Abstract
A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, orori fd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the genetic manipulation of pathogenicNeisseriae are described.
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Amann EB, Ochs B, Abel KJ (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused protein inEscherichia coli. Gene 69:301–315
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1522
Biswas GD, Sox T, Blackman E, Sparling PF (1977) Factors affecting genetic transformation ofNeisseria gonorrhoeae. J Bacteriol 129:983–992
Biswas GD, Burnstein KL, Sparling PF (1986) Linearization of donor DNA during plasmid transformation inNeisseria gonorrhoeae. J Bacteriol 168:756–761
Biswas GD, Lacks SA, Sparling PF (1989) Transformation-deficient mutants of piliatedNeisseria gonorrhoeae. J Bacteriol 171:657–664
Campbell LA, Yasbin RE (1984) Mutagenesis ofNeisseria gonorrhoeae: absence of error-prone repair. J Bacteriol 160:288–293
Carbonetti NH, Simnad VI, Seifert HS, So M, Sparling PF (1988) Genetics of protein I ofNeisseria gonorrhoeae: construction of hybrid porins. Proc Natl Acad Sci USA 85:6841–6845
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the p15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Close TJ, Rodriguez RL (1982) Construction and characterization of the chloramphenicol-resistance gene cartridge: a new approach to the transcriptional mapping of extrachromosomal elements. Gene 20:305–316
Facius D, Meyer TF (1993) A novel determinant (comA) essential for natural transformation competence inNeisseria gonorrhoeae and the effect of acomA defect on pilin variation. Mol Microbiol 10:699–712
Ferrari FA, Nguyen A, Lang D, Hoch JA (1983) Construction and properties of an integretable plasmid forBacillus subtilis. J Bacteriol 154:1513–1515
Geider K, Hohmeyer KC, Haas R, Meyer TF (1985) A plasmid cloning system utilizing replication and packaging functions of the filamentous fd. Gene 33:341–349
Gibbs CP, Reimann BY, Schultz E, Kaufmann A, Haas R, Meyer TF (1989) Reassortment of pilin genes inNeisseria gonorrhoeae occurs by two distinct mechanisms. Nature 338:651–652
Goodman SD, Scocca JJ (1988) Identification and arrangement of the DNA sequence recognized in specific transformation ofNeisseria gonorrhoeae. Proc Natl Acad Sci USA 85:6982–6986
Haas R, Schwarz H, Meyer TF (1987) Release of soluble pilin antigen coupled with gene conversion inNeisseria gonorrhoeae. Proc Natl Acad Sci USA 84:9079–9083
Haas R, Kahrs AF, Facius D, Allmeier H, Schmitt R, Meyer TF (1993a) TnMax — a versatile mini-transposon for the analysis of cloned genes and shuttle mutagenesis. Gene 130:23–31
Haas R, Meyer TF, van Putten JPM (1993b) Aflagellated mutants ofHelicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis. Mol Microbiol 8:753–760
Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989
Kahrs AF, Bihlmaier A, Facius D, Meyer TF (1994) Generalized transposon shuttle mutagenesis inNeisseria gonorrhoeae: a method for isolating epithelial cell invasion-defective mutants. Mol Microbiol 12:819–831
Kathariou S, Stephens DS, Spellman P, Morse SA (1990) Transposition of Tn916 to different sites in the chromosome ofNeisseria meningitidis: a genetic tool for meningococcal mutagenesis. Mol Microbiol 4:729–735
Knapp JS, Zenilman JM, Biddle JW, Perkins GH, DeWitt WE, Thomas ML, Johnson SR, Morse SA (1987) Frequency and distribution in the United States of strains ofNeisseria gonorrhoeae with plasmid-mediated, high-level resistance to tetracycline. J Infect Dis 155:819–822
Koomey JM, Falkow S (1987) Cloning of therecA gene ofNeisseria gonorrhoeae and construction of gonococcalrecA mutants. J Bacteriol 169:790–795
Koomey JM, Gill RE, Falkow S (1982) Genetic and biochemical analysis of gonococcal IgA1 protease: cloning inEscherichia coli and construction of mutants of gonococci that fail to produce the activity. Proc Natl Acad Sci USA 79:7881–7885
Koomey JM, Gotschlich EC, Robbins K, Bergström S, Swanson J (1987) Effects ofrecA mutations on pilus antigenic variation and phase transitions inNeisseria gonorrhoeae. Genetics 117:391–398
Korch CP, Hagblom H, Öhman M, Göransson M, Normark S (1985) Cryptic plasmid ofNeisseria gonorrhoeae: complete nucleotide sequence and genetic organization. J Bacteriol 163:430–438
Kupsch EM, Knepper B, Kuroki T, Heuer I, Meyer TF (1993) Variable opacity (Opa) outer-membrane proteins account for the cell tropisms displayed byNeisseria gonorrhoeae for humanleucocytes and epithelial cells. EMBO J 12:641–650
Legerski RJ, Robberson DL (1985) Analysis and optimization of recombinant DNA joining reactions. J Mol Biol 181:297–312
Maniatis T, Fritsch EF, Sambrooks J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y.
