Cytogenetics

Finals: The Genetics of Bacteria and Their Viruses

Bacteria and their viruses (bacteriophage) have unique and diverse reproductive
systems with multiple and novel mechanisms of genetic exchange.
A high percentage of bacteria isolated from clinical infections are resistant to one or
more antibiotics. The widespread antibiotic- resistance genes almost never originate
from new mutations in the bacterial genome, but they are actually acquired, usually
several at a time, in various forms of Mobile DNA
(sequences in bacteria which are mobile and can be transferred between DNA
molecules and from one cell to another, between individuals and among species).
Plasmids- nonessential DNA molecules that exist inside bacterial cells. They
replicate independently of the bacterial genome and segregate to the progeny when
a bacterial cell divides, so they can be maintained indefinitely in a bacterial lineage.
Many are circular DNA molecules but others are linear. They range in size from a few
kilobases to a few hundred kilobases.
The presence of plasmids can be detected physically by electron microscopy or by
gel electrophoresis of DNA samples. Some can be detected because of phenotypic
characteristics that they confer on the host cell (like antibiotic resistance).

Fig. 7.1

Plasmids rely on the DNA- replication enzymes of the host cell for their reproduction,
but the initiation of replication is controlled by plasmid genes.

High- copy-number plasmids- found in as many as 50 copies per host cell.

Replication is initiated multiple times during replication of the host genome.
Low-copy-number plasmids- present in 1 to 2 copies per cell. Replication is initiated
only once per round of replication of the host genome.
Pilus (plural: pili)- a tube-like structure formed between the cells through which the
plasmid DNA passes as they are being transferred between cells.
Conjugation- the joining of bacterial cells in the transfer process. Plasmids that can
be transferred in this manner are called conjugative plasmids. Not all plasmids
are conjugative.
Most small plasmids
are nonconjugative. They can be
maintained in a bacterial lineage as the cells divide, but they do not contain the
approximately 20 genes necessary for pilus assembly or those for DNA transfer.
Hence they are unable to be transferred on their own.
Fig. 7.2

The pilus between the E. coli cells above is an F pilus whose synthesis results from
the presence of a conjugative plasmid called the F factor (F stands for fertility).
Cells that contain the F plasmid (low-copy number plasmid) are donors and are
designated the F+ cells (F plus); those lacking F are recipients and are designated
the F- cells (F minus.)
Conjugation begins with physical contact between a donor cell and a recipient cell.
Once the pilus contacts the F- cell, the pilus retracts and the cell membranes of the
donor and recipient are brought into close proximity. Then the donor DNA moves
through a pore in the membrane from the donor to the recipient. The transfer is
always accompanied by replication of the plasmid. Contact between an F+ and an
F- cell initiates rolling circle replication which results in the transfer of a singlestranded linear branch of the rolling circle to the recipient cell. During transfer, DNA
is synthesized in both donor and recipient. When transfer is complete, the linear F
strand becomes circular again in the recipient cell. After the transfer, both cells
contain F and can function as donors. The F- cell has been converted into an F+ cell.

Fig. 7.3

Transposable elements are DNA sequences that can jump from one position to
another or from one DNA molecule to another. Bacteria contain a wide variety of
transposable elements. The smallest and simplest are insertion sequences or IS
elements, which are typically 1-3 kb in length and usually encode only the
transposase protein required for transposition and one or more additional proteins
that regulate the rate of transposition.
Other transposable elements in bacteria contain one or more genes unrelated to
transposition that can be mobilized along with the transposable element; this type
of element is called a transposon. Much of the widespread antibiotic resistance
among bacteria is due to the spread of transposons that include one or more
antibiotic- resistance genes. When a transposon mobilizes and inserts into a
conjugative plasmid, it can be widely disseminated among different bacterial hosts
by means of conjugation.
Some transposons have composite structures with antibiotic resistance sandwiched
between insertion sequences, as is the case with the Tn5 element (illustration
below), which terminates in two IS 50 elements in inverted orientation. Transposons
are designated by the abbreviation Tn followed by an italicized number (ex. Tn5).
For example, Tn5( neo- r ble-r str-r) contains genes for resistance to three different
antibiotics: Neomycin, Bleomycin and Streptomycin.

