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BACTERIAL GENETICS Structure of DNA • • • • Double stranded (double helix) Chains of nucleotides 5’ to 3’ (strands are anti-parallel) Complimentary base pairing – A-T – G-C DNA Structure Phosphate-P Sugar-blue Bases-ATGC 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 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) Replication Fork Protein Synthesis • DNA------- mRNA------ protein transcription translation Central Dogma of Molecular Genetics 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 Transcription 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) PHENOTYPIC VARIATION • Change in the colony characters , capsule or flagella GENOTYPIC VARIATION • Change in the genes resulting in change in protein synthesis Mutations – changes in the DNA • Point mutation – addition, deletion or substitution of a few bases • Missense mutation – causes change in a single amino acid • Nonsense mutation – changes a normal codon into a stop codon • Silent mutation – alters a base but does not change the amino acid 17 Mutations – changes in the DNA • Point mutation – addition, deletion or substitution of a few bases • Missense mutation – causes change in a single amino acid • Nonsense mutation – changes a normal codon into a stop codon • Silent mutation – alters a base but does not change the amino acid 18 Mutations • Changes in base sequence of DNA/lethal and inheritable • Can be: – Harmful – Lethal – Helpful – Silent Normal DNA/Missense Mutation Nonsense Mutation/Frameshift Mutation Ames Test 22 General Features of Gene Transfer in Bacteria • Unidirectional – Donor to recipient • Donor does not give an entire chromosome – Merozygotes • Gene transfer can occur between species 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 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 – Transduction • Definition: Gene transfer from a donor to a recipient by way of a bacteriophage Phage Composition and Structure • Composition – Nucleic acid Head/Capsid • Genome size • Modified bases – Protein • Protection • Infection • Structure (T4) – Size – Head or capsid – Tail Contractile Sheath Tail Tail Fibers Base Plate Generalized Transduction • Infection of Donor • Phage replication and degradation of host DNA • • • • Assembly of phages particles Release of phage Infection of recipient Legitimate recombination Transduction • Types of transduction – Generalized - Transduction in which potentially any dornor bacterial gene can be transferred. – Specialized - Transduction in which only certain donor genes can be transferred Specialized Transduction Lysogenic Phage • Excision of the prophage • Replication and release of phage • Infection of the recipient • Lysogenization of the recipient bio gal gal gal bio bio – Legitimate recombination also possible gal bio bio Transduction • Definition • Types of transduction • Significance – Common in Gram+ bacteria – Lysogenic (phage) conversion Conjugation • Definition: Gene transfer from a donor to a recipient by direct physical contact between cells • Mating types in bacteria – Donor Donor • F factor (Fertility factor) – F (sex) pilus – Recipient • Lacks an F factor Recipient Physiological States of F Factor • Autonomous (F+) – Characteristics of F+ x F- crosses • F- becomes F+ while F+ remains F+ • Low transfer of donor chromosomal genes F+ 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 Physiological States of F Factor • Autonomous with donor genes (F’) – Characteristics of F’ x F- crosses • F- becomes F’ while F’ remains F’ • High transfer of donor genes on F’ and low transfer of other donor chromosomal genes Hfr F’ Mechanism of F+ x F- Crosses • Pair formation – Conjugation bridge • DNA transfer – Origin of transfer – Rolling circle replication F+ F- F+ F- F+ F+ F+ F+ Mechanism of Hfr x F- Crosses • Pair formation – Conjugation bridge • DNA transfer Hfr F- Hfr F- – Origin of transfer – Rolling circle replication • Homologous recombination Hfr F- Hfr F- Mechanism of F’ x F- Crosses • Pair formation – Conjugation bridge • DNA transfer F’ F- F’ F- F’ F’ F’ F’ – Origin of transfer – Rolling circle replication Conjugation • Significance – Gram - bacteria • Antibiotic resistance • Rapid spread – Gram + bacteria • Production of adhesive material by donor cells 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 Types of Transposable Genetic Elements • Insertion sequences (IS) – Definition: Elements that carry no other genes except those involved in transposition – Nomenclature - IS1 – Structure GFEDCBA ABCDEFG Transposase – Importance • Mutation •Plasmid insertion •Phase variation Phase Variation in Salmonella H Antigens H1 gene H1 flagella IS H2 gene H2 flagella 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 IS Resistance Gene(s) IS IS Resistance Gene(s) IS Plasmids • Definition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon) • Episome - a plasmid that can integrate into the chromosome Classification of Plasmids • Transfer properties – Conjugative – Nonconjugative • Phenotypic effects – Fertility – Bacteriocinogenic plasmid – Resistance plasmid (R factors) Structure of R Factors • RTF RTF – Conjugative plasmid – Transfer genes • R determinant – Resistance genes – Transposons R determinant Genetic basis of drug resistance • Mutational drug resistance - Chromosomal • Transferable drug resistance – Plasmid mediated 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. • 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 recombinations involving cells of higher organisms. • 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. • PCR - POLYMERASE CHAIN REACTION: Rapid automated method for amplification of specific DNA sequences. A single PCR cycle consists of; • Denaturation of sample DNA to obtain a single strand • Annealing of sequence specific oligonucleotide primers. . Extension of primers by DNA Polymerase enzyme to form a new double stranded DNA. • This cycle is repeated for 20 - 50 cycles in a thermocycler to obtain several thousands of copies of Sample DNA that can be identified later by DNA probes. • Diagnosis of Infectious diseases, genetic disorders, cancer, forensic investigations and biotechnology. • Restriction endonucleases: Enzymes which can cleave DSDNA at specific oligonuleotide seqences. • e.g: Taq I, Hind III. • RECOMBINANT DNA TECHNOLOGY: • Isolation of genes coding for any desired protein from cells or microorganisms – • Synthesis- small sequences; Enzymes - restriction endonucleases. • mRNA - DNA (SS) synthesis by reverse transcriptase & • polymerization by DNA polymerase to yield DS DNA. • Vectors - Plasmids, Bacteriophages;E.coli K12 & Yeast's.