Lecture 6 - Microbial Genetics

Microbial genetics Edet E. Udo PhD Department of Microbiology Faculty of Medicine. Kuwait University. Kuwait.

Microbial genetics • Objectives: • Define genetics and heredity • Discuss the mechanisms of genetic exchange in bacteria • Discuss mutation and mutagenic agents • Discuss the concepts and application of genetic engineering in Medicine

Definitions • 1. Genetics:

– is the science of the study of heredity. It includes the the study of gene replication and transmission.

• 2. Gene :

– a segment of DNA that codes for a functional product or a linear sequence of DNA that forms a functional unit of a chromosome.

– The location of a characteristic is the Locus. – Genes with different information at the same locus are alleles • 3. Genotype: – the genetic composition of an organism- its entire DNA

• 4. Phenotype:

– the expression of the genes– the proteins and the properties they confer on the organism

Bacterial DNA • Bacterial DNA: • DNA stores information used to guide the replications of DNA in preparation for cell division • 1. Chromosomal DNA, • 2. Extra chromosomal DNA----Plasmid DNA

Bacterial DNA • Bacterial DNA Replication: – by the semi conservative mode, – Replication of DNA usually begins at a specific point origin of replication) – During binary fission each daughter cell receives a chromosomal DNA like the one in the parent cell.

• Definition:

Mutations

• Mutations are alterations (changes) in the nucleic acid sequence. – accounts for variations in the genotypes and phenotypes of microorganisms

Types of mutations : • Point mutations: consists of changes in a single nucleotide

• Frameshift mutations: consists of

of the insertion or deletion of a single nucleotide

Mutations • Effects of mutation: Phenotypic variations e.g lethal mutation leading to arrest of protein synthesis – No effect:

• Spontaneous mutation:

• Occurs in the absence of known mutagen • May be due to errors in base pairing

• Induced mutation:

• Produced by agents known as mutagens • Antibiotic resistance, • Generation of new phenotypes.

Mutagens • Chemical agents: •

5-bromouracil, nitrous acid, ethidium bromide, nitroguanidine, acridines, etc.

• Physical agents: • X-rays, UV-rays: causes formation of dimers

• Biological mutants: • Transposons, insertion sequences.

Mutants • Repair of DNA damage: • Light repair: • involves enzymes that is activated by visible light and breaks bonds between pyrimidines of a dimer – ( role in skin cancer)

• Dark repair:

•

involves several enzymes that do nor require light for DNA repair.

• The AMES test: is used to identify possible chemical carcinogens in chemicals applied to human body such as body creams, shampoos etc. • It is based on the ability of bacteria to mutate by reverting to their original synthetic ability (reverse mutation). • E.g. Histidine auxotroph of Salmonella are exposed to potential carcinogen and revertants to the non mutant state shows that the chemical is a potential carcinogen. • It

Genetic transfer and recombination • Definition:

– Gene transfer refers to the movement of genetic information between organisms. – Occurs by transformation, transduction and conjugation.

• Transformation:

– involves the uptake of naked DNA by bacteria. Uptake of DNA . – It was first demonstrated in 1928 by Griffith while studying pneumococcal infections in mice. – Occurs naturally in Streptococcus pneumoniae, Heamophilus species and some Bacillus species

Transformation • Transformation is significant because: – It contributes to genetic diversity – It can be used to introduce DNA into organisms, observe its effect and study gene location – It can be used to create recombinant DNA

Genetic transfer and recombination • Transduction: • mode of transfer involving bacteriophages. • Significance of transduction. – Transfers genetic materials and demonstrates a close evolutionary relationship between host cell DNA and prophage. – Its persistence in a cell suggests a mechanism for viral origins of cancer. – Provides a mechanism for studying gene linkage

• Phages can be virulent (lytic) or temperate. • 1. Virulent phages • destroy a host cell’s DNA, and cause lysis of the host cell in a lytic cycle • 2. Temperate phages (prophages) – produces a repressor substance that prevents destruction of host DNA.

•

Cells containing prophages are called Lysogenic cells because they have the potential to enter the lytic cycle

Phage cycle

Transduction • Transduction can be specialized or • generalized. • Generalized transduction, • the phage can incorporate any part of the chromosomal or plasmid DNA and transfer them.

Transduction • In specialized transduction the phage is incorporated into the chromosome and can transfer only genes adjacent to the phage.e.g. phage lambda in E. coli

Lysogenic or phage conversion • The alteration of a bacterial phenotype resulting from the acquisition of a phage. • It may confer virulence property . Examples include: The production of diphtheria toxin after the acquisition of phage B The acquisition of Shiga-like toxin by E.coli after acquisition of a phage Production of botulinum toxinC by phage Production of scarlet fever toxin by lysogenic Streptococcus pyogenes

Genetic transfer and recombination • Conjugation: A process requiring cell to cell contact. – 1. Contact between donor and recipient cells is required – 2. Larger amount of DNA is transferred. – Mediated by conjugative plasmids or conjugative transposons.

– Sex pilli is involved In E. coli and other Gramnegative bacilli, – Sex pheromones may be involved in Streptococci • Significance of conjugation: – It increases genetic diversity – May represent an evolutionary stage between asexual and sexual reproduction – It provides a means of mapping genes in bacterial chromosome

Conjugation

Conjugatio n in E. coli

Genetic transfer and recombination • Characteristics of plasmids: • Double stranded extra chromosomal DNA. • Plasmids are self-replicating • They are identified by virtue of some recognizable function that they serve in a bacterium e.g. F-plasmids (fertility factors) direct the synthesis of proteins that self-assemble into sex pili • R-plasmids (resistance factors) carry genes that provide resistance to various antibiotics • Other plasmids direct the synthesis of bactericidal proteins called bacteriocin, toxin plasmids ( metabolic plasmids (Tol plasmids)

Plasmids • Classification: based on size, copy number, phenotype, incompatibility • Restriction endonucleases analysis ( physical mapping) • Plasmids are used in the study of epidemiology of pathogenic bacteria. •

Agarose gel electrophoresis of plasmids.

Genetic engineering Definition: the purposeful manipulation of genetic material to alter the characteristics of an organism. • Techniques include genetic fusion, protoplast fusion and recombinant DNA. • Recombinant DNA makes it possible to fuse genes with vectors and clone them in host cells.

• Making Recombinant DNA involves: • The manipulation of DNA in vitro, • The cloning of DNA from other organism s in bacteria DNA with phage or plasmid. • The production of many genetically identical progeny of phages or plasmids.

Applications of Recombinant DNA technology in Medicine • 1. Treatment or management of disease

– Production of growth hormones, insulin, anti sera, blood coagulation proteins etc – Gene therapy

• 2. Diagnosis of disease

– DNA probes for detection of genetic defects, identification of pathogens – DNA amplification (PCR)

• 3. Prevention and control of infections

– Recombinant vaccines – Molecular epidemiology- for the determination of source and extent of spread of an infectious agent

• Safety concerns: • Environmental impact, health effect of recombinant plants and vegetables, • Production of Biological warfare agents, ethical concern with use on human subjects