Nassif X, Puaoi D, So M (1991) Transposition of Tn1543-D3 in the pathogenicNeisseriae: a genetic tool for mutagenesis. J Bacteriol 173:2147–2154
Projan SJ, Monod M, Narayanan CS, Dubnau D (1987) Replication properties of pIM13, a naturally occurring plasmid found inBacillus subtilis, and of its close relative pE5, a plasmid native toStaphylococcus aureus. J Bacteriol 169:5131–5139
Roberts MC, Falkow S (1979) In vivo conjugal transfer of R plasmids inNeisseria gonorrhoeae. Infect Immun 24:982–984
Roberts MC, Knapp JS (1988) Transfer ofβ-lactamase plasmids fromNeisseria gonorrhoeae toNeisseria meningitidis and commensalNeisseria species by the 25.2 megadalton conjugative plasmid. Antimicrob Agents Chemother 32:1430–1432
Roberts MC, Knapp JS (1989) Transfer frequency of various 25.2 Mdal tetracycline-resistant plasmids inNeisseria gonorrhoeae. Sex Trans Dis 16:91–94
Roberts MC, Wagenvoort JHT, van Kligeren B, Knapp JS (1988) TetM andβ-lactamase containingNeisseria gonorrhoeae (tetracycline-resistant and penicillinase-producing) in The Netherlands. Antimicrob Agents Chemother 32:158
Rudel T, van Putten JPM, Gibbs CP, Haas R, Meyer TF (1992) Interaction of two variable proteins (PilE and PilC) required for pilus-mediated adherence ofNeisseria gonorrhoeae to human epithelial cells. Mol Microbiol 6:3439–3450
Rudel T, Facius D, Barten R, Nonnenmacher E, Meyer TF (1995) Role of pili and the phase variable PilC protein in natural competence for transformation ofNeisseria gonorrhoeae. Proc Natl Acad Sci USA 92:7986–7990
Seifert HS, Ajioka RS, Paruchuri D, Heffron F, So M (1990) Shuttle mutagenesis ofNeisseria gonorrhoeae: pilin null mutations lower DNA transformation competence. J Bacteriol 172:40–46
Sharetzsky C, Edlind TD, LiPuma JJ, Stull TL (1991) A novel approach to insertional mutagenesis ofHaemophilus influenzae. J Bacteriol 173:1561–1564
Sparling PF (1966) Genetic transformation ofNeisseria gonorrhoeae to streptomycin resistance. J Bacteriol 92:1364–1370
Stein DC, Silver LE, Clark VL, Young FE (1983) Construction and characterization of a new shuttle vector, pLES2, capable of functioning inEscherichia coli andNeisseria gonorrhoeae. Gene 25:241–247
Stein DC, Gregoire S, Piekarowicz A (1988) Restriction of plasmid DNA during transformation but not conjugation inNeisseria gonorrhoeae. Infect Immun 56:112–116
Swanson J (1973) Studies on gonococcus infection. Pili: their role in attachment of gonococci to tissue culture cells. J Exp Med 137:571–581
Trieu-Cuot P, Gerbaut G, Lambert T, Courvalin P (1985)In vivo transfer of genetic information between Gram-positive and Gram-negative bacteria. EMBO J 4:3583–3587
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Kupsch, EM., Aubel, D., Gibbs, C.P. et al. Construction of hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformableNeisseria mutants. Molec. Gen. Genet. 250, 558–569 (1996). https://doi.org/10.1007/BF02174444
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DOI: https://doi.org/10.1007/BF02174444