BACTERIAL GENETICS

Introduction:•The properties of a living bacterial cell are determined by its metabolic reactions which are controlled by enzymic processes. •All the characteristics are ultimately controlled by the genetic material of the cell i.e. DNA.

In a bacterium e.g. E.coli the DNA is in the form of a single circular chromosome which is a big threadlike molecule consisting of two polynucleotide chains wound around a common long axis to form a double stranded helix.

Bacterial Chromosome: Its structure and Replication: The bacterial chromosome has a molecular weight of approximately 3 x 109 and is a closed circle of approximately 1 mm in circumference (Cairns)? Its structure was determined by the Nobel laurates Watson and Crick (1953). It is made up of a double helix of two complementary strands of polynucleotides that contain purine and pyrimidine bases arranged in a backbone of alternating deoxyribose and phosphate groups.

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The two strands are held together by hydrogen bonds that occur between a purine and a pyrimidine. The bonding is specifically between the pyrimidine thymine and purine adenine (A-T base pair), or between the pyrimidine cytosine and the purine guanine (G-C base pair).

Hence the sequence of bases on one strand always has a complementary sequence of bases on the opposite strand e.g. ATTATCCG will be complementary to TAATAGGC. E.coli has 4 x 106 base pairs.

Along the length of the chomosome, various segments of the DNA represent individual genes of which there are 3000-6000 in E-coli. (1,000 base pairs =1 Kilobase pair (Kbp). Thus E.coli has about 4000 kbp

Continued: The presence of complementary strands allows for faithful replication of the chromosome during division. The two strands separating at a particular origin of replication and each strand acting as its own template and a new strand being made from enzymatic polymerization of the deoxy-ribonucleotide subunits – dATP, dTTP, dCTP and dGTP. Because of the specificity of the bonding, the new strands will be the exact complements of the template strands and genetic information will be faithfully transmitted to the daughter cells. This mode of replication is referred to as semi conservative.

1st generation / / → / / / /

2nd generation / / / / / / / /

In order for each daughter cells to receive a copy of thechromosome at cell division, some control must be maintained over the replication of the chromosome and it has been postulated that the bacterial chromosome has some attachment point on the cell membrane so that replication can be synchronized with cell division. It has been suggested in a replicon model by Jacob & Brenmer (1963) that there is a specific “ori” locus point in the chromosome that is activated by an “initiator protein”. At 370C replication proceeds at 750 base pairs per sec per replication fork.

Translation of Genetic information from Chromosome and from Extra chromosomal DNA

Whereas all the genetic potential of the bacterial cell is contained in the base sequence of the DNA chromosome, many bacteria contain extra chromosomal DNA molecules which can confer additional genetic potential on a cell. In order for this information to be expressed in the cellular behaviours of the cell, the following processes occur:

(i) Transcription of the DNA base sequence into mRNA utilising enzyme RNA polymerase and making use of ribonucleotides (ATP, GTP, CTP and UTP) into a complementary strand of RNA;

e.g. DNA sequence AGTC gives rise to mRNA sequence UCAG. Each discrete DNA segment will specify an RNA message that will

eventually be translated into a protein molecule. The DNA segment is referred to as a gene coding for one protein.

(II) Translation of mRNA message into protein by protein synthesitizing cell machinery including activated amino acids, tRNA & ribosomes.

-tRNA with attached activated amino acid comes on the mRNA on the surface of the ribosome.

-Each tRNA molecule finds its complementary triplet on the mRNA and the amino acid it carries is put into a peptide linkage with the amino acids of the preceding tRNA molecule.

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As the ribosome moves along the mRNA, the peptide grows by the addition of an additional amino acid until the complete mRNA message has been read.

Each three bases in the DNA or mRNA (a codon) specifies a particular amino acid.

VARIATION OF BACTERIAL PROPERTIES

Properties of a bacterium at a particular time (phenotype) depend on its genetic constitution (its genotype) and on environmental factors.

Genotypic Variation

- Completely new properties may arise by mutation but new heritable properties may also be acquired by transfer of genes from other organisms.

Bacterial Sexual Processes

• Eukaryotes have the processes of meiosis to reduce diploids to haploidy, and fertilization to return the cells to the diploid state.

