Molecular analysis of oriT and MobA protein in the 7.4 kb mobilizable beta-lactamase plasmid pSJ7.4 from Neisseria gonorrhoeae.
The mobilization region of the 7.4 kb beta-lactamase plasmid pSJ7.4 from Neisseria gonorrhoeae was characterized. The 3.2 kb HindIII-BamHI fragment of pSJ7.4 was mobilized between Escherichia coli strains by conjugative plasmid RK2. Selected restriction enzyme-generated deletions of this fragment were subcloned in pACYC177 to obtain constructs that were suitable for analysis of the mobilization region. Mating experiments showed that a highly conserved 1.9 kb DNA region within coordinates 4096-5997 is required for mobilization of pSJ7.4. This region contains two genes encoding the mobilization protein MobA, another protein, MobC, putatively involved in mobilization from some hosts, and an intergenic oriT. The 168 bp intergenic sequence also contains the promoters for mobA and mobC in an arrangement that suggests divergent transcription and autoregulation from oriT. The 56 kDa MobA was expressed in E. coli as a (6x)His-Tag fusion protein. Purified MobA specifically induced plasmid relaxation by nicking at the oriT. MobA is exceptional because the N-terminal region alone can mobilize pSJ7.4, albeit at a lower frequency than the full-length protein, even in the absence of MobC. The carboxyl terminal region of MobA did not share homology with other mobilization proteins, but may be involved in promoting efficient transfer of pSJ7.4.
Rodriguez-Bonano
Nydia M
NM
School of Sciences, Mathematics and Technology, Universidad del Este, Carolina, Puerto Rico 00984. nrodrigu@suagm.edu
Torres-Bauza
Luis J
LJ
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000201:enzymology / Q000235:genetics*; D011815:R Factors / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D018741:Replication Origin / Q000235:genetics*; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000235:genetics*; D014170:Transformation, Genetic; D001618:beta-Lactamases / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D001618:beta-Lactamases