Summary
A chimeric β-lactamase encoding plasmid, containing the 4.4 Mdal β-lactamase plasmid and the 2.6 Mdal cryptic plasmid of Neiseria gonorrhoeae has been characterized by physical and biological methods. Digestion with restriction enzymes indicates the presence of the following restriction sites: 1 site: AccI, AvaI, HgiAI, HincII, MstI, PstI, PvuII, XbaI and XorI; 2 sites: HindIII and BamHI; 3 sites: BclI, Sau961 and AvaII; 6 sites: HinfI; >8 sites: AluI, BbvI, DdeI, HhaI, HpaII, MspI, Sau3A, TacI and TaqI. No restriction sites were found for the following: BglI, BglII, BstEII, EcoRI, EcoRII, HpaI, HphII, KpnI, SacI, SalI, SmaI, SstI, SstII, and XhoI. Five plasmid specific proteins have been identified by DNA directed in vitro protein synthesis (43K, 41K, 30K, 16K and 14K). The location on the physical map of the coding regions for each of these proteins has been determined by the following methods: using plasmid DNA restricted by various enzymes in an in vitro protein synthesis system; identifying promoter-containing regions by digesting plasmid DNA with DdeI, adding RNA polymerase and then determining which fragments are retained by nitrocellulose. This plasmid contains both parental phenotypes in that it encodes penicillin resistance and possesses the sequence necessary for uptake in the gonococcus. Transformation data indicate that this plasmid can function in both E. coli and N. gonorrhoeae and that growth in E. coli has no effect on the plasmid's ability to transform the gonococcus.
Similar content being viewed by others
References
Dougherty TJ, Asmus A, Tomaza A (1979) Specificity of DNA uptake in genetic transformation of gonococci. Biochem Biophys Res Comm 86:97–104
Elwell LP, Roberts M, Mayer LW, Falkow S (1977) Plasmid mediated beta-lactamase production in Neisseria gonorrheae. Antimicrob Agents Chemother 11:528–533
Guerry P, LeBlanc DJ, Falkow S (1973) General method for the isolation of plasmid deoxyribonucleic acid. J Bacteriol 116:1064–1066
Heffron F, McCarthy BJ, Ohtsubo H, Ohtsubo E (1979) DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transportation of Tn3. Cell 18:1153–1163
Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–201
Inouye H, Pratt C, Beckwith J, Torriani A (1977) Alkaline phosphatase synthesis in a cell free system using DNA and RNA templates. J Mol Biol 110:75–87
Lovett M, Falkow S (1981) Cloning strategies for gonococci. In: Danielsson D, Normark S (eds) Genetics and immunobiology of pathogenic Neisseria. University of Umea, Umea, Sweden. p 143–144
Morrison DA (1979) Transformation and preservation of competent bacterial cells by freezing. Methods in Enzymol 68:326–331
O'Calloghan CH, Morric A, Kirby SM, Shingler AH (1972) Novel method for the detection of β-lactamase by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother 1:283–288
Phillips I (1976) Beta-lactamase-producing penicillin resistant gonococcus. Lancet II:656–657
Sarubbi FA Jr, Blackman E, Sparling PF (1974) Genetic mapping of linked antibiotic resistance loci in Neisseria gonorrhoeae. J Bacteriol 120:1284–1292
Sparling PF (1966) Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance. J Bacteriol 124:1364–1371
Sparling PF (1977) Antibiotic resistance in the gonococcus. In: Roberts RB (ed) The gonococcus. John Wiley and Sons, New York, p 111–136
Sparling PF, Biswas GD, Sox TE (1977) Transformation of the gonococcus. In: Roberts RB (ed) The gonococcus. John Wiley and Sons, New York, p 155–176
Sparling PF, Biswas G, Graves J, Blackman F (1981) Mechanisms of gene exchange in the gonococcus. In: Danielsson D, Normark S (eds) Genetics and immunobiology of pathogenic Neisseria. University of Umea, Umea, Sweden, p 123–125
West RW, Rodriguez RL (1980) Construction and characterization of E. coli promoter-probe plasmid vectors. II. RNA polymerase binding studies of antibiotic-resistance promoters. Gene 9:175–193
Wilhelm J, Haselkorn R (1971) In vitro protein synthesis directed by RNA from T4-infected Escherichia coli. Methods Enzymol 20:531–536
Zubay G, Chambers DA, Chenong LC (1970) Cell free studies on the regulation of the lac operon. In: Beckwith JR, Zipser D (eds) The lactose operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, p 375–392
Author information
Authors and Affiliations
Additional information
Communicated by H. Boyer
Rights and permissions
About this article
Cite this article
Stein, D.C., Young, F.E., Tenover, F.C. et al. Characterization of a chimeric β-lactamase plasmid of Neisseria gonorrhoeae which can function in Escherichia coli . Molec Gen Genet 189, 77–84 (1983). https://doi.org/10.1007/BF00326058
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00326058