• Bacterial sexual processes are not so regular. • However, they serve the same aim: to mix the genes

from two different organisms together.• The three bacterial sexual processes:

– 1. conjugation: direct transfer of DNA from one bacterial cell to another.

– 2. transduction: use of a bacteriophage (bacterial virus) to transfer DNA between cells.

– 3. transformation: naked DNA is taken up from the environment by bacterial cells.

Mutation:The progeny of a single bacterial cell are not genetically homogeneous but contain a definite proportion of cells which differ from the parent in some heritable property. Mutation is a change in the base sequence of the DNA double helix.

In many such cases, such a base change will result in a change which will result in an altered amino acid sequence of a protein that will in turn alter the normal functioning of that protein.

Such a change will be propagated when the DNA is replicated.Mutation rates vary with different properties and different bacteria but is commonly between 1 in 107 and 1 in 1010

for a single property.

Bacterial Mutants• Mutants in bacteria are mostly biochemical in nature, because we can’t generally see

the cells.

• The most important mutants are auxotrophs. • An auxotroph needs some nutrient that the wild type strain (prototroph) can make for

itself. For example, a trp- auxotroph can’t make its own tryptophan (an amino acid). To grow trp- bacteria, you need to add tryptophan to the growth medium. Prototrophs are trp+; they don’t need any tryptophan supplied since they make their own.

• Chemoauxotrophs are mutants that can’t use some nutrient (usually a sugar) that prototrophs can use as food. For example, lac- mutants can’t grow on lactose (milk sugar), but lac+ prototrophs can grow on lactose.

• Resistance mutants confer resistance to some environmental toxin: drugs, heavy metals, bacteriophages, etc. For instance, AmpR causes bacteria to be resistant to ampicillin, a common antibiotic related to penicillin.

• Auxotrophs and chemoauxotrophs are usually recessive; drug resistance mutants are usually dominant.

Replica Plating• A common way to find bacterial mutants is replica plating, which

means making two identical copies of the colonies on a petri plate under different conditions.

• For instance, if you were looking for trp- auxotrophs, one plate would contain added tryptophan and the other plate would not have any tryptophan in it.

• Bacteria are first spread on the permissive plate, the plate that allows both mutants and wild type to grow, the plate containing tryptophan in this case. They are allowed to grow for a while, then a copy of the plate is made by pressing a piece of velvet onto the surface of the plate, then moving it to a fresh plate with the restrictive condition (no tryptophan). The velvet transfers some cells from each colony to an identical position on the restrictive plate.

• Colonies that grow on the permissive plate but not the restrictive plate are (probably) trp- auxotrophs, because they can only grow if tryptophan is supplied.

Replica Plating, pt. 2

Continued:Total mutants involving a variety of properties is far greater for each replicating bacterial strain.

Rates of mutation can be increased by e.g. X-rays, U-V light, alkylating agents etc.

Chemicals that are carcinogenic for man damage bacterial DNA

leading to mutations.

Bacterial mutagenesis tests are used in screening for carcinogens in drugs, vaccines and other pharmaceutics.

Bacterial mutations may be due to :

(i) Substitution of base pairs(ii) Deletion of bases

or(iii) Insertion of new bases

continued:S-R (smooth-rough) variation is one of most obvious mutations seen in bacteria.

It involves change of colonial appearance from smooth to rough, loss of surface components (somatic & capsular antigens) and a loss or diminution of virulence.

It commonly occurs when bacteria are grown for long periods on artificial media.

This variation can be reversed by repeated passaging the culture through susceptible animals.

S-R variation is best shown in salmonella, shigella and pnemococcus but similar changes occur in most genera although the colonial changes are not so obvious.

Other mutations involve changes in:(i) Cell morphology (size, ability to form spores, capsules and flagella),

(ii) Colonial appearance (size, shape, pigmentation,)

(iii) Biochemical activity (nutritional requirements, fermentative power, enzymic activity)

(iv) antigenic properties (flagella variation,

loss of antigens)

(v) Toxigenicity

(vi) Sensitivity to phages

(vii) Drug sensitivity

Extra chromosomal inheritance:

Many bacteria posses genetic elements which replicate and function independently of the main chromosome.

These are known as plasmids.

They consist of closed circular molecules of DNA commonly 1-2% of the size of the chromosome.

The information they carry is often in the form of transposable genetic elements (transposons).

