Microbial Genetics

Microorganisms have the ability to acquire genes from other organisms and thereby undergo the process

of recombination. In recombination, a new chromosome with a genotype different from that of the parent

results from the combination of genetic material from two organisms. This new arrangement of genes is

usually provides new chemical or physical properties to the organism which undergoes recombination.

Plasmids.

Prokaryotes such as bacteria possess a single chromosome composed of double‐stranded DNA in a loop. The

DNA is located in the nucleoid of the cell and is not associated with protein. In Escherichia coli, the length of

the chromosome, when open, is many times the length of the cell.

Many bacteria (and some yeasts or other fungi) also possess circular DNA in the cytoplasm known as plasmids,

which exist and replicate independently of the nucleoid chromosome. Plasmids have relatively few genes (fewer

than 30). The genetic information of the plasmid is usually not essential to survival of the host bacteria but gives

additional properties like sex pili formation, antibiotic production, antibiotic resistance, pathogenecity, toxin

production and etc.

Certain plasmids, called episomes, may be integrated into the bacterial chromosome. Others contain genes for

certain types of pili and are able to transfer copies of themselves to other bacteria. Such plasmids are referred to

as conjugative plasmids.

A special plasmid called a fertility (F) factor plays an important role in conjugation. The F factor contains

genes that encourage cellular attachment during conjugation and accelerate plasmid transfer between

conjugating bacterial cells. Those cells contributing DNA are called F+ (donor) cells , while those receiving

DNA are the F- (recipient) cells. The F factor can exist outside the bacterial chromosome or may be integrated

into the chromosome.

Plasmids contain genes that impart antibiotic resistance. Up to eight genes for resisting eight different

antibiotics may be found on a single plasmid. Genes that encode a series of bacteriocins are also found on
plasmids. Bacteriocins are bacterial proteins capable of destroying other bacteria. Still other plasmids increase

the pathogenicity of their host bacteria because the plasmid contains genes for toxin synthesis.

Plasmids are extremely valuable tools in the fields of molecular biology and genetics, specifically in the area

of genetic engineering. They play a critical role in such procedures as gene cloning,

recombinant protein production (e.g., of human insulin), and gene therapy research. In such procedures, a

plasmid is cut at a specific site (or sites) using enzymes called restriction endonucleases. A foreign DNA

element (such as the gene for insulin) is then attached into the plasmid. The resulting circular structure,

a recombinant DNA molecule, is then introduced into bacterial cells. The autonomous replication of the plasmid

within the bacterial cells makes it possible to produce large numbers of copies of the recombinant DNA

molecule for experimental manipulation or commercial purposes (such as the production of large amounts of

insulin). Plasmids are well suited to genetic engineering in other ways. Their antibiotic resistance genes, for

example, prove useful in identifying those bacterial cells that have taken up the recombinant DNA molecule in a

high background of untransformed cells. Plasmids are used to deliver the desired gene into the genetically

defective cells to treat the genetic disorders in gene therapy technique.

Transposons

Transposons ("jumping genes") encode enzymes that enable the transposon to move from one DNA location to

another, either on the same molecule of DNA or on a different molecule. Transposons may be found as part of a

bacterium's chromosome (conjugative transposons) or in plasmids and are usually between one and twelve

genes long. A transposon contains a number of genes, such as those coding for antibiotic resistance or other

traits, flanked at both ends by insertion sequences coding for an enzyme called transpoase. Transpoase is the

enzyme that catalyzes the cutting and resealing of the DNA during transposition.
Conjugative transposons, like conjugative plasmids, carry the genes that enable mating pairs to form for

conjugation. Therefore, conjugative transposons also enable mobilizable plasmids and nonconjugative

transposons to be transferred to a recipient bacterium during conjugation.

Many conjugative plasmids and conjugative transposons possess rather promiscuous transfer systems that

enables them to transfer DNA not only to related species, but also to unrelated species. The ability of bacteria to

adapt to new environments as a part of bacterial evolution most frequently results from the acquisition of large

DNA sequences from another bacterium by conjugation.

Bacterial transposable elements are of the following types:

(a) Insertion Sequences or IS Elements:

They are the transposable sequences which can insert at different sites in the bacterial chromosomes. IS

elements are relatively short usually not exceeding 2500 bp. IS-elements contain ITRs (Inverted Terminal

Repeats or Inverted Repeats), these were first observed in [Link]. The ITRs present at the ends of IS-elements

are an important feature which enables their mobility. The ITRs present in the IS-elements of [Link] usually

range between 18-40 bp.

