Introduction to bacterial genetics

Microbial genetics

• Is the study of the inheritance of the characteristics of a microbial cell and how these characteristics can vary

• all phenotypes are encoded in the genome - including virulence factors, what media it grown on, lab tests

• Is used to engineer strains of microbes which have desirable characteristics - bacteria is often used in industrial fermentation to produce useful products. below is example of bacteria cell being engineered to produce human insulin gene - easy to grow to large levels due to fast replication

• Helps us understand how microbes become resistant to antimicrobial agents and how to prevent such resistance emerging

The genetic material of bacteria

• Exists in the BACTERIAL CHROMOSOME which is separate from the cytoplasm but not bound by a nuclear membrane

• Exists in PLASMIDS which are transient circular genetic elements in the cytoplasm - extra chromosomes which are a lot smaller and don’t usually carry essential genes but instead beneficial genes e.g. genes helping with ABR, or a gene allowing virulence

DNA

• Chemically, genetic information is carried by the nucleic acids: DNA and RNA (ribonucleic acid)

• RNA is the intermediate molecule that converts RNA and genetic information to amino acids

• The monomers of nucleic acids (DNA and RNA) are  nucleotides:

Pentose sugar – ribose or 2’deoxyribose

Nitrogen base - ADTG

Molecule of phosphate (PO₄^3-) - sugar phosphate backbone

Bases complement each other:

• Purine with pyrimidine

• Adenine – Thymine - 2 H BONDS

• Guanine – Cytosine - 3 H BONDS

This sequence of bases represents the organism’s genetic code

The two sugar-phosphate chains are antiparallel (5’ to 3’ and 3’ to 5’) and are twisted in a double helix

Each twist of the helix is 10 base pairs

The structure is supercoiled

• bases are attached to C1

• Phosphate is attached to 5 prime Carbon-

Diagram

Replication of the Bacterial Chromosome

Occurs in a semi-conservative manner and is usually bidirectional

The precursor of each new nucleotide in the DNA strand is a deoxynucleoside 5’triphosphate

Nucleotides are added to the new strand by DNA polymerase only in the 5’-3’ end (leading strand)

In the 3’-5’ direction, synthesis is discontinuous in short Okazaki fragments using an RNA primer. Fragments then joined together with DNA ligase (lagging strand)

1. Helix s unwound and a new strand is synthesised complementary to the parent strand which is the template - semi conservative as there is 1 parent strand and 1 new strand

2. Bi-directional so very efficient e.g. E.coli can replicate in 20 minutes

3. Nucleotides can only be added to the new strand by DNA polymerase in the 5 to 3 prime

4. In other strand since it is going in other direction we need Okazaki fragment - lagging or discontinuous strand

Semi conservative replication

• Replication fork - two strands split to allow synthesis of new strands of DNA

• On other strand small RNA primers are synthesised along the length of the strand so that the DNA can add the nucleotide in the 5 to 3 prime direction using DNA polymerase

• to do this two terminal phosphates are removed from the new nucleotide and inner phosphate is attached covalently to the deoxyribose of the growing chain at the three prime carbo. this relies on the free hydroxyl of the three prime carbon - see picture 1 for reference

diagram

• small RNA primer is attached at the beginning and then the DNA polymerase adds nucleotides

• on the lagging strand it is discontinuous. Small RNA primers are synthesised by the DNA primase enzyme - normally 11 base pairs long

• DNA polymerase can then add nucleotides from each of these primers in 5 to 3 direction - stops when it reaches previously synthesised DNA

• after this the primer is removed and is replaced with more nucleotides - these are called okazaki fragments

• DNA ligase seals the end gaps between the fragments

• TAU unit is there to stabilise the complex

• DNA helicase is responsible for unwinding the supercoiled DNA

• DNA gyrase prevents unwanted supercoil occurring

• DNA binding proteins also stabilise the structure

Replication of circular DNA

Map of the Chromosome of E. coli K-12

4,639,221 base pairs

4288 open reading frames (indicate genes

• a gene is the unit of genetic information and the genome is the cells complete set of genes

• many of the genes which encode enzymes of a single biochemical pathway are often clustered into what we call operands

• an operand is a set of adjacent genes which is under the control of one promoter and the mRNA is then synthesised in one piece

• for example if the bacteria needed to make some triptonan it would transcribe this operand to produce mRNA and then that would then produce the proteins for each of the five proteins for tryptophan biosynthesis - makes it more efficient as they are all clustered together

• 40 % of the predictive proteins in E. Coli are still unknown

Transcription

Sections (corresponding to genes) of ONE strand of the DNA molecule are transcribed

This is the process of converting DNA into mRNA and is carried out by RNA polymerase - RIFAMPCIN targets RNA polymerase

There is an initiation site (promoter) and termination site, which are specific nucleotide sequences - promoter region is found upstream while termination site is at the end of the gene where synthesis stops

The RNA polymerase moves down the DNA chain, temporarily opening the double helix and transcribing one of the strands

• RNA polymerase must bind to sigma factor before it can work as it produces the holo enzyme which can then bind to the promoter region on the DNA

• it then moves along the DNA and as it does that RNA is transcribed to MRNA - Sigma factor can dissociate

• it then reaches the termination site and the enzyme is released and can bind to another sigma factor.

