Characterisation of plasmid p31T1 isolated from Aeromonas

[摘要] ENGLISH ABSTRACT: Plasmids are an integral part of the horizontal gene pool and, therefore, are the mainvectors for the spread of antibiotic and heavy metal resistance genes in theenvironment. Functional and taxonomic characterization of novel plasmids is, therefore,central to our general understanding of plasmid biology and their contribution tomicrobial evolution. Two 14-kb mobilizable plasmids, p31T1 and p36T2, conferringresistance to tetracycline were isolated from the opportunistic fish pathogensAeromonas sobria and Aeromonas hydrophila and were found to have indistinguishablerestriction fragment length polymorphism (RFLP) patterns (Marx, MSc Thesis). DNAsequence analysis of the two isogenic plasmids (only p36T2 was sequenced) revealedthe presence of 18 putative open reading frames (ORFs), of which the tetARtetracycline resistance genes, associated with a truncated Tn1721, were the only ORFswith significant similarity to known sequences within the NCBI database. Putativefunctions were assigned to 10 of the ORFs based on their distant homology withproteins of known function. Six of the 18 ORFs, spanning 5.7-kb, were found tocomprise the minimal region required for replication (minimal replicon) by means ofdeletion analysis using derivatives of p31T1. Of the six ORFs, ORF2 and ORF4 werefound to be essential for plasmid replication. Inactivation of ORF3 resulted in anincrease of plasmid copy number (PCN) from ~3 to ~7 plasmids per chromosome and adecrease in plasmid stability from ~80 % to 16 % over approximately 127 generations (7days). Furthermore, by means of β-galactosidase promoter fusion assays it was shownthat ORF3 autoregulated its own promoter. These results, therefore, suggested thatalthough ORF3 was not essential for replication, it may be involved in plasmid copynumber regulation and control. Host range analysis indicated that p31T1 was able toreplicate in two other members of the γ-proteobacteria group (Escherichia coli andPseudomonas putida) but was unable to do so in an α-proteobacterium strain, thussuggesting a limited host range. Furthermore, p31T1 was mobilized only at lowfrequencies (5.4 x 10-5 transconjugants per donor) by an IncP-1 conjugative systemthough it is possible that the mobilization system of these plasmids is adapted to function optimally with alternate conjugative systems. Given the unique PCN, stability,host range and mobilization characteristics determined for p31T1 and that no otherplasmid replication and mobilization systems with significant sequence similarity tothese plasmids have yet been identified, it is likely that these two plasmids are the firstrepresentative members of a new family of plasmids found within aquacultureassociatedAeromonas species and which are involved in the spread of tetracyclineresistance.

[发布机构] Stellenbosch University