Isolation of a New Broad-Host-Range IncQ-Like Plasmid, pTC-F14, from the Acidophilic Bacterium Acidithiobacillus caldus and Analysis of the Plasmid Replicon

doi:10.1128/JB.183.11.3303-3309.2001

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Journal of Bacteriology, June 2001, p. 3303-3309, Vol. 183, No. 11
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.11.3303-3309.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Isolation of a New Broad-Host-Range IncQ-Like Plasmid, pTC-F14, from the Acidophilic Bacterium Acidithiobacillus caldus and Analysis of the Plasmid Replicon

Murray N. Gardner, Shelly M. Deane, and Douglas E. Rawlings^*

Department of Microbiology, University of Stellenbosch, Matieland 7602, South Africa

Received 13 December 2000/Accepted 16 March 2001

A moderately thermophilic (45 to 50°C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain,f, was isolated from a biooxidation process used to treat nickelore. Trans-alternating field electrophoresis analysis of totalDNA from the A. caldus cells revealed two plasmids of approximately14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was clonedand shown by replacement of the cloning vector with a kanamycinresistance gene to be capable of autonomous replication in Escherichiacoli. Autonomous replication was also demonstrated in Pseudomonasputida and Agrobacterium tumefaciens LBA 4404, which suggestedthat pTC-F14 is a broad-host-range plasmid. Sequence analysisof the pTC-F14 replicon region revealed five open reading framesand a replicon organization like that of the broad-host-rangeIncQ plasmids. Three of the open reading frames encoded replicationproteins which were most closely related to those of IncQ-likeplasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB,78%; RepC, 74%). However, the two plasmids were fully compatibleand pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly,asymmetrical incompatibility was found with the less closely relatedIncQ plasmid R300B derivative pKE462 and the IncQ-like plasmidderivative pIE1108. Analysis of the pTC-F14 oriV region revealedfive direct repeats consisting of three perfectly conserved 22-bpiterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 hada 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 wereunable to functionally complement each other's oriV regions, butreplication occurred when the genes for each plasmid's own RepA,RepB, and RepC proteins were provided in trans. Two smaller openreading 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. Thisis the second time a plasmid stability system of this type hasbeen found on an IncQ-likeplasmid.

^* Corresponding author. Mailing address: Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland,South Africa. Phone: 27 21 808 5848. Fax: 27 21 808 5846. E-mail:der{at}land.sun.ac.za.

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