Articulo Microbiologia

Bacterial Genetics
28
Chapter
1. Reproduction Versus Gene

Transfer ................................... 896
2. Fate of the Incoming DNA

After Uptake........................... 897
3. Transformation Is Gene
Transfer by Naked DNA ......898
I n many ways, bacterial genetics underlies molecular
biology. The discovery of gene transfer in bacteria,
and, in particular, the involvement of plasmids in this,
4. Gene Transfer by Virus—
Transduction........................... 904 provided the foundations for molecular cloning. The
5. Transfer of Plasmids Between genetics of bacteria is very different from that of higher
Bacteria................................... 908 organisms. Firstly, bacteria are generally haploid, with
6. Gene Transfer Among Gram- one copy of each gene on a single circular chromosome
Positive Bacteria...................915
(unlike eukaryotes, which are diploid with multiple linear
7. Archaeal Genetics ................917
chromosomes). Secondly, gene transfer in bacteria is nor-
8. Whole-Genome
Sequencing............................. 918 mally unidirectional; that is, a donor cell transfers genes
Review Questions .......................923 to a recipient cell rather than two cells sharing genetic
Further Reading........................... 924 information to generate progeny as seen in the more
familiar forms of reproduction in higher organisms. Gene
transfer in bacteria occurs by three major mechanisms,
which form the main topics of this chapter.
1. Reproduction Versus Gene Transfer

Sex and reproduction are not at all the same thing in all organisms.
In animals, reproduction normally involves sex, but in bacteria, and
in many lower eukaryotes, these are two distinct processes. Bacteria
divide by binary fission. First, they replicate their single chromosome
and then the cell elongates and divides down the middle. No resort-
binary fission Simple form of cell division in which the cell replicates its DNA,
elongates, and divides down the middle.
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2. Fate of the Incoming DNA After Uptake 897
ing of the genes between two individuals (that is, no sex) is involved and so this is
In bacteria, cell division and the
known as asexual or vegetative reproduction. reshuffling of genetic
From a biological perspective, sexual reproduction serves the purpose of reshuf- information are completely
fling genetic information. This will sometimes produce offspring with combinations of separate processes.
genes superior to those of either parent (and, of course, sometimes worse!). Although
bacteria normally grow and divide asexually, gene transfer may occur between bacte-
rial cells. During sexual reproduction in higher organisms, germ-line cells from two
parents fuse to form a zygote that contains equal amounts of genetic information
from each parent. In contrast, in bacteria gene transfer is normally unidirectional and
cell fusion does not occur. Genes from one bacterial cell are donated to another. We Gene transfer between bacteria
thus have a donor cell that donates DNA and a recipient cell that receives the DNA. may involve uptake of naked
The transfer of genes between bacteria fulfills a similar evolutionary purpose to DNA, transport of DNA via
the mingling of genes during sexual reproduction in higher organisms. However, virus particles, or transfer of
mechanistically it is very different. Consequently, some scientists regard bacterial DNA via a specialized cell-to-
gene transfer as a primitive or aberrant form of sex, whereas others believe that it is cell connection.
quite distinct, and that use of the same terminology is misleading.
Molecular biologists use bacteria together with their plasmids and viruses to
carry most cloned genes, whether they are originally from cabbages or cockroaches.
Consequently, a basic understanding of bacterial gene transfer is needed to under-
stand the genetic engineering of plants and animals. Gene transfer in bacteria occurs
by three basic mechanisms. Genes are only transferred by cell-to-cell contact in con-
jugation. In transduction, genes are transferred via virus particles, and in transforma-
tion, bacterial cells take up free molecules of DNA. Before considering these three
mechanisms in detail, we will discuss what happens to the DNA after uptake, as sim-
ilar considerations apply in all three cases.
2. Fate of the Incoming DNA After Uptake

Incoming fragments of DNA
Irrespective of its mode of entry, DNA that enters a bacterial cell has one of three will be destroyed by cells that
possible fates. It may survive as an independent DNA molecule, it may be receive them unless they form a
completely degraded, or part may survive by integration or recombination with the replicon.
host chromosome before the rest is degraded.
For incoming DNA to survive inside a bacterial cell as a self-replicating DNA
molecule, it must be a replicon. In other words, it must have its own origin of repli-
cation and lack exposed ends. For survival in the vast majority of bacteria, this
means that it must be circular. In those few bacteria, such as Borrelia and
Streptomyces (see Chapter 4: Genes, Genomes, and DNA) with linear replicons, the
ends must be properly protected. In eukaryotes, long-term survival of a linear DNA
molecule requires a replication origin, a centromere sequence, and telomeres to pro-
tect the ends (see Chapter 4: Genes, Genomes, and DNA).
A linear fragment of double-stranded DNA that enters a bacterial cell will nor-
mally be broken down by exonucleases that attack the exposed ends. For any of its
genes to survive, they must be incorporated into the chromosome of the recipient cell
by the process of recombination (see Chapter 27: Recombination). For recombination
Incoming fragments of DNA
to occur, crossovers must form between regions of DNA of similar sequence—that is,
may be preserved from
homologous sequences. The two DNA molecules will swap DNA between two cross- destruction by recombination
over points (Fig. 28.01). Consequently, if genes from incoming DNA are incorporated, onto the host chromosome.
the corresponding original genes of the recipient cell are lost.
Such homologous recombination normally only occurs between closely related
molecules of DNA—for example, DNA from two strains of the same bacterial spe-
cies. Unrelated DNA may be incorporated by recombination provided it is
asexual or vegetative reproduction Form of reproduction in which there is no reshuffling of the genes between two individuals.
conjugation Process in which genes are transferred from one bacterium to another by cell-to-cell contact.
donor cell Cell that donates DNA to another cell.
recipient cell Cell that receives DNA from another cell.
sexual reproduction Form of reproduction that involves reshuffling of the genes between two individuals.
transduction Process in which genes are transferred from one bacterium to another via virus particles.
transformation (As used in bacterial genetics) Process in which genes are transferred into a cell as free molecules of DNA.
898 CHAPTER TWENTY EIGHT Bacterial Genetics
FIGURE 28.01 g homologous DNA

omin
Recombination Allows Inc a b c d
Survival of Transformed
DNA c
b d
In most cases, incoming linear
a
DNA molecules are degraded by
Crossover
the host cell exonucleases. If there Host
are homologous regions between chromosome
incoming DNA and the host
chromosome, crossing over may
replace regions of the host
chromosome with part of the
incoming DNA.
HOMOLOGOUS RECOMBINATION
(CROSSING OVER)
c
b d
a
Some incoming
genes are
incorporated
surrounded by sequences that are related (Fig. 28.02). Another possibility is that the
incoming DNA contains a transposon that can function in the recipient cell. If so,
Incoming circular DNA with its then the transposon may survive by abandoning the incoming DNA molecule and
own origin of replication can jumping into the chromosome of the new host cell.
survive without recombination.
If the incoming DNA is a plasmid that can replicate on its own, recombination
into the chromosome is not necessary for survival. For genetic engineering purposes,
it is usually more convenient to avoid adding genes to the bacterial chromosome via
recombination. Consequently, molecular biologists often put the genes they are
working with onto plasmids (see Chapter 23: Plasmids).
In addition to exonuclease attack, incoming DNA is often susceptible to restriction.
This is a protective mechanism designed to destroy incoming foreign DNA. Most bac-
teria assume that foreign DNA is more likely to come from an enemy, such as a virus,
than from a harmless relative, and they cut it into small fragments with restriction
enzymes. This applies to both linear and circular DNA, since the degradative enzymes
are endonucleases that cut DNA molecules in the middle (see Chapter 5: Manipulation
of Nucleic Acids, for details). Only DNA that has been modified by methylating the
appropriate recognition sequences is accepted as friendly. In genetic engineering,
Restriction enzymes degrade restriction negative host strains are used to surmount this obstacle.
unmethylated foreign DNA,
whether linear or circular.
3. Transformation Is Gene Transfer by Naked DNA
The simplest way to transfer genetic information is for one cell to release DNA into the
medium and for another cell to import it. The transfer of “pure” or “naked” DNA
from the external medium into a bacterium is known as transformation (Fig. 28.03). By
“naked,” we mean no other biological macromolecules, such as protein, are present to
enclose or protect the DNA. No actual cell-to-cell contact happens during transforma-
tion, nor is the DNA packaged inside a virus particle. Bacterial cells can often take up
naked DNA molecules and may incorporate the genetic information they carry.
3. Transformation Is Gene Transfer by Naked DNA 899
DNA unrelated FIGURE 28.02

to chromosome Incorporation of Unrelated
DNA
Incoming DNA does not have to
be entirely related to the host in
order for recombination to occur.
Region of In some instances, the incoming
c
homology
d DNA has regions that are related
b
c (purple) and regions that are
a b d
totally unrelated (green). The
regions of homology may be large
a
Crossover
enough to allow recombination,
Host thus integrating an unrelated piece
chromosome
of DNA into the host chromosome.
Receiving new genetic material
may provide the host cell with a
new trait that is desirable to
changing environments. In
organisms that make identical
clones during reproduction, this
HOMOLOGOUS RECOMBINATION strategy is critical to evolutionary
survival.
Some incoming
genes are
incorporated
c
b
a
DESTROY CELL ADD DNA TO