The term ‘Inverted Terminal Repeat’ (ITR) implies that the sequence at 5 end of one strand is identical to the

sequence at 5′ end of the other strand but they run in inverse opposite direction. In [Link] chromosome, a

number of copies of several IS-elements like IS1, IS2, IS3, IS4 and IS5 are present.

(b) Composite transposons:

These are shown by the symbol Tn. It is made up of two IS elements, one present at each end of a DNA

sequence which contains genes whose functions are not related to the transposition process. These transposons

have been found to have inverted repeats at the ends. The length of these inverted repeats ranges from a few

nucleotides to about 1500 bp.

within two insertion sequences thus enabling it to move. Examples of such transposons include the members of

Tn series like Tn1, Tn5, Tn9, Tn10, etc.

(C) Tn3 Elements

Tn3 elements are simply large transposable elements that are not generated by flanking IS elements (as in Tn

elements). They are generally ~5,000 bp, have ~386 bp inverted repeats at both ends, and carry antibiotic

resistant genes.
 Bacterial Recombinations

In microorganisms, several kinds of recombination are known to occur. The most common forms are bacterial

transformation, bacterial Conjugation and bacterial transduction.

Transformation

Transformation is a form of genetic recombination in which a DNA fragment from a dead, degraded bacterium

enters a competent recipient bacterium and is exchanged for a piece of DNA of the recipient. Transformation

usually involves only homologous recombination, a recombination of homologous DNA regions having nearly

the same nucleotide sequences. Typically this involves similar bacterial strains or strains of the same bacterial

species.

A few bacteria, such as Neisseria gonorrhoeae, Neisseria meningitidis, Hemophilus influenzae, Legionella

pneomophila, Streptococcus pneumoniae, and Helicobacter pylori tend to be naturally competent and

transformable. Competent bacteria are able to bind much more DNA than noncompetent bacteria. Some of these

genera also undergo autolysis that then provides DNA for homologous recombination. In addition, some

competent bacteria kill noncompetent cells to release DNA for transformation.

bacterium and bind to DNA binding proteins on the surface of a competent living revipient bacterium.

Depending on the bacterium, either both strands of DNA penetrate the recipient bacterium or only one strand

penetrates another strand will be degraded by nuclease. This DNA fragment from the donor is then exchanged

for a piece of the recipient's DNA by means of RecA proteins and other molecules and involves breakage and

reunion of the paired DNA segments.

Transduction

Transduction involves the transfer of a DNA fragment from one bacterium to another by a bacteriophage. There

are two forms of transduction: generalized transduction and specialized transduction.

During the replication of lytic bacteriophages and temperate bacteriophages, occasionally the phage capsid

accidently assembles around a small fragment of bacterial DNA. When this bacteriophage, called a transducing

particle, infects another bacterium, it injects the fragment of donor bacterial DNA it is carrying into the

recipient where it can subsequently be exchanged for a piece of the recipient's DNA by homologous

recombination. Generalized transduction is summarized as follows.

 Step 1: A bacteriophage adsorbs to a susceptible bacterium.

 Step 2: The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic

machinery to manufacture bacteriophage components and enzymes. Bacteriophage-coded enzymes will

also breakup the bacterial chromosome.

 Step 3: Occasionally, a bacteriophage capsid mistakenly assembles around either a fragment of the

donor bacterium's chromosome or around a plasmid instead of around a phage genome.

 Step 4: The bacteriophages are released as the bacterium is lysed. Note that one bacteriophage is

carrying a fragment of the donor bacterium's DNA rather than a bacteriophage genome.

 Step 5: The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium.

 Step 6: The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium.
  Step 7: Homologous recombination occurs and the donor bacterium's DNA is exchanged for some of

the recipient's DNA.

Generalized transduction occurs in a variety of bacteria, including Staphylococcus, Escherichia, Salmonella,

and Pseudomonas. Plasmids, such as the penicillinase plasmid of Staphylococcus aureus, may also be carried

from one bacterium to another by generalized transduction.

Specialized transduction: This may occur occasionally during the lysogenic life cycle of a temperate

bacteriophage. During spontaneous induction, a small piece of bacterial DNA may sometimes be exchanged for

a piece of the bacteriophage genome, which remains in the bacterial nucleoid. This piece of bacterial DNA

replicates as a part of the bacteriophage genome and is put into each phage capsid. The bacteriophages are
released, adsorb to recipient bacteria, and inject the donor bacterium DNA and phage DNA complex into the

recipient bacterium where it inserts into the bacterial chromosome.

Genetic recombination in which there is a transfer of DNA from a living donor bacterium to a living recipient

bacterium by cell-to-cell contact. In Gram-negative bacteria it typically involves a conjugation or sex pilus.