Translation

The mRNA has triplet sequences of bases – codons

Codons encode the 20 amino acids

Some codons act as start codons (AUG - methionine) and stop codons (UAA, UAG, UGA) which regulate the process of translation

After transcription, the newly-formed mRNA binds to a 30-S subunit of a ribosome - good AB target

Each amino acid associates with a molecule of tRNA. This tRNA contains a triplet base sequence (anticodon) which is complementary to the codons on the mRNA

The anticodon of tRNA for the first amino acid (usually a methionine) binds to its complementary mRNA codon at the appropriate place on the ribosome

A 50-S subunit associates with the 30-S subunit to form a complete working ribosome

 A 2^nd amino acid and its tRNA is added at the appropriate place on the ribosome and a peptide bond forms between the 2 amino acids

The first tRNA molecule then dissociates from the ribosome and can pick up another molecule of its specific amino acid

The ribosome then moves along the mRNA to the next codon and the next tRNA-amino acid complex is added

Growth of the polypeptide chain stops when a stop codon is reached

• 1st stage is that the 502 subunit associates with 302 subunit to form a complete working ribosome

• second amino acid moves in and its trna is added to the mrna and a peptide bond can form between them

• third amino acid - trn

Plasmids

Genetic elements that replicate independently of the host chromosome

Replicates via semi-conservative replication - does so independently to the chromosomal DNA

Small circular molecules

Range in size from 1 kbp to >1 Mbp

Carry a variety of non-essential, but often very helpful, genes

• Can carry genes for AB resistance and virulence factors

Their abundance in the cell is variable

Genotype and phenotype

Genotype

The entire array of genes in the organism, i.e. the cell’s total genetic potential

Phenotype

The characteristics expressed by the cell under a particular set of conditions

Examples of Phenotypes Conferred by Plasmids in Prokaryotes

Phenotype Class	Organism
Resistance
•Antibiotic resistance	Wide range of bacteria
•Resistance to toxic metals	Wide range of bacteria
Virulence
•Bacteriocin production and resistance	Wide range of bacteria
•Animal cell invasion	Salmonella, Shigella, Yersinia
•Coagulase, haemolysin, enterotoxin	Staphylococcus
•Toxins and capsule	Bacillus anthracis
•Enterotoxin, K antigen	Escherichia

AB resistance - some bacteria are able to produce beta lactamases which breaks down beta lactam ring found in penicillin’s and cephalosporins

Mutation and mutants

Mutation

Heritable change in DNA sequence that can lead to change in the phenotype

Mutant

A strain of any cell or virus differing from parental strain in the genotype

Wild-type strain

Typically refers to a strain isolated from nature

Can be good or bad

• beneficial mutation example - bacterial cell showing increased tolerance to an AB

Isolation of mutants

Selectable mutations

Give the mutants a growth advantage under certain environmental conditions

Useful in genetic research

Non-selectable mutations

Have neither an advantage or disadvantage over the parent

Detection of such mutants requires screening a large number of colonies and looking for the difference

• e.g. ability of bacteria to produce a pigment

Molecular basis of mutations

Induced mutations

Made deliberately by using mutagens. Mutagens can be chemical, physical or biological. Examples is X RAY or UV.

Spontaneous mutations

Occur as a result of exposure to natural radiation or oxygen radicals

Occur during replication as a result of errors in base pairing

Point mutations

Result from base-pair substitutions in the DNA or in the insertion/deletion of a base pair

Can lead to single amino acid changes, larger changes or no change at all

Molecular Basis of Mutation (2)

Possible Effects of Base-Pair Substitution

Missense mutation

Amino acid changed

Polypeptide altered

Nonsense mutation

Codon becomes a stop codon

Polypeptide is incomplete

Silent mutation

No affect in amino acid sequence, although there is change in the genotype.