RECIPIENT CELL RECOMBINATION
AND PURIFY DNA
Chromosome
ORIGINAL FRAGMENTS TRANSFORMED RECOMBINANT

BACTERIAL CELL OF DNA CELL CELL
FIGURE 28.03
Gene Transfer by Transformation
Under the right conditions, bacteria can take up pieces of naked DNA from the external environment. The fragment of DNA may pass through
the outer cell layers without the aid of a protein or virus. Once inside the bacteria, the fragment of DNA must recombine with the chromosome
to prevent degradation by exonucleases or restriction enzymes.
In practice, transformation is mostly a laboratory technique. The DNA is

extracted from one organism by the experimenter and offered to other cells in cul-
ture. Cells able to take up DNA are said to be “competent.” Some species of bacte-
ria readily take up external DNA without any pretreatment. Probably they use this
ability to take up DNA under natural conditions. From time to time, bacteria in nat-
ural habitats die and disintegrate, which releases DNA that nearby cells may import.
Cells that have cell walls Other bacteria rarely undergo natural transformation and must first be treated
usually need some sort of to make them competent. Two different laboratory approaches are used to make
treatment before they can take bacteria competent for transformation. One method is to chill the bacterial cells in
up DNA. the presence of metal ions, especially high concentrations of Ca21, that damage their
cell walls and then to heat shock them briefly. This loosens the structure of the cell
walls and allows DNA to enter. Another method is electroshock treatment. Bacteria
are placed in an “electroporator” and zapped with a high-voltage discharge that
opens the cell wall and allows the DNA to get into the cell. Laboratory transforma-
tion techniques are an essential tool in genetic engineering. After genes or other
useful segments of DNA have been cloned in the test tube, it is almost always neces-
sary to put them into some bacterial cell for analysis or manipulation. Escherichia
coli (E. coli) is normally treated by some variant of the Ca21/cold-shock treatment
and does not require electroshock. Yeast cells may also be transformed. Since yeast
has a very thick cell wall, electroshock is used. Conversely, animal cells, which lack
cell walls, often take up DNA readily, and only require a mild chemical treatment.
3.1. Transformation As Proof That DNA Is the Genetic Material

Transformation was first observed by Oswald Avery in 1944 and provided the earli-
est strong evidence that purified DNA carries genetic information and, therefore,
that genes are made of DNA. Pneumococcus pneumoniae (now renamed
Streptococcus pneumoniae) has two variants; one forms smooth colonies when grown
on nutrient agar, the other has a rough appearance. The smooth variant has a cap-
sule that surrounds the bacterial cell wall, whereas the bacteria in the rough colonies
lack the capsule. The ability to make a capsule affects both colony shape and viru-
lence as the capsule protects bacteria from the animal immune system. Thus, if
smooth isolates of S. pneumoniae are injected into a live mouse, it dies of bacterial
pneumonia. In contrast, rough strains are nonvirulent.
Avery exploited this difference to prove that DNA from one strain could “trans-
form” or change the other strain. Avery used DNA extracted from virulent strains
of S. pneumonia. He purified the DNA and added it to harmless strains of the same
bacterial species. Some of the harmless bacteria took up the DNA and were trans-
formed into virulent strains. Hence, Avery named this process transformation
(Fig. 28.04). (Strictly speaking, Avery’s transforming DNA could have interacted in
some unknown way with the host chromosome to promote a genetic change. His
experiment was therefore not absolute proof that DNA is the genetic material.
The transfer of inherited Nonetheless, this is the most obvious interpretation and this observation convinced
characteristics due to the many scientists that genes were very likely made of DNA.)
uptake of pure DNA was part The use of viruses to transfer DNA into a bacterium provided more evidence
of the original proof that DNA that DNA was the genetic material that passed from one generation to the next.
was the genetic information. Special terminology is used when scientists use naked viral DNA during transforma-
tion. In a viral infection, the virus punctures a hole in the bacterial cell wall and
injects DNA from the viral particle into the cytoplasm. The viral DNA induces the
host to manufacture new viral particles. When viruses infect cells naturally, they
often leave their protein coats behind and only the viral genome enters [see
Chapter 24: Viruses, Viroids and Prions]. The term transfection (a hybrid of
competent cell Cell that is capable of taking up DNA from the surrounding medium.
electroporator Device that uses a high-voltage discharge to make cells competent for taking up DNA.
transfection Process in which purified viral DNA enters a cell by transformation. Often used to refer to entry of any DNA, even if not of viral
origin, into an animal cell.
Gene for
capsule DEAD MOUSE
Capsule
layer
SMOOTH
COLONY
INJECT INTO MOUSE
VARIANT
(has capsule) Cell wall
KILL,
Chromosome EXTRACT DNA,
AND PURIFY
ADD DNA TO
ROUGH
VARIANT
LIVE MOUSE
ROUGH
COLONY Cell wall
VARIANT
(no capsule) INJECT INTO MOUSE
Chromosome
DEAD MOUSE
Gene for capsule
INJECT INTO MOUSE
FIGURE 28.04
Avery’s Experiment
Avery isolated DNA from the virulent variant and added it to the rough variant of S. pneumonia. He noticed that the virulent DNA
“transformed” or changed the rough variant into a smooth variant. To confirm that the bacteria were truly transformed, he exposed mice to the
newly created smooth variants, and the mice died. Thus, the transformed bacteria had gained both the smooth appearance and virulence by
taking up DNA from the original virulent strains.
transformation with infection) refers to the use of purified viral DNA in transforma-
tion. In this case, the experimenter purifies the viral genome from the virus particle
and offers it to competent cells (Fig. 28.05). If taken up, purified viral DNA induces
the cell to synthesize virus, illustrating that the virus coat is only necessary to protect
the viral DNA outside the host cell and does not carry any of the virus genetic
information.
Transformation and transfection can also have two other meanings. Cancer spe-
cialists use the term “transformation” to refer to the changing of a normal cell into a
cancer cell, even though in most cases no extra DNA enters the cell. (Note that
alterations in the DNA are indeed involved in creating cancer cells, but as a result
of mutation.) Supposedly to avoid ambiguity, researchers who use animal cells often
use the term “transfection” to refer to the uptake of DNA (by transformation!)
whether it is of viral origin or not.
3.2. Transformation in Nature

More detailed investigation of S. pneumoniae and other gram-positive bacteria,
including Bacillus, shows that they develop natural competence in dense cultures.
competent cell Cell that is capable of taking up DNA from the surrounding medium.
transformation (As used of cancer) Changing a normal cell into a cancer cell, even if no extra DNA enters the cell.
FIGURE 28.05
Transfection
During viral transfection, an
DNA
experimenter first isolates pure
viral DNA from virus particles. In Protein
coat
this diagram, DNA is isolated from
P1 virus. Next, the bacterial cell
wall is made competent to take up P1 virus particles
naked DNA (usually by treating
with calcium ions or by
electroshock). The isolated DNA PURIFY
DNA
and the competent bacteria are
mixed. If the bacteria take up the
P1 DNA, the bacteria will start
producing viral particles and burst
DNA
to release the viral progeny. Thus,
viral DNA alone can give the same
end result as infection with whole
TRANSFECT ONLY
virus particles. DNA INTO CELL
Bacterial
cell
Chromosome
P1
MULTIPLIES
BURST
Cell wall FIGURE 28.06

Chromosome Competence Pheromones
Precursor Dense cultures of Streptococcus

polypeptide pneumoniae start producing
competence pheromones that
induce nearby cells to take up
DNA. First, certain cells of the
Shorter culture produce polypeptide
peptide or precursors, which are digested into
pheromone
a small peptide, or competence
is cut out
pheromone. The small peptide is
PHEROMONE secreted from the producer cell
IS SECRETED and binds to a receptor on a
nearby cell. The receptor then
signals that cell to make proteins
Competence used in DNA uptake.
pheromone
PHEROMONE BINDS
TO RECEPTOR ON
ANOTHER CELL
SIGNAL TO
ACTIVATE Pheromone
GENES receptor
ON
DNA UPTAKE
GENES
EXPRESSED
Endonuclease
DNA receptor
Exonuclease
Competence is induced by competence pheromones. (A pheromone is a hormone

that travels between organisms, rather than circulating within the same organism.)
Competence pheromones are short peptides that are secreted into the culture
medium by dividing bacteria (Fig. 28.06). Only when the density of bacteria is high,
pheromone Hormone or messenger molecule that travels between organisms, rather than circulating within the same organism.
will the pheromones reach sufficient levels to trigger competence. This mechanism is
Transformation occurs among
certain bacteria in the natural presumably meant to ensure that any DNA taken up will come from related bacteria
environment. as competence is only induced when there are many nearby cells of the same species.
Natural competence is not merely due to random entry of DNA, but involves
the induction of a variety of genes whose products take part in DNA uptake. First,
DNA is bound by cell-surface receptors (Fig. 28.07). Then the bound DNA is cut
into shorter segments by endonucleases, and one of the strands is completely
degraded by an exonuclease. Only the resulting short single-stranded segments of
DNA enter the cell. Part of the incoming DNA may then displace the corresponding
region of the host chromosome by recombination.
Note that in the case of artificially induced competence, the mechanism is quite
different. Double-stranded DNA enters the cell through a cell wall that is seriously
damaged. Indeed, many, perhaps the majority, of the cells that are made artificially
competent are killed by the treatment. It is the few survivors who take up the DNA.
Before succumbing to the myth that natural gene transfer is peaceful and tran-
quil compared to brutal laboratory methods, some recent findings deserve attention.
Firstly, in S. pneumoniae competent cells practice “fratricide.” They actively kill and
lyse both noncompetent cells of their own species and those of closely related spe-
cies. This gives them access to their victims DNA, which they then take up. Even
more aggressive is the gram-negative Vibrio cholerae, the causative agent of cholera
(see Box 28.01).
4. Gene Transfer by Virus—Transduction

