Gardner M N, Deane S M, Rawlings D E
Department of Microbiology, University of Stellenbosch, Matieland 7602, South Africa.
J Bacteriol. 2001 Jun;183(11):3303-9. doi: 10.1128/JB.183.11.3303-3309.2001.
A moderately thermophilic (45 to 50 degrees C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids 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 kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida 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 encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14 oriV region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 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 functionally complement each other's oriV regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided 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 sequence 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 been found on an IncQ-like plasmid.
从用于处理镍矿石的生物氧化过程中分离出一株中度嗜热（45至50摄氏度）、高度嗜酸（pH 1.5至2.5）的化能自养嗜酸氧化亚铁硫杆菌菌株f。对嗜酸氧化亚铁硫杆菌细胞的总DNA进行交变电场凝胶电泳分析，发现了两个大小约为14 kb和45 kb的质粒。将14 kb的质粒命名为pTC-F14，进行克隆，并通过用卡那霉素抗性基因取代克隆载体，证明其能够在大肠杆菌中自主复制。在恶臭假单胞菌和根癌农杆菌LBA 4404中也证实了自主复制，这表明pTC-F14是一种广宿主范围的质粒。对pTC-F14复制子区域的序列分析揭示了五个开放阅读框以及与广宿主范围的IncQ质粒相似的复制子结构。其中三个开放阅读框编码的复制蛋白与IncQ样质粒pTF-FC2的复制蛋白关系最为密切（氨基酸序列同一性：RepA，81%；RepB，78%；RepC，74%）。然而，这两个质粒完全兼容，pTC-F14代表了一种新的IncQ样质粒复制子。令人惊讶的是，发现它与亲缘关系较远的IncQ质粒R300B衍生物pKE462和IncQ样质粒衍生物pIE1108存在不对称不相容性。对pTC-F14 oriV区域的分析揭示了五个直接重复序列，由三个完全保守的22 bp重复子组成，两侧分别是23 bp和21 bp的重复子。在大肠杆菌和嗜酸氧化亚铁硫杆菌中，质粒pTC-F14的拷贝数均为每条染色体12至16个拷贝。pTC-F14和pTF-FC2的rep基因产物不能在功能上互补彼此的oriV区域，但当为每个质粒自身的RepA、RepB和RepC蛋白提供反式基因时，复制就会发生。在pTC-F14的repB和repA基因之间发现了两个较小的开放阅读框，它们编码的蛋白质与pTF-FC2的质粒成瘾系统具有较高的氨基酸序列同一性（PasA，81%；PasB，72%）。这是第二次在IncQ样质粒上发现这种类型的质粒稳定性系统。