Fig. 7.4

Nonconjugative and conjugative plasmids typically coexist in the same cell along
with host genomic DNA, and when a transposable element is mobilized, all of the
DNA molecules present are potential targets for insertion. Many nonconjugative and
conjugative plasmids present in a bacterial cell come to carry one or more copies of
the same transposable element. Because these copies are homologous DNA
sequences, they can serve as substrates for recombination. When 2 plasmids
undergo recombination in a region of homology, the result is as shown below. The
recombination forms a composite plasmid called a cointegrate. By this
mechanism, nonconjugative plasmids can temporarily ride along with conjugative
plasmids and be transferred from cell to cell.
Fig. 7.5

In the evolution of multiple antibiotic resistance, bacteria have also made liberal use
of a set of enzymes known as Site- specific recombinases which were present in
bacterial populations and functioned in the evolution of other traits long before the
antibiotic era. Each type of site specific recombinase binds with a specific
nucleotide sequence in duplex DNA. When the site is present in each of two duplex

DNA molecules, the recombinase brings the sites together and catalyzes a
reciprocal exchange between the duplexes. Site specific recombinases are used in
the assembly of multiple antibiotic-resistance units called integrons. An integron
is a DNA element that encodes a site- specific recombinase as well as a recognition
region that allows other sequences with similar recognition regions to be
incorporated into the integron by recombination. The elements that integrons
acquire are known as cassettes. In the

context of integrons, a cassette is a circular antibiotic- resistance- coding region

flanked by a recognition region for an integron. Because the site specific
recombinase integrates cassettes, the integron recombinase is usually called an
integrase.
Fig. 7.7

In nature, a conjugative plasmid can accumulate different transposons containing

multiple independent antibiotic- resistance genes, or transposons containing
integrons that have acquired multiple antibiotic- resistance cassettes with the result
that the plasmid confers resistance to a large number of completely unrelated
antibiotics. These multiple resistance plasmids are called R plasmids. The
evolution of R plasmids is promoted by the use of and overuse of antibiotics which
selects for resistant cells because in the presence of antibiotics, resistant cells have
a growth advantage over the sensitive cells. The presence of multiple antibiotics in
the environment selects for multiple drug resistance. Serious complications result
when plasmids resistant to multiple drugs are transferred to bacterial pathogens or
agents of disease.
Transduction- bacterial DNA is transferred from one bacterial cell to another by a
phage particle containing the DNA. Such a particle is called a transducing phage.
Two types of transducing phages are known:
1. Generalized transducing phage- produces some particles that contain only
DNA obtained from the host bacterium, rather than phage DNA; the bacterial
DNA fragments can be derived from any part of the bacterial chromosome.
2. Specialized transducing phage- produces particles that contain both phage
and bacterial genes linked in a single DNA molecule, but the bacterial genes
are obtained from a particular region of the bacterial chromosome.
Bacteriophage life cycles:
Lytic cycle- the reproductive cycle of a phage. Phage DNA enters a cell and
replicates repeatedly, bacterial ribosomes are used to produce phage protein
components, the newly synthesized phage DNA molecules are packaged into
protein shells to form progeny phage, and the bacterium is split open (lysis),
releasing the progeny phages from the cell.
Lysogenic cycle- The alternative to the lytic cycle. No progeny particles are
produced, the infected bacterium survives, and a phage DNA molecule is
transmitted to each bacterial progeny cell when the cell divides. All phage species
can undergo a lytic cycle. Those phages that are also capable of the lysogenic cycle
are called temperate phage , and those capable of only the lytic cycle are called
virulent phage. In the lysogenic cycle, a replica of the infecting phage DNA becomes
inserted, or integrated into the bacterial chromosome. The inserted DNA is called a
prophage, and the surviving bacterial cell is called a lysogen.