When a virus succeeds in infecting a bacterial cell, it manufactures more virus parti-
cles, each of which should contain a new copy of the virus genome. Occasionally,
Transduction is when viruses viruses make mistakes in packaging DNA, and fragments of bacterial DNA get pack-
pick up fragments of current aged into the virus particle. From the viewpoint of the virus, this results in a defective
host DNA and carry them to particle. Nonetheless, such a virus particle, carrying bacterial DNA, may infect
another host cell. another bacterial cell. If so, instead of injecting viral genes, it injects DNA from the
previous bacterial victim. This mode of gene transfer is known as transduction.
Bacterial geneticists routinely carry out gene transfer between different but related
strains of bacteria by transduction using bacterial viruses, or bacteriophages (phages
for short). If the bacterial strains are closely related the incoming DNA is accepted as
“friendly” and is not destroyed by restriction. In practice, transduction is the simplest
way to replace a few genes of one bacterial strain with those of a close relative.
To perform transduction, a bacteriophage is grown on a culture of the donor
bacterial strain. These bacteria are destroyed by the phage, leaving behind only
DNA fragments that carry some of their genes and that are packaged inside phage
particles. If required, this phage sample can be stored in the fridge for weeks or
months before use. Later, the phage is mixed with a recipient bacterial strain and
the viruses infect the bacteria, injecting their DNA. Most recipients get genuine
phage DNA and are killed. However, a few get donor bacterial DNA and are suc-
cessfully transduced (Fig. 28.09).
4.1. Generalized Transduction

There are two distinct types of transduction. In generalized transduction, fragments
of bacterial DNA are packaged more or less at random in the phage particles. This is
the case for bacteriophage P1 as described earlier (Fig. 28.09). Consequently, all
genes have roughly the same chance of being transferred. In specialized transduction,
certain regions of the bacterial DNA are carried preferentially—discussed later.
generalized transduction Type of transduction where fragments of bacterial DNA are packaged at random and all genes have roughly the same
chance of being transferred.
specialized transduction Type of transduction where certain regions of the bacterial DNA are carried preferentially.
4. Gene Transfer by Virus—Transduction 905
Long piece of FIGURE 28.07

double-stranded DNA Mechanism of Natural
Competence
Endonuclease A cell that is naturally competent

Receptor takes DNA into its cytoplasm by a
protein-mediated process. First, the
Exonuclease long molecule of double-stranded
DNA is recognized by a receptor
on the surface of the competent
cell. A cell-surface endonuclease
digests the DNA into small
fragments. An exonuclease then
DNA BOUND degrades one strand of the DNA.
The remaining single-stranded
fragment is taken into the
DNA is cut by cytoplasm of the bacterium.
endonuclease
ADDITIONAL DNA CUTS MAKE

SMALL FRAGMENTS
EXONUCLEASE DEGRADES ONE STRAND

OF HELIX AND OTHER STRAND ENTERS
SHORT SINGLE-STRANDED
DNA IS TAKEN INTO CELL
Box 28.01 Vibrio Stabs Other Cells to Death to Steal Their DNA
V. cholerae is notorious as the causative agent of cholera. Every so often new strains of cholera with altered characteristics
emerge and spread causing new epidemics. These are the result of horizontal gene transfer (HGT) between different Vibrio strains
in their natural habitat—coastal waters and estuaries.
Some HGT is due to the integration of bacteriophages into the host cell genome. Indeed, the genes for choleratoxin are car-
ried on the CTX phage that integrates into the bacterial chromosome. Other HGT events are due to transformation. These include
surface alterations that protect from the human immune system, such as converting V. cholerae El Tor to V. cholerae Bengal. This
new strain began a worldwide epidemic starting in 1992 in India.
The mechanism of transformation in Vibrio is extremely aggressive and involves assassination of the cell that is the source of the
DNA. The victim is stabbed by a structure known as a type VI secretion system (T6SS). This kills the target cell and results in the
release of its DNA. The DNA is then imported into the attacking cell by a type IV pilus together with other components (Fig. 28.08).
DNA-uptake machinery
chitin
PilA
pilABCD pilMNOPQ
TfoX
CRP cAMP
qstR comEA comEC ComEA
External
ComEC DNA
HapR QstR
protein
VipA/B
High cell
T6SS genes
density
IM OM
Type VI secretion system
FIGURE 28.08
Vibrio Natural DNA Uptake
Natural uptake of DNA in Vibrio depends on the possession of a type VI secretion system (T6SS, purple) as well as dedicated DNA uptake
machinery consisting of a type IV pilus (blue) plus two competence proteins (green). Genes encoding these components are shown as wide,
color-coded arrows. High cell density plus the presence of chitin induce these structures via the TfoX and QstR regulators. Activation of the
Tfox protein also needs cyclic AMP to bind via CRP protein (see Chapter16: Regulation of Transcription in Prokaryotes, Section 16.6 for
details of the cyclic AMP system). Solid black arrows indicate positive regulation. Dashed arrow indicates expression of QstR protein.
Credit: Fig. 1 in Metzger, L.C., Blokesch, M., 2016. Regulation of competence-mediated horizontal gene transfer in the natural habitat of
Vibrio cholerae. Curr. Opin. Microbiol. 30, 17.
Bacteria possess a variety of secretion systems that can both export and/or import proteins and/or nucleic acids. The T6SS is
structurally related to the contractile tails of certain bacteriophage and operates in a similar manner. The T6SS has a contractile
sheath that pushes the spike into the victim upon contraction. Toxic proteins then enter and kill the victim.
The structures that take part in this process are all induced in response to chitin. Remember that chitin is the polymer found in
the hard coats of insects and crustaceans. Because of this, chitin is the most common polymer in aquatic habitats. Vibrio often
associates with the surfaces of marine crustaceans and can digest chitin using secreted enzymes. As might be expected, it is actu-
ally the soluble fragments of chitin breakdown that are responsible for inducing transformation, not the original insoluble polymer.
As Fig. 28.08 indicates, the induction of the DNA uptake machinery also requires high cell density, as monitored by the
HapR regulator. This ensures that plentiful DNA donor cells are nearby before major resources are invested.
For a bacterial virus to transduce, several conditions must be met. In particular,

the phage must not degrade the bacterial DNA. For example, phage T4 normally
destroys the DNA of E. coli after infection. However, mutants of T4 that have lost
the ability to degrade host DNA work well as transducing phages. The packaging
4. Gene Transfer by Virus—Transduction 907
PHAGE INFECTS AN OCCASIONAL PHAGE PHAGE WITH BACTERIAL DONOR DNA ENTERS
DONOR CELL PACKAGES BACTERIAL DNA DNA INFECTS RECIPIENT CELL
RECIPIENT CELL
FIGURE 28.09
Principle of Transduction
Occasionally, when a phage infects a bacterium, one of the virus coats will be packaged with host bacterial DNA (pink). The defective phage
particle infects a nearby cell where it injects the bacterial DNA. This cell will survive since it is not injected with viral DNA. The incoming DNA
may recombine with the host chromosome, thus this cell may gain new genetic information.
mechanism is also critical. Some phages, such as lambda, use specific recognition
sequences when packaging their DNA into the virus particle and so will not package
random fragments of DNA (see Section 28.4.2). In other cases, packaging depends
Some viruses can carry
on the amount of DNA the head of the virus particle can hold. Such “headful pack-
fragments of host DNA. In
aging” is essential for generalized transduction.
generalized transduction,
Two examples of generalized transducing phages are P1, which works on random pieces of the host DNA
E. coli, and P22, which infects Salmonella. The ratio of transducing particles to live are packaged and injected into
virus is about 1:100 in both cases; that is, for every 100 virus particles made, one will a different host.
contain bacterial host DNA. The likelihood of the transduced DNA recombining
into the recipient chromosome is roughly 12 in 100. P1 can package approximately
2% of the E. coli chromosome (about 90 kb of DNA), whereas P22 is smaller and
can carry only 1% of the Salmonella chromosome. Taken all together, about 1 in
500,000 P1 particles will successfully transduce any particular gene on the E. coli
chromosome. This may seem a low probability, but as both typical bacterial cultures
and preparations of P1 contain about 109 per mL, transduction happens at useful fre-
quencies in practice. P1 can also transduce DNA from E. coli into certain other
gram-negative bacteria, such as Klebsiella.
4.2. Specialized Transduction