Conjugation is encoded by plasmids or transposons. It involves a donor bacterium that contains a conjugative

plasmid and a recipient cell that does not. A conjugative plasmid is self-transmissible, in that it possesses all the

necessary genes for that plasmid to transmit itself to another bacterium by conjugation. Conjugation genes

known as tra genes enable the bacterium to form a mating pair with another organism, while oriT (origin of

transfer) sequences determine where on the plasmid DNA transfer is initiated by serving as the replication start

site where DNA replication enzymes will nick the DNA to initiate DNA replication and transfer. In addition,

mobilizable plasmids that lack the tra genes for self-transmissibility but possess the oriT sequences for

initiation of DNA transfer may also be transferred by conjugation if the bacterium containing them also

possesses a conjugative plasmid. The tra genes of the conjugative plasmid enable a mating pair to form, while

the oriT of the mobilizable plasmid enable the DNA to moves through the conjugative bridge.
Mobilizable plasmids, that lack the tra genes for self-transmissibility but possess the oriT sequences for

initiation of DNA transfer, may also be transferred by conjugation if the bacterium containing them also

possesses a conjugative plasmid. The tra genes of the conjugative plasmid enable a mating pair to form while

the oriT quences of the mobilizable plasmid enables the DNA to move through the conjugative bridge.

a. General mechanism of transfer of conjugative plasmids by conjugation in Gram-negative bacteria

In Gram-negative bacteria, the first step in conjugation involves a conjugation pilus (sex pilus or F pilus) on the

donor bacterium binding to a recipient bacterium lacking a conjugation pilus. Typically the conjugation pilus

retracts or depolymerizes pulling the two bacteria together. A series of membrane proteins coded for by the

conjugative plasmid then forms a bridge and an opening between the two bacteria, now called a mating pair.

Using the rolling circle model of DNA replication, a nuclease breaks one strand of the plasmid DNA at the

origin of transfer site (oriT) of the plasmid and that nicked strand enters the recipient bacterium. The other

strand remains behind in the donor cell. Both the donor and the recipient plasmid strands then make a

complementary copy of themselves. Both bacteria now possess the conjugative plasmid.

This is the mechanism by which resistance plasmids (R-plasmids), coding for multiple antibiotic resistance and

conjugation pilus formation, are transferred from a donor bacterium to a recipient. This is a big problem in

treating opportunistic Gram-negative infections such as urinary tract infections, wound infections, pneumonia,

and septicemia by such organisms as E. coli, Proteus, Klebsiella, Enterobacter, Serratia, and Pseudomonas, as

well as with intestinal infections by organisms like Salmonella and Shigella.

There is also evidence that the conjugation pilus may also serve as a direct channel through which single-

stranded DNA may be transferred during conjugation.

This results in the transfer of an F+ plasmid possessing tra genes coding only for a conjugation pilus and mating

pair formation from a donor bacterium to a recipient bacterium. One strand of the F+ plasmid is broken with a

nuclease at the origin of transfer (oriT) sequence that determines where on the plasmid DNA transfer is initiated

by serving as the replication start site where DNA replication enzymes will nick the DNA to initiate DNA

replication and transfer. The nicked strand enters the recipient bacterium while the other plasmid strand remains

in the donor. Each strand then makes a complementary copy. The recipient then becomes an F + male and can

make a sex pilus .

In addition, mobilizable plasmids that lack the tra genes for self-transmissibility but possess the oriT sequences

for initiation of DNA transfer, may also be transferred by conjugation. The tra genes of the F+ plasmid enable a

mating pair to form and the oriT sequences of the mobilizable plasmid enable the DNA to moves through the

conjugative bridge.

c. Hfr (high frequency recombinant) conjugation

Hfr conjugation begins when an F+ plasmid with tra genes coding for mating pair formation inserts or integrates

into the chromosome to form an Hfr bacterium. (A plasmid that is able to integrate into the host nucleoid is

called an episome.) A nuclease then breaks one strand of the donor's DNA at the origin of transfer (oriT)

location of the inserted F+ plasmid and the nicked strand of the donor DNA begins to enter the recipient

bacterium. The remaining non-nicked DNA strand remains in the donor and makes a complementary copy of

itself.

The bacterial connection usually breaks before the transfer of the entire chromosome is completed so the

remainder of the F+ plasmid seldom enters the recipient. As a result, there is a transfer of some chromosomal

DNA, which may be exchanged for a piece of the recipient's DNA through homologous recombination, but not

the ability to form a conjugation pilus and mating pairs.