MICROBIAL GENETICS PLASMIDS OTHER MOBILE GENETIC ELEMENTS

1. 1 MICROBIAL GENETICS PLASMIDS OTHER MOBILE GENETIC ELEMENTS

2. 2 MICROBIAL GENETICS - PLASMIDS / OTHER MOBILE GENETIC ELEMENTS PLASMIDS Characteristics, Resistance Factors, Resistance Transfer Factors Drug Resistance Mechanisms Penicillin Resistance, Penicillinase, Beta-lactamase Multiple Drug Resistance Transformation by Plasmids OTHER MOBILE GENETIC ELEMENTS Insertion Sequences Transposons Integrons Superintegrons Conjugative Transposons Genomic Islands The Busy Genome

3. 3 PLASMIDS: SMALL, CIRCULAR, DOUBLE-STRAND DNA MOLECULES ~ 5 - 50 GENES, CYTOPLASMIC LOCATION, NOT ESSENTIAL (NORMALLY) REPLICATION GENES AND SITES 1 - 20 COPIES EACH / CELL SEVERAL DIFFERENT PLASMIDS / CELL RESISTANCE FACTORS - PLASMIDS WHICH CARRY GENES WHICH ENCODE PROTEINS WHICH MAKE THE BACTERIAL HOST RESISTANT TO ANTIBIOTIC - CALLED DRUG RESISTANCE RESISTANCE TRANSFER FACTORS - ALL ABOVE PLUS ABILITY TO TRANSFER PLASMID IN MATING [CONJUGATION] OTHER PLASMID GENES: HYDROCARBON CATABOLISM TOXIN PRODUCTION MINERAL UPTAKE

4. 4 MECHANISMS OF DRUG RESISTANCE MUTATION RESULTS IN ALTERED BACTERIAL PROTEIN. IT NO LONGER RECOGNIZES ANTIBIOTIC BUT CONTINUES TO PERFORM NORMAL FUNCTION IN BACTERIAL GROWTH EX: STREPTOMYCIN BACTERIA ACQUIRE NEW GENE WHICH CODES FOR ENZYME WHICH DESTROYS ANTIBIOTIC EX: PENICILLINASE DESTROYS PENICILLIN BACTERIA ACQUIRE NEW GENE WHICH CODES FOR ENZYME WHICH PUMPS ANTIBIOTIC BACK OUTSIDE CELL EX: TETRACYCLINE RESISTANCE

5. 5 PENICILLIN CLEAVAGE [INACTIVATION] BY PENICILLINASE b - LACTAM RING PENICILLINASE [b - LACTAMASE] INACTIVE

6. 6 MULTIPLE DRUG RESISTANCE: JAPAN, 1957, SHIGELLA DYSENTERIAE, DYSENTERY RESISTANT TO: SULFONAMIDES, STREPTOMYCIN, CHLORAMPHENICOL, NEOMYCIN JAPAN: 1957 - FIRST CASE; 1964 - 50% LONDON: 1962 - 3% 1964 - 61% NEISSERIA GONORRHOEAE 1945: PENICILLIN-SENSITIVE ~1975: PENICILLIN-RESISTANT, BUT SPECTINOMYCIN-SENSITIVE ~1985 - 1990: STREPTOMYCIN-RESISTANT, CEFTRIAXONE-SENSITIVE

7. NEISSERIA GONORRHOEAE 1940s: SULFANILAMIDES 1945 : PENICILLIN-SENSITIVE (70 MG, ORAL; CHEAP; THEN UP TO 3 GM OVER TIME) 1970s: PENICILLIN-RESISTANT, SHIFT TO SPECTINOMYCIN (PROTEIN SYNTHESIS INHIBITOR BUT INJECTON, EXPENSIVE SHIFT TO TETRACYLCINE 1980s: TETRACYCLINE-RESISTANT, SHIFT TO CIPROFLOXACIN – FLUORO- QUINOLONE – TOPOISOMERASE INHIB. 2007: CIPRO –RESISTANT, SHIFT TO CEFTRIAXONE A CEPHALOSPORIN – BETA LACTAM BUT NOT INACTIVATED BY BETA-LACTAMASE 2011: NEISSERIA GONORRHOEAE SUPER-BUG- THROAT OF JAPANESE CSW- RESISTANT TO ALL CEPHALOSPORINS 7

8. 8 BACTERIUM RESISTANT TO PENICILLIN PENICILLINASE GENE FOR PENICILLINASE [b - LACTAMASE] PLASMID PHENOTYPE: ORGANISM GROWS ON MEDIUM CONTAINING PENICILLIN

9. 9 TRANSFORMATION BY PLASMIDS PENICILLIN RESISTANCE PLASMID PENICILLIN - SENSITIVE CELLS MIX, SOME PLASMIDS ENTER CELLS PENICILLIN - RESISTANT TRANSFORMANT SELECT: PLATE ON MEDIUM AND PENICILLIN PLASMID ONLY NO COLONIES RECIPIENT CELLS ONLY NO COLONIES RECIPIENT CELLS AND PLASMIDS COLONIES PENICILLIN - R