During specialized transduction, certain specific regions of the bacterial chromosome
are favored. This is due to integration of the bacteriophage into the host chromosome
(see Chapter 24: Viruses, Viroids, and Prions). If the virus enters its lytic cycle and
manufactures virus particles, those bacterial genes nearest the virus integration site
are most likely to be incorrectly packaged into the viral particles. As discussed in
Chapter 24, Viruses, Viroids, and Prions, when bacteriophage lambda (or λ) infects
E. coli, it sometimes inserts its DNA into the bacterial chromosome (Fig. 28.10). This
occurs at a single specific location, known as the lambda attachment site (attλ), which
headful packaging Type of virus packaging mechanism that depends on the amount of DNA the head of the virus particle can hold (as opposed
to using specific recognition sequences).
lambda (or λ) Specialized transducing phage of E. coli that may insert its DNA into the bacterial chromosome.
lambda attachment site (attλ) Site where lambda inserts its DNA into the bacterial chromosome.
P1 A generalized transducing phage of E. coli.
P22 A generalized transducing phage of Salmonella.
lies between the gal and bio genes. The integrated virus DNA is referred to as a
prophage.
When lambda is induced, it excises its DNA from the chromosome and goes
into lytic mode. The original donor cell is destroyed and several hundred virus parti-
cles containing lambda DNA are produced. Just like generalized transducing phages,
BACTERIAL
CHROMOSOME a small fraction of lambda virus particles contain bacterial DNA. There are, how-
ever, two major differences. First, only chromosomal genes next to the lambda
attachment site are transduced. Second, the specialized transducing particles contain
bio a hybrid-DNA molecule comprising both lambda and chromosomal DNA
e
ge en
ne lg (Fig. 28.11). This hybrid molecule results from mistakes during excision of the
ga
a tt λ
lambda prophage. Chromosomal DNA to the right or to the left of the prophage,
but not both, may be included in the transducing phage. In practice, this means that
+ either the gal or bio genes are picked up.
Mistakes in excision of lambda only occur at a rate of 1 in a million relative to
Lambda DNA
correct excision. Furthermore, the defective excision must generate a segment of
DNA approximately the same length as the lambda genome in order for it to fit into
the phage head. Consequently, specialized transducing particles arise only at
extremely low frequency. However, once a lambda-transducing phage has been cre-
ated, it may reintegrate its DNA into the chromosome of another host cell. This
may occur either in the attλ site or into the chromosomal copy of the gene (usually
gal or bio) carried by the lambda-transducing phage. Inducing this defective pro-
phage DNA will give a second generation of transducing phage particles at a much
higher frequency.
The properties of lambda-transducing phages depend on which lambda genes
were lost in exchange for chromosomal DNA. The λdgal-transducing phages lack
lambda genes needed for making head and tail components and instead, the virus
bio
contains the E. coli gal gene. These are therefore “defective” (hence, the “d” in
ne
gen
ge
λdgal). Defective phage may be grown together with a wild-type lambda as a helper
e
l
ga
at
t R att
L
phage, which provides the missing functions. In the case of λdgal, helper phage
Lambda DNA
would make the head and tail components. Conversely, the λpbio-transducing
phages lack the lambda int gene, which integrates the phage DNA into the attλ site,
FIGURE 28.10 and instead contains the bio gene from E. coli. Since the phage cannot integrate,
Integration of Lambda into λpbio must enter the lytic phase and are thus obligate plaque formers (hence, the
the E. coli Chromosome “p” in λpbio). If wild-type helper phage is added, the int function is restored, and
the phage forms lysogens. Cloning vectors derived from lambda are widely used in
When bacteriophage lambda
infects a host E. coli cell, it can
genetic engineering (see Chapter 7: Cloning Genes for Synthetic Biology). Since the
integrate its phage DNA into the cloned DNA replaces many of the lambda genes, such vectors need to be grown in
chromosome. The phage DNA will the presence of helper phages.
only integrate at a site called attλ,
which is found between the bio
5. Transfer of Plasmids Between Bacteria
gene and gal gene of the
chromosome. Once integrated, the The transfer of genetic information between two bacterial cells may occur via cell-
phage is referred to as a prophage.
to-cell contact. This process is known as bacterial conjugation and typically depends
on the presence of plasmids. Transferability is the ability of certain plasmids to
Specialized transduction occurs move from one bacterial cell to another. Many medium-sized plasmids, such as the
in viruses that integrate into
F-type and P-type plasmids, are able to move and are referred to as Tra1 (transfer-
host DNA at specific sites in the
positive) or self-transferable. For transfer to occur, the bacterial cell containing the
host chromosome. Only
adjacent genes to the plasmid must make physical contact with a suitable recipient cell. During bacterial
integration site are transduced. conjugation DNA moves in one direction only, from the plasmid-carrying donor to
defective phage Mutant phage that lacks genes for making virus particles.
helper phage Phage that provides the necessary genes so allowing a defective phage to make virus particles.
prophage Virus DNA that is integrated into the host chromosome.
Tra1 Transfer-positive (refers to a plasmid capable of self-transfer).
transferability Ability of certain plasmids to move themselves from one bacterial cell to another.
5. Transfer of Plasmids Between Bacteria 909
INFREQUENT
gal gene att L Lambda DNA
ge
ne PACKAGING
l
ga
L
att
Lambda DNA
BACTERIAL FREQUENT Lambda DNA
CHROMOSOME PACKAGING
tR
at
e
g en
bio INFREQUENT
Lambda DNA att R bio gene
PACKAGING
FIGURE 28.11
Packaging of Host DNA During Transduction by Lambda
When lambda phage enters its lytic cycle and makes phage particles, it usually packages the lambda DNA between the attL and attR sites.
Occasionally, a mistake will occur, and part of the bacterial chromosome DNA will be packaged. Since lambda DNA normally integrates
between the gal and bio genes of the E. coli chromosome, the defective lambda particles will most likely contain one or other of these genes.
the recipient (Fig. 28.12). The donor cell manufactures a sex pilus that binds to the
recipient and draws the two cells together. Next, a conjugation bridge forms Transferable plasmids move
from one cell to another via the
between the two cells and provides a channel for DNA to move from donor to
conjugation bridge.
recipient. In real life, mating bacteria tend to cluster together in groups of 510
(Fig. 28.13).
The genes for formation of the sex pilus and conjugation bridge and for oversee-
ing the DNA transfer process are known as tra genes and are all found on the plas-
mid itself. Since plasmid transfer requires over 30 genes, only medium or large
plasmids possess this ability. Very small plasmids, such as the ColE plasmids, do not
have enough DNA to accommodate the genes needed.
The most famous self-transferable plasmid is the F-plasmid of E. coli, which is
approximately 100 kbp long. Donor cells are sometimes known as F1 or “male” and
recipient cells as F2 or “female” and conjugation is sometimes referred to as bacte-
rial mating. Note, however, that the “sex” of a bacterial cell is determined by the
presence or absence of a plasmid and that DNA transfer is unidirectional, from
donor to recipient. When a recipient cell has received the F-plasmid, it becomes F1.
From a human perspective it has been transmuted from “female” into a “male”!
Thus, bacterial mating is not at all equivalent to sexual reproduction among higher
organisms.
Although we talk about “plasmid transfer,” in reality both the donor cell and
the recipient cell end up with a copy of the plasmid. Thus the transfer mechanism
includes the synthesis of a second copy of the plasmid. Plasmid DNA transfer
involves replication by the rolling circle mechanism (Fig. 28.14). First, one of the
two strands of the double-stranded DNA of the plasmid opens up at the origin of
transfer. This linearized single strand of DNA moves through the conjugation bridge
A single strand of newly made
from the donor into the recipient cell. An unbroken single-stranded circle of plasmid DNA is transferred from the
DNA remains inside the donor cell. This is used as a template for the synthesis of a donor to the recipient cell
new second strand to replace the one that just left. As the linear single strand of during conjugation.
plasmid DNA enters the female cell, a new complementary strand of DNA is made
conjugation bridge Junction that forms between two cells and provides a channel for DNA to move from donor to recipient during conjugation.
F-plasmid Fertility plasmid that allows E. coli to donate DNA by conjugation.
sex pilus Protein filament made by donor bacteria that binds to a suitable recipient and draws the two cells together.
FIGURE 28.12 FORMATION OF MATING PAIRS

Bacterial Conjugation Basal structure
Chromosome
1
Certain plasmids, called Tra or Sex pilus
transfer-positive, are able to move Transferable
a copy of their DNA into a plasmid
different cell through a mechanism
Chromosome
called bacterial conjugation. First,
the cell containing a Tra1 plasmid
manufactures a rod-like extension
on the surface of the outer
membrane called a sex pilus. The
DONOR
sex pilus binds to a nearby cell CELL
and pulls the two cells together by RECIPIENT
retracting. Once the cells are in CELL
contact, the basal structure of the
pilus makes a connection between
the two cells known as the
conjugation bridge. This connects
the cytoplasm of the two cells, so
the plasmid can transfer a copy of
itself to the recipient cell.
FORMATION OF A CONJUGATION BRIDGE
DONOR
CELL
Conjugation bridge
Plasmid transfers
as a single
strand of DNA
RECIPIENT
CELL
using the incoming strand as template. Thus, only one strand of plasmid DNA is
transferred from the donor to the recipient.
The detailed physical mechanism of DNA transfer via the conjugation bridge
was only solved relatively recently. The earliest proposals were that DNA traveled
FIGURE 28.13
Conjugating Cells of E. coli
False-color transmission electron
micrograph (TEM) of a male E. coli
bacterium (bottom-right)
conjugating with two females. This
male has attached two F-pili to
each of the females. The tiny
bodies covering the F-pili are
bacteriophage MS2, a virus that
attacks only male bacteria and
binds specifically to F-pili.
Magnification: 3 11,250. Credit:
Dr. L. Caro, Photo Researchers,
Inc.
REPLICATION FIGURE 28.14

Plasmid Transfer Involving
Origin of Single-strand Single-strand Rolling Circle Replication
transfer nick is made enters recipient cell
5'-end (A) During bacterial conjugation,
the F-plasmid of E. coli is
transferred to a new cell by rolling
circle replication. First, one strand
F-plasmid F-plasmid of the F-plasmid is nicked at the
origin of transfer. The two strands
start to separate and synthesis of a
new strand starts at the origin
Synthesis of new DNA (green strand). (B) The single-
Double-stranded
complementary to strand of F-plasmid DNA that is
DNA
A unbroken strand
displaced (pink strand) crosses the
conjugation bridge and enters the
recipient cell. The second strand of
TRANSFER the F-plasmid is synthesized inside
DONOR the recipient cell. Once the
F- plasmid
CELL complete plasmid has been
Complementary transferred, it is re-ligated to form
strand synthesized a circle once again.
in donor
Chromosome
5'
3'
5'
RECIPIENT
Complementary CELL
strand synthesized
in recipient
B
FIGURE 28.15
Basal Structure of the Sex
Pilus
Shaft of pilus
The basal structure of the sex pilus
resembles a type IV secretion
system. It crosses both the inner Outer layer
and outer membranes and its
central channel is large enough for
Outer membrane
the transit of proteins or DNA. The
details of individual components
vary somewhat between
organisms, depending on the Peptidoglycan
specific role of the system. This
diagram is a simplified version
Inner membrane
showing the common core
structures.
Inner layer
Coupling protein
uses ATP
through the central channel of the sex pilus itself. Although this is incorrect, the
DNA does in fact travel through the central channel of the basal structure on which
the pilus is built. When the sex pilus is assembled, its protein subunits travel through
the channel in the basal structure (also known as the transfer apparatus). After
donor and recipient have made contact, the pilus is retracted and the pilus subunits
return through the same channel. This brings the two cells into close contact and
leaves the basal structure bridging the inner and outer membranes of the donor and
in contact with the recipient. DNA then moves through the channel of the basal
structure into the recipient. The basal structure belongs to the family of type IV
secretion systems. These are used by a variety of bacteria for protein secretion as
well as DNA uptake and DNA transfer (Fig. 28.15).
Although small plasmids such as ColE are not self-transferable, they are often
mobilizable (Mob1). A transferable plasmid, such as the F-plasmid, can mobilize
the ColE plasmid if they both inhabit the same cell. The F-plasmid oversees conju-
Plasmids unable to transfer gation and forms the conjugation bridge and the ColE plasmid is transferred through
themselves may be able to this. The mob (mobilization) genes of the ColE plasmid are responsible for making
hitchhike using the transfer a single-stranded nick at the origin of transfer of ColE and for unwinding the strand
systems of other plasmids. to be transferred. Other small plasmids are neither self-transferable nor mobilizable.
5.1. Transfer of Chromosomal Genes Requires Plasmid Integration