These are discrete segments of DNA, usually containing several genes with property of being readily transferable into the DNA of the bacterial chromosomes, plasmids and infecting phages.

Genetic recombination resulting from the transfer of transposon DNA can occur through conjugation, transformation and transduction.

Plasmids may be transmissible by cellular conjugation or non-transmissible

Transmissible plasmids:• Mainly found in intestinal bacteria ; their activity include formation of

fine, hair like projections (sex pili) from the donor cell surface. • DNA is transmitted through such pili by conjugation, a process

resembling sexual reproduction. • Transmissible plasmids are classified according to the property by

which they were first recognized including:(a) Fertility (F) plasmids (F+ - F-)

Transmissible plasmids contd:(b) Colicinogenic (Col)(c ) Drug-resistance transfer (R ) (d) Others – fermentative

- resistance to bactericidal action of serum antigen- toxin or surface antigen production- adherence to surface of animal cells

Transformation:• With some bacterial species, it is possible to transfer

properties by means of purified DNA obtained from another culture of the same species of bacterium.

• The DNA of the donor strain is incorporated in the recipient strain where it functions genetically e.g. R→S transformation in pneumococci.

Transformation contd

• Transformation is very important for recombinant DNA work.

• The essence of recombinant DNA technology is to remove DNA from cells, manipulate it in the test tube, then put it back into living cells.

• In most cases this is done by transformation. • In the case of E. coli, cells are made “competent” to be

transformed by treatment with calcium ions and heat shock.

• E. coli cells in this condition readily pick up DNA from their surroundings and incorporate it into their genomes.

• Transformation of pneumococci was first described by Griffith in 1928; before then the mechanism was a mystery.

• Drug resistance can be transferred by transformation.

Transduction:• Occurs when a phage incorporates part of the genetic

material of a host bacterium and carries it to another bacterial cell usually of the same species.

• Bacterial antibiotic resistance, antigenic & biochemical properties can be transduced between closely related strains of salmonellae.

Transduction contd

• Transduction is the process of moving bacterial DNA from one cell to another using a bacteriophage.

• Bacteriophage or just “phage” are bacterial viruses. They consist of a small piece of DNA inside a protein coat.

• The protein coat binds to the bacterial surface, then injects the phage DNA.

• The phage DNA then takes over the cell’s machinery and replicates many virus particles.

• Two forms of transduction:– 1. generalized: any piece of the bacterial genome can be

transferred– 2. specialized: only specific pieces of the chromosome can be

transferred.

General Phage Life Cycle

• 1. Phage attaches to the cell and injects its DNA.

• 2. Phage DNA replicates, and is transcribed into RNA, then translated into new phage proteins.

• 3. New phage particles are assembled.

• 4. Cell is lysed, releasing about 200 new phage particles.

• Total time = about 15 minutes.

Generalized Transduction

• Some phages, such as phage P1, break up the bacterial chromosome into small pieces, and then package it into some phage particles instead of their own DNA.

• These chromosomal pieces are quite small: about 1 1/2 minutes of the E. coli chromosome, which has a total length of 100 minutes.

• A phage containing E. coli DNA can infect a fresh host, because the binding to the cell surface and injection of DNA is caused by the phage proteins.

• After infection by such a phage, the cell contains an exogenote (linear DNA injected by the phage) and an endogenote (circular DNA that is the host’s chromosome).

• A double crossover event puts the exogenote’s genes onto the chromosome, allowing them to be propagated.

Transduction Mapping

• Only a small amount of chromosome, a few genes, can be transferred by transduction.

• The closer 2 genes are to each other, the more likely

they are to be transduced by the same phage. • Thus, “co-transduction frequency” is the key parameter

used in mapping genes by transduction.

• Transduction mapping is for fine-scale mapping only. • Conjugation mapping is used for mapping the major

features of the entire chromosome.

Specialized Transduction

• Some phages can transfer only particular genes to other bacteria.

• Phage lambda (λ) has this property. To understand specialized transduction, we need to examine the phage lambda life cycle.

• lambda has 2 distinct phases of its life cycle. The “lytic” phase is the same as we saw with the general phage life cycle: the phage infects the cell, makes more copies of itself, then lyses the cell to release the new phage.

