An investigation into the replicon of a broad host range mobilizable plasmid from the moderately thermophilic bacterium Acidithiobacillus caldus

[摘要] ENGLISH ABSTRACT:The moderately thermophilic (45 to 50DC), highly acidophilic (pH 1.5 to 2.5),chemolithoautotrophic Acidithiobaci/lus caldus strain f' was isolated from abiooxidation process used to treat nickel ore concentrates. Trans-Alternating FieldElectrophoresis (TAFE) analysis of total DNA from the At. caldus cells revealed twoplasmids of approximately 14 and 45-kb. The 14-kb plasmid, designated pTC-F14,was cloned and shown by replacement of the cloning vector with a kanamycinresistance gene to be capable of autonomous replication in Escherichia coli.Autonomous replication was also demonstrated in Pseudomonas putida andAgrobacterium tumefaciens LBA 4404 which suggested that pTC-F14 was a broadhost-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed fiveopen-reading frames, and a replicon organization like that of the broad host-rangeIncQ plasmids. Three of the open-reading frames encoded replication proteins withamino acid sequence identities similar to that of the IncQ-like plasmid pTF-FC2(RepA, 81%; RepB, 78%; RepC, 74%). This high level of relatedness suggested thatthe two replicons had evolved from a common ancestor. Since closely relatedreplicons are usually incompatible, the compatible replicons of pTC-F14 and pTFFC2raised the question of how the replicons of the two sister plasmids had evolvedsuch that they can now co-exist in the same host cell line. Further incompatibilitytesting with the IncQ-like plasmid pIEll08 and the IncQ prototype plasmidRSF10101R11621R300B determined that pTC-F14 was compatible with pIEI108, butincompatible with the IncQ prototype plasmid. It was found that the RepB and RepAreplication proteins ofpTF-FC2 and pIEll08 were able to complement the pTC-FI4orthologs only ifpTC-F14 RepC was present in trans. The RepC protein ofpTC-F14was thus plasmid-template specific, while the RepA and RepB proteins were lessplasmid-template specific. A five nucleotide possible iteron-discriminating region inthe direct repeats of IncQ-like plasmid oriV regions has been identified (Tietze, E.(1998) Plasmid 39: 165-181). The iteron sequence ofpTC-F14 differs from pTF-FC2and pIE 1108 by three nucleotides in this iteron-discriminating region. It wastherefore proposed that co-evolution of the iterons and the RepC protein to a pointwhere the RepC protein no longer recognizes the iteron sequence of a closely relatedsister plasmid is the mechanism by which replicons evolve to become compatible in the same host cell. The incompatibility determinant of the IncQ prototype plasmidRSFlOlOIR11621R300B was also sought, and subsequently localized to the regionencoding the IncQ prototype plasmid's repAC genes. Interference with the initiationof pTC-F14 replication by the IncQ prototype plasmid was demonstrated by growthinhibition of a replication-deficient M13 bacteriophage into which oriVpTC-F14 hadbeen cloned. Secondly, the IncQ prototype derivative pKE462 displaced a ColEloriVpTC-F14 construct in complementation assays, and a construct containing only thepTC-F14 repBAC genes similarly displaced the pKE462 plasmid. As the oriVRSFIOIOregion was not incompatible with a pTC-F14 replicon, this suggested that it was notthe oriV region which was expressing incompatibility, but the products of the IncQprototype plasmid repAC genes. It is proposed that incompatibility between pTC-F14and the IncQ prototype plasmid was the consequence of the repAC gene productsbinding to the iterons of the related rep licon, and that these products are unable toinitiate replication. The compatible phenotypes expressed by members of the IncQplasmid family indicates the inadequacy of using plasmid incompatibility as aclassification system. Alignment of the amino acid sequences of the three replicationprotein orthologs clearly showed that the IncQ plasmid family was divided into twogroups. To account for replication protein relatedness and the incompatibilityphenotype expressed, it is now proposed that that members of the IncQ family beclassified into subdivisions that reflect the different IncQ-like replicons identified inthis study. Investigation of pTC-F14 replicon regulation identified a putativepromoter sequence which is believed to regulate the initiation of a 5.l-5.7-kbpolycistronic transcript that encodes all the replication proteins of the pTC-F14replicon and the MobB and MobA proteins of the IncP-type mobilization module.The large polycistronic transcript appears to regulated by the RepB protein of thepTC-F14 replicon, and is not subject to cross-regulation by related IncQ plasmids.This suggested that the RepB primase function was not plasmid specific, but that itsregulatory function was replicon specific. A second putative promoter sequenceidentified upstream of the pTC-F14 pasAB operon was, however, cross-regulated bythe closely related pTF-FC2 plasmid. The pTC-F14 pas operon encodes two proteinswith high amino acid sequence identity (PasA, 81 %; PasB, 72 %) to the plasmidaddiction system ofpTF-FC2. This is the second time a plasmid addiction system ofthis type has been found on an IncQ-like plasmid.

[发布机构] Stellenbosch University