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Unraveling the Basics of Bacterial Genetics

Explore the structure of DNA, DNA replication, protein synthesis, mutation types, gene transfer mechanisms, and more in bacterial genetics. Learn about bacterial transformation, mutations, and DNA repair.

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Unraveling the Basics of Bacterial Genetics

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  1. BACTERIAL GENETICS Dr.Rouchelle Tellis Associate Prof, Microbiology

  2. Structure of DNA • Double stranded (double helix) • Chains of nucleotides • 5’ to 3’ (strands are anti-parallel) • Complimentary base pairing • A-T • G-C

  3. DNA Structure Phosphate-P Sugar-blue Bases-ATGC

  4. DNA Replication • Bacteria have closed, circular DNA • Genome: genetic material in an organism • E. coli • 4 million base pairs • 1 mm long (over 1000 times larger that actual bacterial cell) • DNA takes up around 10% of cell volume

  5. DNA Replication-occurs at the replication fork • 5’ to 3 ‘ • DNA helicase-unzips + parental DNA strand that is used as a template • Leading stand (5’ to 3’-continuous) *DNA polymerase-joins growing DNA strand after nucleotides are aligned (complimentary) • Lagging strand (5’ to 3’-not continuous) *RNA polymerase (makes short RNA primer) *DNA polymerase (extends RNA primer then digests RNA primer and replaces it with DNA) *DNA ligase (seals Okazaki fragments-the newly formed DNA fragments)

  6. Replication Fork

  7. Protein Synthesis • DNA------- mRNA------ protein transcription translation Central Dogma of Molecular Genetics

  8. Transcription • One strand of DNA used as a template to make a complimentary strand of mRNA • Promoter/RNA polymerase/termination site/5’ to 3’ • Ways in which RNA & DNA differ: • RNA is ss • RNA sugar is ribose • Base pairing-A-U

  9. Transcription

  10. Translation • Three parts: • Initiation-start codon (AUG) • Elongation-ribosome moves along mRNA • Termination: stop codon reached/polypeptide released and new protein forms • rRNA=subunits that form the 70 S ribosomes (protein synthesis occurs here) • tRNA=transfers amino acids to ribosomes for protein synthesis)

  11. PHENOTYPIC VARIATION • Change in the colony characters , capsule or flagella • Phenotypic change = Physical change

  12. GENOTYPIC VARIATION • Change in the genes resulting in change in protein synthesis

  13. Mutations – changes in the DNA • Point mutation – addition, deletion or substitution of a few bases • Mis-sense mutation – causes change in a single amino acid • Non-sense mutation – changes a normal codon into a stop codon • Silent mutation – alters a base but does not change the amino acid

  14. Mutations • Changes in base sequence of DNA/lethal and inheritable • Can be: • Harmful • Lethal • Helpful • Silent

  15. Normal DNA/Missense Mutation

  16. Nonsense Mutation/Frameshift Mutation

  17. Ames test: is based on the ability of auxotrophic bacteria to mutate by reverting to their original synthetic ability. • Used for screening chemicals for mutagenic properties, which indicate potential carcinogens

  18. Spontaneous mutations occur in the absence of any known mutagen, due to errors in base pairing during DNA replication. • Induced mutations are produced by agents called mutagens that increase the mutation rate. • Chemical Mutagens: Alkylating agents, deaminating agents, arcidine derivatives. • Radiations: • Repair of DNA Damage Many bacteria have enzymes that can repair certain damages to DNA • (1) Light repair uses an enzyme that is activated by visible light and that breaks bonds between pyrimidines of a dimer. • (2) Dark repair use several enzymes that do not require light for activation; they excise defective DNA and replace it with DNA complementary to the normal DNA strand.

  19. Bacterial gene transfer TYPES AND SIGNIFICANCE OF GENE TRANSFER: • Movement of genetic information between organisms. • Vertical gene transfer passes genes from parent to offspring. • Lateral gene transfer passes genes to other cells in the same generation. • Mechanisms of bacterial gene transfer: • Transformation • Transduction • Conjugation • Transposition • Gene transfer increases genetic diversity within a population, increasing the likelihood that some members of population will survive environmental changes.

  20. Transformation • Definition: Gene transfer resulting from the uptake of DNA from a donor. • Factors affecting transformation • DNA size and state • Sensitive to nucleases • Competence of the recipient (Bacillus, Haemophilus, Neisseria, Streptococcus) • Competence factor • Induced competence

  21. BACTERIAL TRANSFORMATION: discovered in 1928 by Griffith, who showed that a mixed culture of live rough and heat-killed smooth pneumococci could produce live smooth Pneumococci capable of killing mice. • Avery showed that capsular polysaccharide was responsible for virulence and that DNA was the substance responsible for transformation. • Mechanism of Transformation: Transformation involves the release of naked DNA fragments and their uptake by other cells at a certain stage in their growth cycle: • (l) Uptake of DNA requires a protein called competence factor to make recipient cells ready to bind DNA

  22. Transformation • Steps • Uptake of DNA • Gram + • Gram - • Recombination • Legitimate, homologous or general • recA, recB and recC genes • Significance • Phase variation in Neiseseria • Recombinant DNA technology

  23. The Significance of Transformation • It contributes to genetic diversity • It can be used to introduce DNA into an organism, observe its effects, and study gene locations • It can be used to create recombinant DNA.

