Genetic Exchange and Variation The dynamic nature of DNA Terry D. Connell, Ph.D. Professor Dept. of Microbiology and Immunology [email protected] PresentationSlide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Slide 49Slide 50Slide 51Slide 52Slide 53Slide 54Slide 55Slide 56Slide 57Slide 58Slide 59Slide 60Genetic ExchangeGenetic Exchangeand Variationand VariationThe dynamic nature of DNAThe dynamic nature of DNATerry D. Connell, Ph.D.Terry D. Connell, Ph.D.ProfessorProfessorDept. of Microbiology and ImmunologyDept. of Microbiology and [email protected]@buffalo.eduAn excellent mystery:By what mechanism(s) did a clinically-serious, antibiotic-resistant,sexually-transmitted disease globally emerge?emerge?(Or, why molecular genetics really matters!!)(Or, why molecular genetics really matters!!)Adverse environmental conditionsexert selective pressures on bacterial populationsA bacterium that survived phagocytosis due to acquisition of a favorable, adaptive mutationSelective advantage:a preferential outgrowth of selected clonesOften promoted by acquisition of new genetic informationthat provides new traits which are beneficialin particular environmentsGenetic change in Neisseria gonorrhoeae brought about byChromosomal mutationsEx. - Antibiotic resistanceAntibiotic Gene symbolPenicillin penAErythromycin eryRifampicin rifTetracycline tetStreptomycin strChloramphenicol catGenetic change in Staphylococcus aureus brought about by:Extrachromosomal elementsAntibiotic Gene Symbol ElementPenicillin bla conjugal plasmidErythromycin ermA transposonTrimethoprim dfrA plasmidTetracycline tet plasmidStreptomycin aadA plasmidChloramphenicol cat plasmidGenetic change brought about byMobilizable Genetic ElementsEx. - Virulence determinantsDeterminant Vector Mode of TransferCorynebacterium diphtheriae Diphtheria toxin (DTx) bacteriophage Lysogenic conversionEscherichia coli K-88 adhesin plasmid ConjugationStaphylococcus aureus Exfoliative toxin plasmid TransductionGenetic change brought about byPhase and/or Antigenic conversionTranscriptionInitiation site(Translation)Central DogmaThree general mechanisms forpromoting Genetic Variation:I. Random mutations that alter the genome of a single bacteriumII. Non-random (programmed) changes that alter the genome of a single bacteriumIII. Direct or indirect transmission of genetic material between two bacteria that introduces new information into the recipient cellThree general mechanisms forpromoting Genetic Variation:I. Random mutations that alter the genome of a single bacteriumII. Non-random (programmed) changes that alter the genome of a single bacteriumIII. Direct or indirect transmission of genetic material between two bacteria that introduces new information into the recipient cellWhat are “Mutations”?1. Heritable alterations in the nucleotide sequence of the bacterial DNA2. May alter the sequences of the protein or RNA encoded by the DNA3. May in RARE cases provide the cell a selective advantage4. Arise due to errors made during DNA replication•DNA polymerase has a very low error rate (frequency of 10-7 to 10-11 per base pair)•Infrequent failure in “proof-reading” capacity of DNA polymerases•“Point mutations” are the most common ‘error’Types of Point mutationsCan change the amino acid sequence of the encoded proteinProtein X …Lys Ser Ala Lys … … AAT AAGT GCT AGG …… AAT TGT GCT AGG … Lys Cys Ala Lys (Missense mutation)… AAT GTG CTA GG … Lys Val Leu … AAT TGA GCT AGG … Lys STOP (Nonsense mutation)… AAT CAG TGC TAG … Lys Gln Cys STOP (Nonsense mutation)(Frameshift mutation)Deletion of an ASubstitution of a T for an ASubstitution of T for AAddition of a CChromosomal mutations: Antibiotic resistanceAntibiotic Gene symbolPenicillin penAErythromycin eryRifampicin rifTetracycline tetStreptomycin strChloramphenical catChromosomal mutations: Antibiotic resistanceAntibiotic Gene symbolPenicillin penAErythromycin eryRifampicin rifTetracycline tetStreptomycin strChloramphenical catSingle nt mutation in a geneencoding the S12 subunit ofthe ribosome(Lysine42 Arginine)Three general mechanisms forpromoting Genetic Variation:I. Random mutations that alter the genome of a single bacteriumII. Non-random (programmed) changes that alter the genome of a single bacteriumIII. Direct or indirect transmission of genetic material between two bacteria that introduces new information into the recipient cellMultiple antigenic variants of Opa can be expressed by NeisseriaFA1090 – 11 Opa genesNeisseria gonorrhoeaeN. meningitidis(CTCTT)n : n = 5-17 repeatsPhase and Antigenic variation of Opa proteins Depends upon alterations in the numbers of CTCTT repeats in the Signal Peptide-encoding region of the geneNeisseria gonorrhoeaeHow does the pathogen elude the immune system?Between 8-11 opa genes in the chromosomeOpaA OpaB OpaC OpaA/OpaB OpaB/OpaE etc. Opa+ Opa-opaAopaBopaCopaDopaFopaGopaENeisserial chromosomeSignal peptide-encoding regionNucleic acid sequence of an opa geneA series of pentameric repeats: CTCTT in DNA (CUCUU in mRNA)out-of-frame STOP codonFrameshift mutations regulate Opa expressionin Neisseria spp.“Mistakes in replication”*Correct reading frame leads to expression of Opa protein* Opa mRNAAUG CUC UUC UCU UCU CUU AAG …UAA Opa Protein: Met Leu Phe Ser Sert Leu Lys … STOP *Loss (or gain) of one pentanucleotide (CTTCT) in opa gene* Opa mRNAAUG CUC UUC UCU UUA Opa protein: Met Leu Phe Ser STOPPH2PH1H1 flagellin gene H2 flagellin geneH1 repressor geneSalmonella typhimurium flagellar locusThree genes: H1 flagellum geneH1 repressor geneH2 flagellin geneTwo promoters: PH2PH1H1 flagellum? H2 flagellum?How is the system regulated?Two antigenic types of flagellae in Salmonella: H1 and H2Flagellar DNA inversion enables the bacteriumto avoid targeting immune responses to H1 or H2XX XAntigenic variation of flagellar proteinsSalmonella typhimuriumPhinPhinThree general mechanisms forpromoting Genetic