Although many plasmids allow the cells carrying them to conjugate, usually only the
plasmid itself is transferred through the conjugation bridge. But occasionally, plas-
mids mediate transfer of the host chromosome when they move from one bacterial
cell to another. Plasmids, such as the F-plasmid of E. coli, that enable a cell to
donate host chromosomal DNA are called fertility plasmids. In order to transfer
chromosomal genes, a plasmid must first physically integrate itself into the chromo-
Transferable plasmids some of the bacterium. This event requires pairs of identical (or nearly identical)
sometimes move chromosomal DNA sequences, one on the plasmid and the other on the chromosome. For exam-
DNA from one cell to another. ple, the F-plasmid uses insertion sequences (see Chapter 25: Mobile DNA) for inte-
gration into the chromosome of E. coli (Fig. 28.16).
A variety of different insertion sequences are found on the chromosome of
E. coli and in its plasmids and viruses. The F-plasmid has three insertion sequences
(Fig. 28.17): Two copies of IS3 and a single copy of IS2. The chromosome of a typi-
cal laboratory strain of E. coli has 13 copies of IS2 and 6 copies of IS3 scattered
fertility plasmid Plasmid that enables a cell to donate DNA by conjugation.

insertion sequence A simple transposon consisting only of inverted repeats surrounding a gene that encodes transposase.
5. Transfer of Plasmids Between Bacteria 913
IS
IS
Bacterial
chromosome
d s D NA
IS
IS
F-plasmid
ds DNA
F-plasmid DNA
FIGURE 28.16
Integration of F-Plasmid Into Chromosome
If recombination occurs between two insertion sequences, one on the F-plasmid and one on the host bacterial chromosome, the entire F-plasmid
becomes integrated into the chromosome.
IS 3 FIGURE 28.17
Tn1
00
0
Insertion Sequences
n es on F-Plasmid and
ge
Chromosome
r
fe
ns
Tra
Insertion sequences are scattered

IS3
throughout the F-plasmid and

chromosome of E. coli. The F-
IS2
F-plasmid plasmid has two IS3 elements and

100 kb one IS2 element. Even more copies
of IS2 and IS3 are found on the
chromosome (not shown). A
ori T recombination event between any
Origin of of the chromosomal IS2 or IS3
Re
transfer elements and the corresponding
pli
cat element on the F-plasmid will
io n gen
es ori V integrate the entire F-plasmid into
the chromosome. Tn1000 (also
Origin of
vegetative known as γδ) is another insertion
replication sequence, although not generally
involved in F-plasmid integration in
E. coli.
around more or less at random. Integration of the F-plasmid may occur in either ori-
entation at any of these 19 sites.
When an F-plasmid that is integrated into the chromosome transfers itself by
conjugation, it drags along the chromosomal genes to which it is attached
(Fig. 28.18). Just as for the unintegrated F-plasmid, only a single strand of the DNA
moves and the recipient cell has to make the complementary strand itself. Bacteria
with an F-plasmid integrated into the chromosome are known as Hfr strains because
they transfer chromosomal genes at high frequency. A prolonged mating of 90 min-
utes or so is needed to transfer the whole chromosome of E. coli. More often, bacte-
ria break off after a shorter period of, say, 1530 minutes, and only part of the
In order to mobilize
chromosome is transferred. Since different Hfr strains have their F-plasmids inserted chromosomal DNA, the
at different sites on the bacterial chromosome, transfer of chromosomal genes plasmid must first integrate into
begins at different points. In addition, the F-plasmid may be inserted in either orien- the chromosome.
tation. Consequently, chromosomal gene transfer may be either clockwise or coun-
terclockwise for any particular Hfr strain.
Hfr-strain Bacterial strain that transfers chromosomal genes at high frequency due to an integrated fertility plasmid (F-plasmid).
FIGURE 28.18 f e d c
b
Transfer of Chromosomal a
Genes by F-Plasmid A ori T
F-
origin of
pla
al
An integrated F-plasmid can still transfer
om
sm
induce bacterial conjugation and
Chromos
id
rolling circle transfer of DNA into INTEGRATED
another bacterial cell. Since rolling F-PLASMID
circle replication does not stop Single-stranded
nick is made
until the entire circle is replicated, at ori T
the attached chromosome is also
transferred into the recipient cell.
First, a single-stranded nick is
made at the oriT, or transfer origin
of the integrated plasmid. The free
50 end (black triangle) enters the
recipient cell through the
SINGLE STRAND IS UNROLLED
conjugation bridge. Notice that the AND ENTERS RECIPIENT
transfer of the single-stranded
DNA does not end with the F-
plasmid DNA and continues into Front of
F-plasmid
the chromosomal DNA. Genes f e d c b
a
closest to the site of plasmid
integration are transferred first (in A
D N
the order a, b, c, d, e, f, in this

al
om
example). The amount of

Chromos
chromosomal DNA that is ori T

transferred depends on how long enters recipient
the two bacteria remain attached first
by the conjugation bridge.
Rear of
F-plasmid
Hfr strains were used in earlier times to identify the order of genes on the
E. coli chromosome. To monitor whether the recipient has received a particular
gene, the donor and recipient strains must have different alleles of this gene that can
be distinguished phenotypically, usually by their growth properties. For example, the
recipient might have a mutation in the lac operon that prevents growth on lactose as
carbon source. The donor Hfr strain would have an allele that restores the ability to
use lactose. Using this method, genetic maps were constructed by two major
approaches. First, the cotransfer frequency of two genes was measured. If two genes
were close to each other, a donor Hfr strain would transfer them together at high
frequency. Conversely, if two genes were far apart on the chromosome, an Hfr strain
would usually only transfer one of them, and the cotransfer frequency would be low.
Secondly, time-of-entry measurements were made to determine gene order. Hfr
strains transfer chromosomal genes starting where the F-plasmid is integrated and
proceeding sequentially around the circular chromosome (Fig. 28.19). The length of
time it takes for a gene to enter the recipient gives an estimate of its relative dis-
tance from the origin of transfer of the Hfr strain. For time of entry mapping the
site and orientation of the F-plasmid must be known. In addition, mutations in
the genes being studied (a, b, c, and d) must give recognizable phenotypes. Finally,
the recipient must be resistant to some antibiotic (e.g., streptomycin) so that it can
be selected on medium that prevents growth of the Hfr strain. Different Hfr strains
will transfer the same genes in different orders and at different times, depending on
their location relative to the integration site of the F-plasmid.
cotransfer frequency Frequency with which two genes remain associated during transfer of DNA between bacterial cells.
6. Gene Transfer Among Gram-Positive Bacteria 915
Hfr 1 Hfr 2
0/100 0/100 F FIGURE 28.19

a
Time of Entry by
b a
b Conjugation
c
To determine the time of entry by
d
75
Bacterial
25 75
Bacterial
25 conjugation, the Hfr strain is mixed
chromosome chromosome with a recipient strain carrying a
F defective copy of a particular
gene, “a.” After conjugation has
proceeded for a specific time, a
sample of the mixture is removed.
50 50
This is plated on agar, which
prevents growth of the Hfr and
only allows growth of strains
Number of recipients
Number of recipients
carrying the wild-type version of
receiving gene
receiving gene
gene “a.” Survivors are derivatives
ed
ea
of the recipient that have gained
b
n
n
c
the wild-type version of gene “a”
ge
ge
ne
ne
c
eb
ge
e
from the Hfr. This is repeated for
ge
gen
ed
gen
ea
several time points. The whole
ge n
ge n
procedure is then repeated for the

0 5 10 15 20 0 5 10 15 20 other genes. In strain Hfr 1 (left
Time (minutes) Time (minutes) panel), the integrated F-plasmid is
closest to gene “d” and only
begins transferring gene “a” after
F-plasmids can excise themselves from the chromosome by reversing the inte- about 20 min. In strain Hfr 2 (right
gration process. Sometimes they excise carrying pieces of chromosomal DNA, which panel), the F-plasmid is integrated
creates F0 - or F-prime plasmids. This typically occurs by recombination between a closer to gene “a,” which therefore
different pair of IS sequences than used during integration. Such F0 -plasmids may be begins to appear in the recipient
transferred to F-minus recipients, carrying with them the chromosomal segment as early as 5 min after transfer
begins.
from their previous host. If the chromosomal segment is homologous, the F0 can
reintegrate via homologous recombination. Historically, F-primes were used to carry
part of the lacZ gene in the alpha-complementation method for screening recombi-
nant plasmids (see Chapter 7: Cloning Genes for Synthetic Biology).
6. Gene Transfer Among Gram-Positive Bacteria

