Bacterial Genetics

The rapid development of molecular genetics, in conjunction with the ability of scientists to manipulate and transfer genes, has elicited considerable interest among physicians, scientists, and the public in general. The ability to manipulate the hereditary material of microorganisms and even our own species has far reaching consequences.

Gene Mutation Any change in the nucleotide sequence of a gene. It may result from base substitutions, deletions, insertions and rearrangement. Transition Vs transversion of nucleotides Mismatch repair by proof reading enzymes (SOS system) Mutations in bacterial populations can pose a problem, bacteria have mechanisms by which genes can be transferred to other bacteria. Thus, a mutation arising in one cell can be passed on to other cells.

Types of Mutation 1) Silent mutation 2) Missense mutation 3) Nonsense mutation 4) Frame-shift mutation 5) Null mutation 6) Suppressor mutation (intragenic or extragenic)

Spontaneous Mutation 10-4 (hot spots) to 10-11. Generally 10-7 in progeny of a single cell. Mechanism = electrochemical rearrangement, or moving around of electrons in the nucleotide molecule. Shift from a keto state to the enol form ( tautomerization) promotes abnormal hydrogen bonding between nucleotides. Thymine (keto) → adenine Thymine (enol) → guanine

Induced Mutation Chemical Agents Mutagens (Chemical and Physical agents) Chemical Agents 5-Bromouracil: analogue of thymine binds to G. 2-Aminopurine: analogue of adenine binds to C. Nitrous acid (HNO2): Nucleotide deamination Alkylating agents (eg ethyl-methyl sulfonate) Acridine dyes (Proflavin): insertion or deletion leading to frame-shift mutation.

Physical Agents Heat: deamination of nucleotides. UV light: Pyrimidine dimmers (thymine), hydrated pyrimidines. Gamma and x-rays: breaks in the sugar phosphate backbone.

Gene Transfer Transfer of DNA from a donor cell to a recipient cell with the resultant recombination. Gene transfer in bacteria is unidirectional: Donor → recipient It requires DNA homology A small part of DNA is transferred It is achieved by three mechanisms: Transformation, Transduction and Conjugation.

Transformation Uptake of naked fragmented single stranded DNA Most primitive mechanism of gene transfer It occurs in only a few genera Bacillus subtilis Streptococcus pneumoniae Hemophilus influenzae Neisseria gonorrhea E. coli (in the presence of a high concentration of calcium salts and high temperature)

The probability that transformation has contributed much to genetic variety is very small. Factors affecting transformation: - DNA size and state - Competence of the recipient Steps: - Uptake of DNA - Legitimate/Homologous/General recombination

Transduction Transfer of DNA via a bacteriophage Lytic Vs lysogenic (temperate) phage life cycle. Restricted Vs generalized transduction Not affected by nucleases in the environment Phages that mediate generalized transduction generally breakdown host DNA into smaller pieces and package their DNA by a "head-full" mechanism. Examples: diphtheria toxin, erythrogenic toxin and resistance to penicillin in staphylococci.

Phage Composition and Structure

Plasmid- Mediated Conjugation PIASMIDS - Circular ds DNA molecules ranging in size from 1/1000 to 1/10 of the chromosome. - They are usually autonomous (replicons). - As few as 1 to as many as 100/cell. - May become integrated (episomes). - Two classes: conjugative and nonconjugative

Conjugation Requires the sex (F) factor Donor cell (F+) and recipient cell (F- ) F factor is a ds DNA that is 1/40 of the chromosome in size, not under the control of the bacterial chromosome, ant it can replicate autonomously. It possesses several genes that code for the formation of sex pili.

Conjugation: During the conjugation process, a copy of the sex factor is made and one of its (single) strands is transferred to the recipient cell. The complementary strand of the donor single strand is synthesized in the recipient cell which is now converted to an F+ cell.

Mechanism of F+ x F- Crosses Pair formation Conjugation bridge F+ F- DNA transfer Origin of transfer Rolling circle replication

Physiological States of F Factor Integrated (Hfr) Characteristics of Hfr x F- crosses F- rarely becomes Hfr while Hfr remains Hfr High transfer of certain donor chromosomal genes F+ Hfr

Functions of Plasmids R Plasmids: Antimicrobial resistance, may be multiple but clustered. Resistance to heavy metals (Ag, Co, Hg) in Staphylococci, coexist with R plasmids. Virulence determinants: toxins Bacteriocinogenic plasmids

Mechanisms of Action of Resistance plasmids Modification of antibiotic Alteration of target site Alteration of uptake Replacement of a sensitive pathway

Transposable genetic Elements Move from one location to another (jumping genes) 1) Random movement (not totally=preferable sites) 2) Not capable of self replication 3) Transposition mediated by site-specific recombination (mediated by transposases) no homology is required 4) Transposition can be accompanied by duplication

Types 1) Insertion sequences (Is): Small stretches of DNA that have at their ends repeated sequences 2) Transposons: (Tn) Carry extra genes

Genetic Engineering Cloning (recombinant DNA) - RE sticky ends (passenger DNA) - RE sticky ends (vector DNA) of plasmids - Annealing (ligase) - Insertion to a cell (transformation)