Lysogenic Phase• The “lysogenic” phase of the lambda life cycle starts the same way:

the lambda phage binds to the bacterial cell and injects its DNA. Once inside the cell, the lambda DNA circularizes, then incorporates into the bacterial chromosome by a crossover, similar to the conversion of an F plasmid into an Hfr.

• Once incorporated into the chromosome, the lambda DNA becomes quiescent: its genes are not expressed and it remains a passive element on the chromosome, being replicated along with the rest of the chromosome. The lambda DNA in this condition is called the “prophage”.

• After many generations of the cell, conditions might get harsh. For lambda, bad conditions are signaled when DNA damage occurs.

• When the lambda prophage receives the DNA damage signal, it loops out and has a crossover, removing itself from the chromosome. Then the lambda genes become active and it goes into the lytic phase, reproducing itself, then lysing the cell.

More Lysogenic Phase

Conjugation• Conjugation is the closest analogue in

bacteria to eukaryotic sex.• The ability to conjugate is conferred by

the F plasmid. • A plasmid is a small circle of DNA that

replicates independently of the chromosome. Bacterial cells that contain an F plasmid are called “F+”. Bacteria that don’t have an F plasmid are called “F-”.

• F+ cells grow special tubes called “sex pilli” from their bodies. When an F+ cell bumps into an F- cell, the sex pilli hold them together, and a copy of the F plasmid is transferred from the F+ to the F-. Now both cells are F+.

• Why aren’t all E. coli F+, if it spreads like that? Because the F plasmid can be spontaneously lost.

Hfr Conjugation• When it exists as a free

plasmid, the F plasmid can only transfer itself. This isn’t all that useful for genetics.

• However, sometimes the F plasmid can become incorporated into the bacterial chromosome, by a crossover between the F plasmid and the chromosome. The resulting bacterial cell is called an “Hfr”, which stands for “High frequency of recombination”.

• Hfr bacteria conjugate just like F+ do, but they drag a copy of the entire chromosome into the F- cell.

Intracellular Events in Conjugation

• The piece of chromosome that enters the F- from the Hfr is linear. It is called the “exogenote”.

• The F- cell’s own chromosome is circular. It is called the “endogenote”.

• Only circular DNA replicates in bacteria, so genes on the exogenote must be transferred to the endogenote for the F- to propagate them.

• This is done by recombination: 2 crossovers between homologous regions of the exogenote and the endogenote. In the absence of recombination, conjugation is ineffective: the exogenote enters the F-, but all the genes on it are lost as the bacterial cell reproduces.

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Recombinant DNA techniques (Genetic engineering)

Engineering is:

– Is the utilization of science to social requirements.

– Bacterial genetic engineering has transformed biology.

Definition:

Recombinant DNA techniques are artificial techniques which molecular biologists utilise to bring about genetic changes in bacteria by using restriction enzymes which cleave DNA at defined oligonucleotide sequences to create restriction fragments which can be bound on plasmid “vectors” before insertion to other bacterial cells.

• With such techniques, it is possible to take a length of DNA from a bacterium, virus or animal cell and introduce it into the genetic apparatus of a living bacterium where

Continued:it will replicate and instruct the cell to produce a specific protein.

The new bacterial cells are known as recombinants and the methods used to create them are referred to collectively as recombinant – DNA techniques or simply as cloning.This ability is due to presence of insertion sequences which are DNA segments with specific base sequences (800-1400) which allow for recombination i.e. movable from one position in a segment genome to another on the same or different genomes.

Applications of recombinant-DNA techniques include:Designing and creation of novel strains of E.coli capable of manufacturing non bacterial proteins e.g.

– the envelope antigen of hepatitis B virus (vaccine)– the haemagglutinin of influenza virus (vaccine)– human growth hormone,– human insulin

and – human interferon

Phenotypic variation:– Is non-heritable variation representing a temporary adjustment to

the environment, normally involves the cell population as a whole.

– Such organisms show variation in size, shape, staining reactions, metabolism & susceptibility to drugs at different phases of their growth cycle.

– The variation in properties may be induced by environment e.g. – S.aureus makes its golden pigment most abundantly at room temperature and not at 37oC.

– S. aureus produces penicillinase only when exposed to penicillin.

– Conversely, some bacteria may fail to synthesize certain enzymes when the end-product of that pathway is present in the culture medium (enzyme repression).

In all above, variation reversion to normal type occurs when the environmental stimulus is removed.