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Journal of Bacteriology, June 2001, p. 3303-3309, Vol. 183, No. 11
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
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.
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
*
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.
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.
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