Isolation of a new broad-host-range IncQ-like plasmid, pTC-F14, from the acidophilic bacterium Acidithiobacillus caldus and analysis of the plasmid replicon

A moderately thermophilic (45 to 50 degreesC), highly acidophilic (pH 1.5 t o 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating fie ld electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids of approximately 14 and 45 kb, The 14 kb plasmid, designated p TC-F14, was cloned and shown by replacement of the cloning vector with a ka namycin resistance gene to be capable of autonomous replication in Escheric hia coli. Autonomous replication was also demonstrated in Pseudomonas putid a and Agrobacterium tumefaciens LBA 4404, which suggested that pTC-F14 is a broad host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids, Three of the open reading frames encod ed replication proteins which were most closely related to those of IncQ-li ke plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; R epC, 74%), However, the two plasmids were fully compatible and pTC-F14 repr esents a new IncQ-like plasmid replicon, Surprisingly, asymmetrical incompa tibility was found with the less closely related IncQ plasmid R300B derivat ive pKE462 and the IncQ-like plasmid derivative pIE1108, Analysis of the pT C-F14 oriV region revealed five direct repeats consisting of three perfectl y conserved 22-bp iterons flanked by iterons of 23 and 21 bp, Plasmid pTC-F 14 had a copy number of 12 to 16 copies per chromosome in both E. coli, and A. caldus. The rep gene products of pTC-F14 and pTF-FC2 were unable to fun ctionally complement each other's oriV regions, but replication occurred wh en the genes for each plasmid's own RepA, RepB, and RepC proteins were prov ided in trans. Two smaller open reading frames were found between the repB and repA genes of pTC-F14, which encode proteins with high amino acid seque nce identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF- FC2. This is the second time a plasmid stability system of this type has be en found on an IncQ-like plasmid.