Molecular Basis of Mutation (3)

Shifts in the reading frame of mRNA

Deletions and insertions cause more dramatic changes in DNA

Lead to frameshift mutations, often leading to complete loss of gene function

Reversal of mutations

Some mutations are notoriously unstable and the mutants quickly revert back to the wild-type

Can be due to a back mutation:

Reverts back to the original base sequence

Can be due to a suppressor mutation:

A second mutation arises in the genome restoring the phenotype to that exhibited by the WT

Genetic recombination

The physical exchange of DNA between genetic elements

Homologous recombination:

Process of genetic exchange between homologous DNA from two different sources

• Occurs during meiosis in sexually reproducing organisms.

• Involves the exchange of genetic material between homologous chromosomes (chromosomes of the same type, one inherited from each parent).

• Results in gametes (sperm or egg cells) with unique combinations of alleles.

DNA sequences have the same or nearly the same sequence allowing base pairing

Genetic Exchange in Prokaryotes

In prokaryotes, genetic recombination is observed because fragments of homologous DNA from a donor chromosome or plasmid are transferred to a recipient cell by one of three processes:

Transformation - picks up plasmids or chromosomal DNA

Transduction - tends to be chromosomal DNA

Conjugation - tends to be a plasmid

It is after this transfer, when the DNA fragment from the host is in the recipient cell, that homologous recombination may occur

Transformation

Short fragments of linear DNA bind to cell wall of competent cells

The bound DNA enters the cell and is attached to small proteins which prevent degradation

ssDNA incorporates into homologous regions of cell’s chromosome by recombination using RecA

Transduction

Transfer of donor DNA from one cell to another is mediated by a bacteriophage

Two modes:

1. Generalised transduction

DNA derived from any part of the host genome becomes part of the DNA of a mature virus particle in place of virus genome.

2. Specialised transduction

DNA from a specific region of the host genome is integrated directly into virus genome usually replacing some of the virus genes.

Generalised Transduction

Occurs during lytic cycle of phage replication

During production of new copies of phage DNA, the bacterial chromosome degrades into fragments and can be taken up by new particles during assembly

When these transducing phages infect new bacterial cells, they inject bacterial DNA which can recombine with the host chromosome

Specialised Transduction

Involves the lysogenic cycle of phage reproduction

Prophage integrates into bacterial chromosome, and during reversion to lytic cycle prophage separates from bacterial chromosome but takes some adjacent bacterial genes with it

The new phages assemble with mix of viral and bacterial DNA, which is transferred to a new host

New host survives because mixed DNA cannot be replicated, but can recombine with new host chromosome

Conjugation

Transfer of genetic material between bacterial cells requiring cell-to-cell contact

In Gram-negative cells, union is via a sex pilus

In Gram-positive cells, cells aggregate together by the recipients producing pheromones and the donors producing adhesion proteins

Important in plasmid transfer

Definitions

• antimicrobials

• antibiotics

• antiseptic

• disinfectant

• preservative

• selective toxicity

• bacteriostatic

• bactericidal

AB susceptibility

Susceptibility of individual m/o to individual antimicrobial agents varies significantly

e.g. Differences in Gram positives and Gram negatives to the penicillins

Certain broad spectrum antibiotics, such as tetracycline, are effective against both groups

Wider medical use than narrow spectrum antimicrobial

Antibiotics with limited spectrum however, may have valuable activity

Measuring susceptibility

Minimum inhibitory concentration (MIC)

Lowest concentration of antibiotic / antimicrobial needed to inhibit growth of an organism

Minimum bactericidal concentration (MBC)

Lowest concentration of antibiotic / antimicrobial needed to kill an organism

This may be equal to or higher than MIC

Principles of antimicrobial therapy

Clinical diagnosis

Signs and symptoms

Microbiological diagnosis

Microscopic, cultural, serology, molecular biology

Sensitivity tests

Provides a guide to choice of agent

Laboratory control of treatment

Factors affecting agent choice

Known or predicted in vitro sensitivity of the m/o

Patient factors

Urgency for correct therapy

Bactericidal or bacteriostatic action

Drug pharmacokinetics

Combinations

Cost

Combination therapy

Advantages	Disadvantages
Achieve a broad and bactericidal spectrum in severe and undiagnosed infection	Increased likelihood of side-effects and toxicity
Treat pathogens at multiple sites in the body	Superinfection and hospital cross-infection by antibiotic-resistant strains
Achieve synergy	Possible antagonism between agents
Prevent resistance emerging	Increased cost
Treat mixed infections
Prevent fungal overgrowth

Mode of actions

Antimicrobials/antibiotics usually act in one of five major ways:

1.Inhibition of cell wall synthesis

2.Inhibition of protein synthesis

3.Inhibition of nucleic acid synthesis

4.Injury to the plasma membrane

5.Inhibition of essential metabolite synthesis

Inhibition of cell wall synthesis

Beta-lactam antibiotics

Penicillin

Penicillin G from P. chrysogenum (1929 – Flemming)

Florey (1939) – large scale production

Active primarily against Gram positives

Modification of structure led to semi-synthetic antibiotics (some with broader spectrums of activity)

Cephalosporins

Produced by fungus Cephalosporium sp.