Traditionally, the bacteria are divided into two major groups: The gram-negative
and the gram-positive bacteria. This division was originally based on their response
to the gram stain. The differences in staining reflect differences in the chemical com-
position and structure of the cell envelope. The envelope of gram-negative bacteria
consists of the following layers (from inside to outside): Cytoplasmic membrane, cell
Gram-negative bacteria,
wall (peptidoglycan), and outer membrane (Fig. 28.20). The envelope of gram- including E. coli, have an extra
positive bacteria is simpler and lacks the outer membrane. Both kinds of bacteria outer membrane.
sometimes have an extra protective layer, the capsule, on the very outside.
The gram-negative bacterium E. coli is widely used as a host for cloning and
expressing genes from a variety of other organisms. The synthesis of large amounts
of a purified recombinant protein from a cloned gene is often desirable. Secretion of
recombinant protein into the culture medium would be very convenient as this
avoids the need to purify it away from all the other proteins inside the bacterial cell.
However, the complex envelope of gram-negative bacteria is a major hindrance in
alpha-complementation Assembly of functional beta-galactosidase from N-terminal alpha fragment plus the remaining part of the protein.
F0 or F-prime plasmid F-plasmid of E. coli that has excised itself from the host chromosome and contains segments of the host DNA in addition
to the regular plasmid DNA.
gram-negative bacteria Major division of bacteria that possess an extra outer membrane lying outside the cell wall.
gram-positive bacteria Major division of bacteria that lack an extra outer membrane lying outside the cell wall.
outer membrane Extra membrane lying outside the cell wall in gram-negative but not gram-positive bacteria.
FIGURE 28.20 GRAM-NEGATIVE ENVELOPE

Differences in Envelopes of
Gram-Negative and
Gram-Positive Bacteria Lipopoly-
saccharide
The outer surfaces of gram-positive
and gram-negative bacteria have
different structures. (A) In gram-
negative bacteria, such as E. coli, Outer
membrane
there are three surface layers. The
outermost layer, called the outer
membrane, is a lipid bilayer that
contains various proteins Lipoprotein Periplasmic
Peptidoglycan
embedded within the lipids, and (single layer wall) space
an outer coating of
lipopolysaccharide. Next, within
the periplasmic space, the cell wall
contains a single layer of Protein Inner
peptidoglycan. Lipoproteins membrane
connect this cell wall to the outer
membrane. The layer closest to the
cytoplasm, called the inner
membrane, is a lipid bilayer A Phospholipids
embedded with various proteins.
(B) The outer surface of gram- GRAM-POSITIVE ENVELOPE
positive bacteria only has two
layers, a thick wall of
peptidoglycan plus teichoic acid Several layers
of peptidoglycan
surrounding the cell membrane. plus teichoic
acid (red)
Inner
Protein membrane
B Phospholipids
the export of proteins into the culture medium. In contrast, secretion across the sim-
pler gram-positive envelope is easier. Indeed, many gram-positive bacteria, such as
Bacillus, excrete proteins into the culture medium naturally. As a result, there is
Transfer of plasmids between considerable interest in using gram-positive bacteria as hosts in genetic engineering.
gram-positive bacteria is often Unfortunately, the genetics of gram-positive bacteria is far behind that of the inten-
promoted by pheromones. sively studied E. coli and its relatives. Nonetheless, mechanisms of gene transfer are
available in gram-positive bacteria.
Self-transmissible plasmids are widespread among gram-positive bacteria and
many of these plasmids are rather promiscuous. Since the cell envelope is simpler in
gram-positive bacteria, plasmid transfer is also simpler and a sex pilus is not needed.
Apparently, barely a dozen genes are required to encode the transfer functions.
Some gram-positive bacteria, such as Enterococcus, secrete mating pheromones into
the culture medium. These are short peptides (7 or 8 amino acids long) that induce
the tra genes of transferable plasmids by binding to a pheromone receptor protein.
Only bacteria that lack a particular plasmid, secrete the corresponding pheromone,
consequently the plasmid only expresses its transfer genes when a suitable recipient
is nearby. Different pheromones are specific for different plasmids.
In Enterococcus, two different peptide signal molecules, the pheromone and an
inhibitor peptide, compete for binding to the same site on the PrgX receptor pro-
tein. Cells lacking the plasmid secrete the pheromone and plasmid-bearing cells
7. Archaeal Genetics 917
Mating FIGURE 28.21

