Genetic analysis and molecular characterization of the naturally-occurring penicillinase-producing plasmids in Neisseria gonorrhoeae.

Title: Genetic analysis and molecular characterization of the naturally-occurring penicillinase-producing plasmids in Neisseria gonorrhoeae.
Authors: Yeung, Kwok-Him.
Date: 1989
Abstract: Two naturally-occurring beta-lactamase producing plasmids from Neisseria gonorrhoeae, 7.2 kilobases (kB) (Asian-type, pJD4) and 5.1 kb (African-type, pJD5) in size, were analysed and mapped by restriction endonuclease analysis and DNA-DNA heteroduplex analysis. The two plasmids were similar except for a 2.1 kb fragment missing in pJD5, as compared to pJD4, which was located 1.74 kb counter-clockwise from the unique PstI site. In addition, a novel penicillinase-producing plasmid, 4.9 kb (pJD7, 'Toronto-type') in size was discovered during the study and was compared to pJD4 and pJD5. Except for a 2.3 kb fragment, pJD7 was shown to be similar to pJD4 and probably derived from pJD4. Plasmid pJD7 was compared to a plasmid of reportedly smaller size, pGO4717, isolated in the Netherlands and was found to be identical to it. Part of the large BamHI fragment of pJD4 was sequenced and compared to that of Tn3. It was shown that after base 79 from the BamHI site, the sequence starts to differ. The presence of two replication regions on pJD4 has been identified. Through the construction of mini-plasmids (pJD8, pJD9), the replication region of pJD4 was located on a 1.5 kb fragment, designated region 'a', which included the unique HindIII site. However, fragment 'a' was absent in pJD5. A 1.5 kb fragment (region 'b') was found to be required for the replication of this plasmid. In pJD4, the nucleotide sequence of region 'b' was interrupted by region 'a'. Incompatibility studies showed that, in vitro derived deletion-derivatives from pJD4 and pJD5, containing either region 'a' or region 'b', were compatible. Sequence analysis of region 'a' indicated that this region shares many characteristics of plasmids such as pSC101, P1, F, and R6K. Ten conjugative plasmids were tested for their ability to mobilize plasmid pJD4 between E. coli strains. Plasmid pJD4 was mobilized by RP4 (IncP), R124 (IncIV), pBG791 (IncI$\alpha)$ and R100 (IncFII), but not by R621a (IncI$\gamma$), R199 (IncN), R6K (IncX), R1drd16 (IncFII), TP124 (IncH$\sp{\rm e}$) and S-a (IncW). The 2.4 kb BamHI fragment of pJD4, containing the beta-lactamase gene and a 1.0 kb BamHI- HinfI fragment designated region 'M', was cloned into pACYC184, and was shown to be required in cis for the mobilization of the recombinant plasmid by pBG791. The entire DNA sequence of region 'M' was analysed, and contained sequences that were found to be essential for the conjugal transfer of other plasmids. Mobilization of deletion derivatives of the 7.2 kb and the 5.1 kb penicillinase-producing plasmids of N. gonorrhoeae by pBG791 indicated another origin of transfer, located within one of the two replication regions in pJD4.
CollectionTh├Ęses, 1910 - 2010 // Theses, 1910 - 2010
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