Semisynthetic cephalosporins have broader activity than penicillins

Beta-lactam antibiotics – mode of action:

Beta-lactams (penicillin G, ampicillin, cephalosporins & carbapenems)

Structure based on b-lactam ring

Act by binding to enzymes involved in peptidoglycan synthesis: penicillin binding proteins (PBPs)

Inhibition of cell wall synthesis leads to death by cell lysis or by a non-lytic mechanism

Glycopeptides

Vancomycin

Interferes with cross-linking of peptidoglycan preventing cell wall synthesis

Often used as a last line of defense against MRSA

Isoniazid

Important growth factor analog with a very narrow spectrum of activity

Effective only against Mycobacteria as interferes with synthesis of mycolic acid

Inhibition of protein synthesis

Antimicrobials act by interacting with the ribosome and disrupting translation

Specific and may involve binding to rRNA

Many antibiotics specifically inhibit ribosomes of m/o from one phylogenetic domain

Aminoglycosides

Bactericidal drugs (including streptomycin & gentamicin)

Act by binding to 30S subunit of ribosome

Distorting its shape and preventing translation of mRNA to the protein

Tetracyclines

Actively taken up by bacterial but not mammalian cells

Bacteriostatic drugs that bind to 30S subunit of ribosome halting protein synthesis

Chloramphenicol

Bacteriostatic drug that binds to 50S ribosomal subunit inhibiting peptidyl transferase activity

This prevents elongation of the polypeptide by blocking peptide bond formation

  Macrolides

Bacteriostatic drugs that include erythromycin

Bind to 50S ribosomal subunit

Mode of action thought to be via inhibition of translocation

Inhibition of nucleic acid synthesis

Chloramphenicol

Bacteriostatic drug that binds to 50S ribosomal subunit inhibiting peptidyl transferase activity

This prevents elongation of the polypeptide by blocking peptide bond formation

  Macrolides

Bacteriostatic drugs that include erythromycin

Bind to 50S ribosomal subunit

Mode of action thought to be via inhibition of translocation

Quinolones

Antibacterial compounds interfering with DNA Gyrase – bactericidal

Fluoroquinolones (e.g. ciprofloxacin) can treat both Gram negative and Gram positive infections

Rifamycins

Inhibit transcription by inhibiting RNA synthesis

Rifampicin binds to β-subunit of RNA polymerase

Disruption of Plasma Membrane Integrity

Polymyxins

Interact with cytoplasmic membrane leading to structural distortion, segments released and integrity destroyed

Polymyxin B, Colistin (Polymyxin E)

Often used as last resort due to side effects

Inhibition of Essential Metabolite Synthesis

Sulphonamides

Synthetic antimicrobial drugs

Competitively inhibit folic acid synthesis due to structural similarity to para-aminobenzoic acid (PABA)

Antifungal agents

Eukaryotes

Pose special problem for successful chemotherapy

Drug toxicity a problem

Can target unique fungal structures or metabolic processes

Fungal infections in immunocompromised people are an increasing problem.

Examples of antifungal agents

Category	Target	Examples	Formulaton
Azoles	Ergosterol synthesis	Clotrimazole Fluconazole	Topical Oral
Polyenes	Ergosterol	Amphotericin B Nystatin	Oral, IV Oral, topical
Nucleic Acid analogues	DNA synthesis	5-fluorocytosine	Oral

Antiviral agents

Design of successful antivirals is difficult because:

Acellular

Agents in use are restricted

Most successful approach to controlling viral infections has been through vaccination

Many viral infections clear up spontaneously in immunocompetent patients

Examples

Category	Target	Example	Virus
Nucleoside Analogues	Viral polymerase inhibitors Reverse transcriptase inhibitors	Acyclovir Lamivudine	Herpes, Varicella zoster HIV, hep B
Synthetic amines	Viral uncoating blocker	Amantadine	Influenza A
Protease Inhibitors	Viral protease inhibitor	Amprenavir Indinavir	HIV HIV

Aciclovir – Selective Toxicity

Nucleoside analog that resembles the nucleoside guanosine

When it is phosphorylated by thymidine kinase it blocks viral DNA synthesis by inhibiting viral DNA polymerase

It is only phosphorylated in cells infected with herpes viruses because of viral induction