pheromone Pheromones Induce
Pheromone binds
Mating in Gram-Positive
to receptor
Bacteria
Tra+ In gram-positive bacteria such as
plasmid Enterococcus, cells without
plasmids secrete pheromones to
Chromosome
attract bacteria with transferable
(Tra1) plasmids. Mating
pheromones bind to receptors on
the surface of cells containing Tra1
Gram + Gram + plasmids. Binding the receptor
cell without cell with
plasmid transferable activates the transfer genes to form
plasmid a conjugation bridge and transfer
MATING PAIR FORMS; the plasmid by rolling circle
PLASMID IS TRANSFERRED replication. Each pheromone is
specific and only attracts bacteria
with certain plasmids.
5'
secrete the inhibitor. When the inhibitor peptide binds to PrgX, it acts as a repressor
and prevents transcription from the promoter of prgQ. Conversely, when the phero-
mone binds to PrgX, it dissociates from the promoter and the prgQ gene is de-
repressed and activates the plasmid-transfer system. This occurs when a plasmid free
recipient cell comes close to a cell carrying a plasmid. The final result is cell aggre-
gation and plasmid transfer (Fig. 28.21).
Gram-positive bacteria also harbor conjugative transposons (e.g., Tn916 of
Enterococcus). These can transfer themselves from one bacterial cell to another (see
Chapter 25: Mobile DNA). These elements excise themselves temporarily from the
chromosome of the donor cell before conjugation. Once inside the recipient, they
reinsert themselves into the bacterial chromosome.
7. Archaeal Genetics
There are two genetically distinct lineages of prokaryotes, the “normal” bacteria
(previously Eubacteria) and the Archaea (previously Archaebacteria). Although
both are prokaryotic cells without a nucleus, the bacteria and Archaea are not
related to each other genetically. Together with the eukaryotes (strictly, the nuclear
genome of eukaryotes) they comprise the three domains of life. (See Chapter 29:
Molecular Evolution, for further discussion of these relationships.) The bacteria
include most typical bacteria found in normal environments, including both the
gram-negative and gram-positive bacteria discussed earlier. The Archaea include the Archaea are genetically distinct
from Bacteria and often live
methane producing “bacteria,” or methanogens, and a variety of less well-known
under unusual or extreme
prokaryotes found in extreme environments. Many have strange biochemical path-
conditions.
ways and are adapted to extremes of temperature, pH, or salinity. This makes the
Archaea an attractive source of novel enzymes or proteins with unusual properties
Archaea (or Archaebacteria) Type of prokaryote forming a genetically distinct domain of life. Includes many cells that grow under extreme
conditions.
conjugative transposon Transposon that can transfer itself from one bacterial cell to another by conjugation.
Eubacteria Officially Bacteria—the typical bacteria that have peptidoglycan in their cell walls and form a separate domain from the Archaea.
and/or resistance to extreme conditions. There are many possible industrial uses for
enzymes capable of withstanding extreme temperatures, for example.
Although many complete genome sequences are available for members of the
Archaea, development of systems for gene transfer has lagged way behind the bacte-
ria. There are many practical problems, including the need to grow many Archaea
under extreme conditions. For example, most extreme thermophiles grow at tem-
peratures high enough to melt agar. Obtaining colonies on solid media has required
the development of alternative materials.
Another major problem is choice of a selectable marker. Most standard antibio-
tics do not affect Archaea due to their unusual biochemistry. For example, Archaea
do not have cell walls made of peptidoglycan and are therefore not susceptible to
penicillin derivatives. In addition, many resistance proteins from normal organisms
are denatured at the extremes of temperature, salinity, or pH under which many
Archaea grow. Novobiocin (a DNA gyrase inhibitor—see Chapter 10: Cell Division
and DNA Replication) and mevinolin (an inhibitor of the isoprenoid pathway) have
been used to inhibit halophiles, and puromycin and neomycin (both protein synthe-
sis inhibitors—see Chapter 13: Protein Synthesis) will inhibit methanogens.
It has been possible to express the lacZ reporter gene in methanogens under
Gene transfer in Archaea is control of an archaeal promoter. However, staining of β-galactosidase with Xgal
widespread but still poorly requires exposure to air, which kills methanogens! Consequently, colonies must first
understood. be replicated and one set sacrificed for analysis.
Transformation procedures now exist for getting DNA into several Archaea.
Some rely on removal of divalent cations, especially Mg21, which results in the
disassembly of the glycoprotein layer surrounding many archaeal cells. (Note the
contrast with the corresponding procedures for typical bacteria, which involve cold-
shock in the presence of excess divalent cations!) However, those Archaea that can
survive the removal of high salt or being exposed to oxygen can often be trans-
formed by electroporation.
Viruses have been discovered that infect many Archaea. So far, only one, the
ΨM1 phage of Methanobacterium thermoautotrophicum, has been shown to trans-
duce the genes of its host bacterium. Unfortunately, this is of no practical use
because of the low burst size—about six phage particles are liberated per cell after
infection. The SSV1 phage of Sulfolobus solfataricus integrates into the bacterial
chromosome and may be of future use.
Plasmids have been found in several Archaea and some have been developed
into cloning vectors (Fig. 28.22). However, only the plasmids of Sulfolobus (and
related Archaea) are self-transferable, as far as is known. Conjugation in Sulfolobus
relies on type IV pili to bring the cells together. The process of DNA transfer is still
mysterious although it occurs between genetically identical cells and may be bidirec-
tional. This differs from typical bacterial conjugation, which involves one-way trans-
fer of DNA from a cell containing a plasmid to a recipient cell that lacks a plasmid.
The recently discovered CedAB protein complex is located in the in Sulfolobus
cell membrane and takes part in DNA uptake. Whether single or double-stranded
DNA is transferred in Sulfolobus is unknown; however, DNA that is transferred can
take part in homologous recombination. The CedB protein uses hydrolysis of adeno-
sine triphosphate (ATP) to energize DNA uptake.
Conjugation has also been reported in some halobacteria that form conjugation
bridges without the participation of fertility plasmids. This system is still obscure.
Neither type of archaeal conjugation has so far been developed into the sort of use-
ful gene-transfer system found in well-studied bacteria such as E. coli or Bacillus.
8. Whole-Genome Sequencing
The techniques for gene transfer described in this chapter have allowed the con-
struction of detailed genetic maps and convenient gene transfer systems for E. coli
and a few other well-investigated bacteria. However, for the vast majority of
8. Whole-Genome Sequencing 919
Crenarchaeota Euryarchaeota
Halobacterium
Haloferax
Haloarcula
Halorubrum
Archaeoglobus
Sulfolobus Methanobacterium
Methanospirillum
Thermoproteus Pyrococcus
Methanosarcina
Desulfurococcus Methanolobus
Methanococcus
Pyrodictium
Methanopyrus
Transformation
Archaea
Eubacteria Eukaryotes Transduction
Conjugation
FIGURE 28.22
Groups of Archaea and Their Gene-Transfer Mechanisms
Phylogenetic tree of the Archaea lineage illustrating that different types of gene transfer can occur. The green zone contains salt tolerant
organisms, the blue zone indicates methane producers and the red zone contains Archaea that grow at extremely high temperatures. Some
Archaea use transformation whereas others use conjugation. Rare cases of viral transduction also occur. The modes of gene transfer seen within
each family do not correlate well with either lifestyle or evolutionary relationships. The Crenarchaeota and the Euryarchaeota are the two major
branches of the Archaea.
microorganisms, no “classical” genetics exists. Nowadays these are largely being inves-
tigated by more modern techniques, such as gene cloning and DNA sequencing.
Since the development of rapid automated techniques for sequencing DNA (see The bacterium Hemophilus
Chapter 8: DNA Sequencing) many whole genomes have been totally sequenced. influenzae was the first
The first genome sequence to be finished was from the bacterium Hemophilus influ- organism to have its DNA
enzae in 1995. Since then, thousands of bacterial genomes have been sequenced. completely sequenced.
Sequence comparison with genes of well-investigated organisms allows provisional
identification of many genes. However, even in E. coli, the function of about a third
of the genes remains uncertain.
Virulence genes are often
Whole-genome sequencing of pathogenic bacteria and comparison with their clustered together forming
harmless relatives often reveals extra blocks of genes responsible for causing disease. “islands.”
Many virulence genes are carried on plasmids as discussed in Chapter 23, Plasmids.
Others are found clustered together in regions of the chromosome known as
“pathogenicity islands.” Most genes of Salmonella, as well as their order around the
chromosome, correspond to those of its close relative E. coli, as would be expected.
However, extra segments of DNA are found in Salmonella that are lacking in
E. coli. Some of these are pathogenicity islands (Fig. 28.23). Such extra regions are
often flanked by inverted repeats, implying that the whole region was inserted into
the chromosome by transposition at some period in the evolutionary past. In agree-
ment with this idea, such islands are often found in some strains of a particular spe-
cies but not others. In addition, these islands tend to have different G/C to A/T
ratios and/or codon usage frequencies from the rest of the chromosome, suggesting
their origin in some other organism. Conversely, E. coli possesses a few DNA
pathogenicity island Region of bacterial chromosome containing clustered genes for virulence.
FIGURE 28.23 g h
Pathogenicity Islands of f
Salmonella
i
e
Comparison of the E. coli genome
j
with its close relative, Salmonella, Harmless
d
reveals large regions of DNA that bacterium
e.g., E. coli
k
have no homology (orange). The
c
remaining regions have similar
l
genes that are in identical order. b
For example, Salmonella genes d
m
a
through j are clustered together in
the exact same order as E. coli Bacterial
genes d through j. Since chromosomes
Salmonella is pathogenic and
E. coli is not, the regions of no f g h Inverted
homology probably encode the e i repeats
genes required for pathogenicity; d j
therefore, they are termed
pathogenicity islands. The islands
are flanked by inverted repeats,
suggesting the DNA may have Related
been acquired through pathogenic
relative
transposition. (Note: This figure is
e.g., Salmonella typhi
not drawn to scale; the
pathogenicity islands are greatly
exaggerated relative to the rest of
the chromosome for purposes of
illustration.) Pathogenicity
islands
c
k
b l
a m
segments missing in Salmonella. Interestingly, one of these is the area including the
Differences in G/C and A/T
ratios reveal segments of
lac operon and a few surrounding genes. Thus, the classic lac operon, the most-
chromosomes with foreign studied “typical” gene of the “standard organism” is probably a relatively recent
origins. intruder into the E. coli genome!
Pathogenicity islands are simply the best-known case of “specialization islands.”
These are blocks of contiguous genes, presumed to have a “foreign” origin, which
contribute to some specialized function that is not needed for simple survival. Not
Horizontal transfer of genes is surprisingly, medical relevance has drawn most human interest. Other examples
especially significant in include genes encoding pathways for the biodegradation of aromatic hydrocarbons,
bacteria. herbicides, and other products of human industry and pollution.
Movement of genes “sideways” is designated lateral or horizontal gene transfer
in distinction to the “vertical” transfer of genes from ancestors to their direct des-
cendants. HGT can occur by conjugation, natural transformation, viral transduction,
or transposon jumping. HGT may occur between closely related organisms or those
far apart taxonomically. Estimates suggest that in typical bacteria around 5% of the
genes have been obtained by lateral gene transfer, and in rare cases up to 25%.
Thermotoga is a bacterium adapted to life at very high temperatures and which con-
sequently shares its habitat with several Archaea. Thermotoga has apparently gained
around 25% of its genes by transfer from thermophilic Archaea such as
Archaeoglobus and Pyrococcus. When we remember that the F-plasmid of E. coli
horizontal gene transfer Movement of genes sideways between unrelated organisms. Same as lateral gene transfer.
8. Whole-Genome Sequencing 921
can mediate DNA transfer into yeast (see Chapter 23: Plasmids), these results are
perhaps not so surprising.
8.1. Bacterial Genome Assembly and Transplantation

The Venter Institute has performed an intriguing set of genetic manipulations
intended to pave the way for the synthesis of artificial life. They have shown that it
is possible to transform a whole bacterial genome into a suitable recipient cell. For
** Elements for yeast propagation
* **
and genome transplantation
BssH II
Asc I * *
811–
9 900
–79 94D
701
****
WM4
BssH II
1,077,947 1
90
1–
*
10
0
70
00
*
1–
* Oligonucleotide
60
A G C T synthesizer
TG
CT A
CG
CG
ATCAA CGCA
TA
GA TG Oligonucleotides
CAACT
AG
TC GCAT 0
,00
1001–1
AT CATG 200
1,080 bp cassettes (1,078)
501–600
(Assemble 109X)
104
Yeast 10,080 bp assemblies (109)
800,000 (Assemble 11X)
100,000 bp assemblies (11)

(Assemble 1X)
2–1
Asc I 00
00
1,077,947 bp
–5
40
1
40
0,
WM2
00
*
0
0
,00
600
BssH II 10
20
1– WM3
0 0
40
BssH II 1–
30 *
BssH II 201–300 * **
Asc I
* WM1
BssH II
FIGURE 28.24
Assembly of Synthia Venter
The whole genome for a synthetic organism called Mycoplasma mycoides was assembled in yeast. First, segments of DNA about 1000 bp in
length were chemically synthesized using an oligonucleotide synthesizer. These were combined into groups of 10 to create the 10 kb fragments
(blue arrows). The 10 kb pieces were combined into groups of 10 to create 100 kb fragments (green arrows). Finally, the 11 fragments were
combined into one genome (red circle). The pieces were assembled using homologous recombination in yeast. Credit: Gibson, D.G., et al.,
2010. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 5256.
this they used the bacteria of the genus Mycoplasma, which has one of the smallest
bacterial genomes (just under 600,000 nucleotides in length). The genome from one
species of Mycoplasma was purified and then transformed into a cell of another
Mycoplasma species. The incoming chromosome was selected by antibiotic resis-
tance and displaced the resident chromosome. Technically, this “genome transplan-
tation” converted one species of the genus Mycoplasma to another.
The next step was to synthesize the whole genome of Mycoplasma chemically
and then insert it into a cell. This was done in several hierarchical stages. First,
about 100 segments of DNA of 50007000 bases long were chemically synthesized.
These had overlapping sequences at their ends that allowed them to be joined
together by recombination in E. coli. Assembly proceeded via units of 24, 72, and
144 kb (quarter genomes) all carried on bacterial artificial chromosomes. Final
assembly of the four quarters into a complete genome was performed in yeast. The
genome was then transplanted into a Mycoplasma host cell and selected as before.
Artificial “watermark” sequences were included in the artificially assembled genome
to verify its presence. Several variants of this procedure have been carried out. One
of the most recent versions (Fig. 28.24) contains the rather larger genome (1.08
megabases) of Mycoplasma mycoides chemically synthesized and modified to con-
tain “watermark” sequences including the authors’ names and famous quotes, one
being “What I cannot build, I cannot understand,” by the physicist Richard
Feynman. This cell has been nicknamed Synthia Venter!
The next issue to be tackled by whole genome assembly was the question of the
minimal genome. That is: What is the fewest number of genes needed for a self-
replicating cell? Starting from Mycoplasma mycoides JCVI-syn1.0 (as depicted in
Fig. 28.24), as many genes as possible were deleted. Genes were retained based on
two major factors. Firstly, knowledge of molecular biology provided a list of genes
needed to synthesize and process macromolecules. Secondly, exhaustive mutagenesis
of the Mycoplasma genome by transposon insertions revealed which genes are essen-
tial. A third generation construct, JCVI-syn3.0 had a genome smaller than any natu-
rally existing cell. JCVI-syn3.0 has 531 kbp of DNA with 473 genes (438 encoding
proteins and 35 encoding RNA). Note that Mycoplasma mycoides JCVI-syn3.0 is
grown in rich medium that supplies most small molecules needed for growth.
Consequently, many genes that would be essential for growth in minimal medium or
that would be needed to use growth substrates other than glucose have been deleted.
Of the 438 protein-encoding genes, 149 (31% of the genome!) are of unknown
function but were found to be essential by transposon mutagenesis. Many of these
genes have homologs in other organisms, implying that they encode proteins essen-
tial for a wide range of organisms, despite their presently unknown function.
Key Concepts
G In bacteria reproduction and sex are two distinct processes.

