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Applied and Environmental Microbiology, September 2008, p. 5686-5694, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.01235-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Construction of arsB and tetH Mutants of the Sulfur-Oxidizing Bacterium Acidithiobacillus caldus by Marker Exchange{triangledown}

Leonardo J. van Zyl, Jolanda M. van Munster, and Douglas E. Rawlings*

Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa

Received 4 June 2008/ Accepted 15 July 2008

Acidithiobacillus caldus is a moderately thermophilic, acidophilic bacterium that has been reported to be the dominant sulfur oxidizer in stirred-tank processes used to treat gold-bearing arsenopyrite ores. It is also widely distributed in heap reactors used for the extraction of metals from ores. Not only are these bacteria commercially important, they have an interesting physiology, the study of which has been restricted by the nonavailability of defined mutants. A recently reported conjugation system based on the broad-host-range IncW plasmids pSa and R388 was used to transfer mobilizable narrow-host-range suicide plasmid vectors containing inactivated and partially deleted chromosomal genes from Escherichia coli to A. caldus. Through the dual use of a selectable kanamycin resistance gene and a hybridization probe made from a deleted portion of the target chromosomal gene, single- and double-recombinant mutants of A. caldus were isolated. The functionality of the gene inactivation system was shown by the construction of A. caldus arsB and tetH mutants, and the effects of these mutations on cell growth in the presence of arsenic and by means of tetrathionate oxidation were demonstrated.

* Corresponding author. Mailing address: Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa. Phone: 27-21-808 3071. Fax: 27-21-808 3680. E-mail: der{at}sun.ac.za

{triangledown} Published ahead of print on 25 July 2008.

Applied and Environmental Microbiology, September 2008, p. 5686-5694, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.01235-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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