10. 10 ~108 PEN - S CELLS [RECIPIENT] ~1µg PLASMID [PENICILLIN - R] ~108 RECIPIENT CELLS PLUS ~1µg PLASMID PEN - R TRANSFORMANTS MEDIUM AND PENICILLIN

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16. 14 PLASMID R1 REAL - NATURAL RESISTANCE GENES ON TRANSPOSONS KANAMYCIN NEOMYCIN Tn5 STREPTOMYCIN IS1 SULFONAMIDE TETRACYCLINE Tn3 PENICILLIN Tn10 IS3 IS3 Tn4 IS2 NOTE: SEVERAL INSERTION SEQUENCES IS1 TRANSFER GENES REPLICATION GENES AND SITES 100,000 BP

17. 15 INTEGRONS - GENE CAPTURE ELEMENTS • CODE FOR INFORMATION NECESSARY TO INSERT HOST GENE INTO ITSELF • INTEGRASE TO MOVE GENE • ATTACHMENT SITE TO PUT THE GENE INTO • HAVE A PROMOTER TO EXPRESS CAPTURED GENE • ACQUIRE VARIOUS HOST GENES • EXIST IN MANY FORMS - DIFFER IN NUMBER AND IDENTITY OF CAPTURED GENES

18. PROMOTER FOR INSERTED GENE ATT SITE FOR INCOMING GENES INTEGRASE PROMOTER FOR INTEGRASE GENE FOR DRUG RESISTANCE OR TOXIN HOST GENE 1 HOST GENE 2 16 • INTEGRON • INTEGRON WITH INSERTED HOST GENE • INTEGRON WITH MULTIPLE INSERTIONS OF HOST GENES

19. 17 • SUPER INTEGRON - VIBRIO CHOLERAE CHROMOSOME 179 INSERTED GENES • HOW ARE INTEGRONS INVOLVED IN MOBILITY OF DNA? • BECAUSE INTEGRONS CAN BE LOCATED ON TRANSPOSONS AND ON CONJUGATIVE PLASMIDS, WHEN THEY CATALYZE MOVEMENT OF HOST GENES INTO THEMSELVES, THE OVERALL RESULT IS MOVEMENT OF GENES INTO TRANSPOSONS AND CONJUGATIVE PLASMIDS

20. 18 CONJUGATIVE TRANSPOSONS • MOVE FROM FIRST HOST (DONOR) GENOME SECOND HOST (RECIPIENT) GENOME • CELL-CELL CONTACT • SOME MOVE TO NEW SITES • SOME MOVE TO IDENTICAL SITES • 20 - 100 KB WITH GENES FOR: • INTEGRATION AND EXCISION • CONJUGATION • ANTIBIOTIC RESISTANCE • TRANSFER VIA CIRCULAR INTERMEDIATE AND THEN INTEGRATE

21. 19 TRANSFER ORIGIN = oriT DRUG RESISTANCE TRANSFER OPERON INTEGRASE EXCISIONASE oriT Tn916 TET-R EXCISION (IN DONOR CELL) • SINGLE STRAND TRANSFERRED TO RECIPIENT • CIRCULARIZES IN RECIPIENT • COPIED INTO DOUBLE STRAND • INTEGRATES INTO RECIPIENT CHROMOSOME oriT

22. 20 GENOMICISLANDS LINEAR, 10-200 KB, IN CHROMOSOMES INTEGRATION / TRANSFER ENZYMES BOUNDARIES ARE DIRECT REPEATING SEQUENCES DIFFER FROM HOST CHROMOSOME IN G AND C CONTENT OF DNA - INDICATES ORIGINATION IN ANOTHER GENUS PERMIT EVOLUTION IN QUANTUM LEAPS GENOMIC ISLAND DIRECT REPEAT INTEGRATION GENE DIRECT REPEAT (SEQUENCE)

23. 21 BRING IN GENES WHICH HELP SURVIVAL (OF BACTERIA)

24. 22 UROPATHOGENIC E. COLI MOST COMMON CAUSE OF BLADDER AND KIDNEY INFECTIONS E. COLI (06: K15: H31) FOUR PATHOGENICITY ISLANDS

25. 23 THE BUSY GENOME: ELEMENTS OF HORIZONTAL EXCHANGE GENOMIC ISLANDS e.g. ESCHERICHIA COLI COMMON: 4.1 MB K12 ISLANDS: 0.53 MB 0157:H7 ISLANDS: 1.34 MB PROPHAGES CONJUGATIVE TRANSPOSONS MINIMAL SPECIES GENOMIC BACKBONE SUPER INTEGRONS INSERTION SEQUENCES INTEGRONS TRANSPOSONS