  24. Transduction • Definition: Gene transfer from a donor to a recipient by way of a Bacteriophages • Phages can be virulent or temperate. • Virulent phages: destroy a host cell's DNA and cause lysis of the host cell in the lytic cycle • Temperate phages: can replicate themselves as a prophage- part of a bacterial chromosome, or eventually produce new phage particles and lyse the host cell. • Persistence of the phage in the cell without the destruction of the host cell is called Lysogeny.

  25. Head/Capsid Contractile Sheath Tail Tail Fibers Base Plate Bacteriophage composition and Structure • Composition • Nucleic acid • Genome size • Modified bases • Protein • Protection • Infection • Structure (T4) • Size • Head or capsid • Tail

  26. Generalized Transduction • Infection of Donor • Phage replication and degradation of host DNA • Release of phage • Assembly of phages particles • Infection of recipient • Legitimate recombination

  27. Transduction Transduction can specialized or generalized: In specialized transduction, the phage is incorporated into the chromosome and can transfer only genes adjacent to the phage In generalized transduction, the phage exists as a plasmid and can transfer any DNA fragment attached to it.

  28. The Significance of Transduction • Transduction is significant because it transfers genetic material and demonstrates a close evolutionary relationship between prophage and host cell DNA. • Also, its persistence in a cell suggests a mechanism for the viral origins of cancer, and it provide a possible mechanism for studying gene linkage

  29. CONJUGATION • Large quantities of DNA are transferred from one organism to another between donor and recipient cells during contact • Conjugation was discovered by Lederberg in 1946 when he observed that mixing strains of E. coli with different metabolic deficiencies allowed the cells to overcome deficiencies • Plasmids are extra-chromosomal DNA molecules.

  30. Mechanisms of Conjugation • 3 mechanisms of conjugation: • In the transfer of F plasmids, a piece of extra-chromosomal DNA plasmid is transferred. • In high-frequency recombination parts of F plasmids that have been incorporated into the chromosome (the initiating segment) are transferred along with adjacent bacterial genes. • An F plasmid incorporated into the chromosome and subsequently separated becomes an F' plasmid and transfers chromosomal genes attached to it.

  31. The Significance of Conjugation • It increases genetic diversity, & may represent an evolutionary stage between asexual and sexual reproduction and provides a means of mapping genes in bacterial chromosomes.

  32. Transposable Genetic Elements • Definition: Segments of DNA that are able to move from one location to another • Properties • “Random” movement, Not capable of self replication • Transposition mediated by site-specific recombination • Transposase • Transposition may be accompanied by duplication

  33. Segment of DNA that is capable of independently replicating itself and inserting the copy into a new position within the same or another chromosome or plasmid. Referred to as transposons or jumping genes. • In bacteria, the transposable elements can be grouped into two classes, insertion sequences and transposons. • Transposons encode resistance to many antibiotics & toxic metals, chemicals. • Some transposons have the ability to direct the synthesis of proteins that metabolize carbohydrates, petroleum, and pesticides.

  34. IS Resistance Gene(s) IS IS Resistance Gene(s) IS Types of Transposable Genetic Elements • Transposons (Tn) • Definition: Elements that carry other genes except those involved in transposition • Nomenclature - Tn10 • Structure • Composite Tns • Importance • Antibiotic resistance

  35. Plasmids • Definition: Plasmids are circular, self-replicating. Double-stranded extra-chromosomal DNA that carries information that is usually not essential for cell growth • Episome - a plasmid that can integrate into the chromosome

  36. Classification of Plasmids • Transfer properties • Conjugative • Nonconjugative • Phenotypic effects • Fertility • Bacteriocinogenic plasmid • Resistance plasmid (R factors)

  37. RTF Tn 10 Tn 8 Tn 9 Tn 21 R determinant Structure of R Factors • RTF • Conjugative plasmid • Transfer genes • R determinant • Resistance genes • Transposons

  38. Genetic basis of drug resistance • Mutational drug resistance – Chromosomal • Transferable drug resistance – Plasmid mediated

  39. Genetic engineering • Genetic engineering is the manipulation of genetic material to alter the characteristics of an organism. • Genetic fusion: allows transposition from one location on a chromosome to another, sometimes deleting a portion, thereby causing the joining of genes from two different operons. • Protoplast Fusion: combines protoplasts ( organisms without cell walls) and allows mixing of genetic information.

  40. Gene Amplification: involves the addition of plasmids to microorganisms to increase yield of useful substances . • Recombinant DNA Technology: is DNA produced when genes from one kind of organism are introduced into the genome of a different kind of organism. The resulting organism is transgenic, or recombinant organism. • Recombinant DNA has proven especially useful in medicine, industry and agriculture. • Hybridisms: are genetic recombination's involving cells of higher organisms.

  41. DNA PROBES: Labeled (Radioactive, Biotin etc.) copies of single-stranded DNA fragments containing unique nucleotide sequences, which are used to detect homologous DNA by hybridization. Highly specific detects even lninute amounts of target DNA.

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