of the
relevant genes. Partial diploids may provide one copy of the
gene on the chromosome and a second copy of the gene on a
plasmid. For example, the partial diploid lacZ/lacZ þ has both
a copy of the lacZ gene and second copy with the lacZ þ gene.
If the functional copy of these genes encodes a protein that
can diffuse through the cell to perform its function, and the
second copy has a loss-of-function mutation, the functional
copy of the gene will be dominant over the mutant copy. Thus,
even though the lacZ gene on the chromosome cannot pro-
duce beta-galactosidase, the plasmid-bone lacZ þ gene makes
beta-galactosidase so the cell is phenotypically Lac þ . The
mutant is complemented by the wild-type gene, indicating
that the mutation is recessive. In contrast, the partial diploid
lacZ/lacZ cannot make beta-galactosidase so the cell is
phenotypically Lac. If two recessive mutants fail to comple-
ment, they affect the same gene (Figure 6).
Complementation analysis in bacteria usually follows the
grouping of mutations by genetic mapping experiments. This
reduces the number of partial diploids that must be con-
structed because genes that map far from each other are clearly
different. In addition, complementation analysis should in-
volve partial diploids with an equal number of copies of each
gene. If one of the genes is present in excess (for example, with
genes cloned on multicopy plasmids) artifacts can occur
which may be very misleading.
Portable Regions of Homology
Although most genes have a specific, defined location in a
bacterial genome, the genome is by no means static. Re-
combination between repeats of homologous DNA sequences
can result in the duplication, deletion, or inversion of the
...TACGCCATTAGCTA...
...ATGCGGTAATCGAT...
...TACGCCATTAGCTA...
...ATGCGGTAATCGAT...
...TACGCCATTAGCTA...
...ATGCGGTAATCGAT...
...TACGCCATTAGCTA...
...ATGCGGTAATCGAT...
Figure 6 Recombination is the breaking and joining of two
homologous DNA molecules. The exchange occurs between two
homologous base pairs, generating a join point between the two
parental DNA molecules. Because the recombination can occur
between any of the base pairs in homologous sequences, the further
apart two genes are, the more likely there will be a recombination
event between them.
intervening DNA. It is possible to select for insertion of such
homologous sequences at strategic positions in the genome.
Such ‘portable regions of homology’ can be generated by
transposable elements, which are mobile segments of DNA that
move to new locations at low frequency. Alternatively, having
specific DNA fragments on a circular DNA molecule (e.g., a
plasmid or phage) can permit the targeted insertion of DNA
into a particular site on the chromosome. These two types of
insertions have a wide variety of genetic uses. A few examples
include: construction of insertion mutants with complete loss
of function; construction of deletion mutations with defined
endpoints; construction of duplication mutations for com-
plementation studies; integration of other genetic elements at
particular sites in the genome by homologous recombination;
transfer of mutations by selection for an associated genetic
marker (e.g., antibiotic resistance); isolation of linked genetic
markers with a selectable phenotype; and construction of
operon or gene fusions.
Transposable Elements
Transposable elements in bacteria include insertion sequences
and transposons. Insertion sequences are short elements
(typically less than 5 kb) that only encode functions required
for their own transposition. Transposons are typically longer
(45 kb) and encode other gene products (e.g., antibiotic re-
sistance) in addition to the functions required for transposi-
tion. The frequency of transposition of these elements is
typically low, although the frequency varies over a wide range
(107–102 per generation). Transposition requires both cut-
ting the DNA at each end of the transposon and the target
DNA site, joining the ends of the transposon and target DNA
molecules, and DNA replication. Transposition can occur by a
replicative mechanism (requiring replication of the entire
transposon) or nonreplicative mechanism (requiring only
replication of short fragments at the end of the insertion site).
Both mechanisms are independent of the homologous
recombination machinery of the host.

Transposable elements play an important role in bacterial
evolution, including the transfer of antibiotic resistance genes
between bacteria and promoting chromosome rearrange-
ments. In addition, transposable elements are useful tools in
bacterial genetics because they provide selectable markers and
portable regions of homology that can be used to facilitate
genetic recombination.
Integration of Circular DNA Molecules
In addition to transposons, it is possible to construct small
repeats of homologous DNA by integration of a circular DNA
molecule with a cloned fragment of chromosomal DNA. This
approach is useful for construction of defined duplications for
complementation analysis and for construction of insertion
mutations on the chromosome. Recombination between the
homologous sequences of the resulting duplication can result
in allele exchange, moving a mutation from a cloned sequence
onto the chromosome DNA.
Summary
A hallmark of bacterial genetics is the ability to analyze very
large populations of cells to identify rare genetic events.
Bacterial Genetics 325
Although a wide variety of genetic tricks have been developed
for specific purposes in particular bacteria, bacterial genetics
relies on a relatively small core of tools for dissection of the
structure and function of genes. The essential tools include the
isolation of mutations, the ability to transfer genes between
bacterial strains, the ability to isolate recombinants, and the
ability to do complementation tests. These tools have been
finely honed for a select group of model bacteria, including
E. coli, Salmonella enterica, and Bacillus subtilis. The concepts
developed for these model bacteria are readily applicable to
other bacteria as well, although the experimental details
typically require adaptation for each particular species of
bacteria.
References
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Maloy S, Hughes K, and Casadesus J (2011) The Lure of Bacterial Genetics.
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Maloy S, Stewart V, and Taylor R (1996) Genetic Analysis of Pathogenic Bacteria.
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Miller JH (1992) A Short Course in Bacterial Genetics. Plainview, New York: Cold
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