G Gene transfer between bacteria may occur by uptake of unprotected DNA,
movement of DNA inside virus particles, or specialized cell-to-cell DNA
transfer.
G DNA that enters a bacterial cell may survive on its own if it is a complete
replicon. Otherwise, it will be degraded unless it is recombined into the
host chromosome.
G Gene transfer by the uptake of unprotected or “naked” DNA is known as
transformation.
G The transfer of inherited characters by transformation was part of the origi-
nal proof that DNA (not protein) is the genetic material.
G Transformation occurs in certain bacteria under natural conditions.
Review Questions 923
G Gene transfer between bacteria by DNA in virus particles is known as

transduction.
G In generalized transduction, random fragments of bacterial DNA are car-
ried by virus particles.
G In specialized transduction, specific regions of the bacterial chromosome
are preferentially packaged in virus particles.
G Many plasmids can transfer themselves between bacterial cells by a process
known as conjugation.
G Transfer of chromosomal genes by plasmids requires integration of the plas-
mid into the bacterial chromosome.
G Mating pheromones secreted into the culture medium often regulate plas-
mid transfer between gram-positive bacteria.
G Conjugative transposons can both transpose between DNA molecules and
transfer themselves between gram-positive bacteria by conjugation.
G Gene transfer in Archaea is common but still poorly investigated. Both
plasmids and viruses exist that can transfer genes in these organisms.
G For most bacteria, genetic information has been gathered by sequencing the
whole genome.
G Genome islands are blocks of contiguous genes usually with a “foreign” origin
that perform some specialized function, such as virulence or biodegradation.
G Whole bacterial genomes have been chemically synthesized and successfully
inserted into bacterial cells.
Review Questions
1. What is the difference between reproduction and gene transfer
2. What are the major genetic differences between bacteria and higher organisms?
3. How does gene transfer in bacteria differ from that in higher organisms?
4. What are the three mechanisms of bacterial gene transfer? Briefly define each type of
transfer.
5. What are the three possible fates of incoming DNA fragments during gene transfer?
6. What is required for an incoming piece of DNA to survive without a recombination
event? What types of DNA molecules can do this?
7. What type of DNA must be incorporated into the chromosome in order to survive?
8. What are “competent” cells? What are two ways to make cells “competent”?
9. Describe Oswald Avery’s experiment to prove that DNA is the genetic material. Why
was his experiment not absolute proof that DNA is the genetic material?
10. What term is used to describe the uptake of naked viral DNA? What is the viral DNA
alone able to do?
11. What does the term “transformation” mean to a cancer specialist?
12. What is the purpose of competence pheromones? How and when do these compounds
work?
13. Compare and contrast the mechanisms of natural competence and artificially induced
competence.
14. How does Vibrio cholerae kill other bacterial cells and steal their DNA?
15. Describe transduction. How is transduction performed in a laboratory? What conditions
are required for transduction?
16. Describe the two types of transduction. Give examples of bacteriophage that perform
each type of transduction.
17. What is meant by the term “headful packaging”? For which type of transduction is
“headful packaging” essential?
18. What is attλ (att-lambda)? Between which genes and in which organism is this site
located?
19. What are the two major differences between generalized and specialized transduction
with regard to bacterial DNA mistakenly carried by bacteriophages?
20. Why do specialized transducing particles arise at extremely low frequencies?

21. What are helper phage and defective phage?
22. What is the term used to describe the ability of certain plasmids to move themselves
from one bacterial cell to another?
23. Give some examples of families of plasmids that have this ability.
24. What is a sex pilus and a conjugation bridge? How does plasmid DNA move from one
cell to another during conjugation?
25. How is the plasmid DNA replicated during transfer?
26. What are “tra genes” and where are they located?
27. Why cannot very small plasmids transfer by conjugation? What does mobilizable mean?
28. How can plasmids mediate the transfer of chromosomal genes by conjugation?
29. What are Hfr-strains? Why were they useful in the early days of bacterial genetics?
Why is it possible for gene transfer to be either clockwise or counterclockwise?
30. Why is the presence of insertion sequences on the F plasmid significant? What are F-
prime (or F’) plasmids?
31. What are the major structural differences between gram-negative and gram-positive
bacteria?
32. What usually promotes the transfer of plasmids between gram-positive bacteria?
33. What are conjugative transposons and how are they transferred?
34. What types of environments do Archaea often thrive in?
35. What are some of the problems associated with the development of genetic systems
within Archaea?
36. Compare and contrast the transformation of genetic material in Archaea versus
Eubacteria.
37. What are “pathogenicity islands”? What usually flanks these regions? What do these
flanking regions indicate?
38. What do differences in GC to AT ratios between “pathogenicity islands” and the rest of
the chromosome indicate? What else gives a similar indication?
39. Give an example of an extra segment of DNA that E. coli has that is missing in
Salmonella?
40. Why can “pathogenicity islands” best be described as “specialization islands”? What are
three properties that describe them?
41. What is horizontal or lateral gene transfer? By which mechanisms may this transfer
occur?
42. What is genome transplantation?
43. What was the first organism to have its genome completely synthesized? How was this
achieved?
Further Reading
Berry, J.L., Pelicic, V., 2015. Exceptionally widespread nanomachines composed of type IV
pilins: the prokaryotic Swiss Army knives. FEMS Microbiol Rev 39, 134154.
Borgeaud, S., Metzger, L.C., Scrignari, T., Blokesch, M., 2015. The type VI secretion system
of Vibrio cholerae fosters horizontal gene transfer. Science. 347, 6367.
Cook, L.C., Dunny, G.M., 2014. The Influence of Biofilms in the Biology of Plasmids.
Microbiol Spectr 2 (5).
Danchin, A., Fang, G., 2016. Unknown unknowns: essential genes in quest for function.
Microb Biotechnol. 9, 530540.
Hutchison 3rd, C.A., et al., 2016. Design and synthesis of a minimal bacterial genome.
Science. 351 (6280), aad6253.
Matthey, N., Blokesch, M., 2016. The DNA-Uptake Process of Naturally Competent Vibrio
cholerae. Trends Microbiol. 24, 98110.
Metzger, L.C., Blokesch, M., 2016. Regulation of competence-mediated horizontal gene
transfer in the natural habitat of Vibrio cholerae. Curr Opin Microbiol 30, 17.
Pohlschroder, M., Esquivel, R.N., 2015. Archaeal type IV pili and their involvement in
biofilm formation. Front Microbiol. 6, 190.
Suzuki, Y., et al., 2015. Bacterial genome reduction using the progressive clustering of
deletions via yeast sexual cycling. Genome Res. 25, 435444.
van Wolferen, M., Wagner, A., van der Does, C., Albers, S.V., 2016. The archaeal Ced system
imports DNA. Proc Natl Acad Sci U S A 113, 24962501.

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Bacterial Genetics

1. Reproduction Versus Gene

2. Fate of the Incoming DNA

1. Reproduction Versus Gene Transfer

Molecular Biology. DOI: https://doi.org/10.1016/B978-0-12-813288-3.00028-8

2. Fate of the Incoming DNA After Uptake

FIGURE 28.01 g homologous DNA

DNA unrelated FIGURE 28.02

DESTROY CELL ADD DNA TO

ORIGINAL FRAGMENTS TRANSFORMED RECOMBINANT

In practice, transformation is mostly a laboratory technique. The DNA is

3.1. Transformation As Proof That DNA Is the Genetic Material

INJECT INTO MOUSE

3.2. Transformation in Nature

Cell wall FIGURE 28.06

Precursor Dense cultures of Streptococcus

Competence is induced by competence pheromones. (A pheromone is a hormone

4. Gene Transfer by Virus—Transduction

4.1. Generalized Transduction

Long piece of FIGURE 28.07

Endonuclease A cell that is naturally competent

ADDITIONAL DNA CUTS MAKE

EXONUCLEASE DEGRADES ONE STRAND

For a bacterial virus to transduce, several conditions must be met. In particular,

4.2. Specialized Transduction

FIGURE 28.12 FORMATION OF MATING PAIRS

FORMATION OF A CONJUGATION BRIDGE

REPLICATION FIGURE 28.14

5.1. Transfer of Chromosomal Genes Requires Plasmid Integration

fertility plasmid Plasmid that enables a cell to donate DNA by conjugation.

Insertion sequences are scattered

throughout the F-plasmid and

F-plasmid plasmid has two IS3 elements and

the order a, b, c, d, e, f, in this

example). The amount of

chromosomal DNA that is ori T

0/100 0/100 F FIGURE 28.19

the wild-type version of gene “a”

several time points. The whole

procedure is then repeated for the

6. Gene Transfer Among Gram-Positive Bacteria

FIGURE 28.20 GRAM-NEGATIVE ENVELOPE

Mating FIGURE 28.21

8.1. Bacterial Genome Assembly and Transplantation

** Elements for yeast propagation

100,000 bp assemblies (11)

G In bacteria reproduction and sex are two distinct processes.

G Gene transfer between bacteria by DNA in virus particles is known as

20. Why do specialized transducing particles arise at extremely low frequencies?

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