ORIGINAL PAPER Phylogenetic analysis of different isolates of Sulfobacillus spp. isolated from uranium-rich environments and recovery of genes using integron-specific primers Received: 19 November 2002 / Accepted: 30 November 2002 / Published online: 30 August 2003 Ó Springer-Verlag 2003 Abstract The isolation and phylogenetic characterization of acidophilic moderate thermophilic bacteria from dif- ferent locations of uranium mines and a uranium pro- cessing mill in Pakistan is reported. The dominant culturable bacteria found were related to Sulfobacillus thermosulfidooxidans in all the samples analyzed. Dif- ferent strains displayed different levels of identity (95– 97%) to16S rDNA of known strains of this species, indicating group heterogeneity. Genomic DNA from five isolates was subjected to amplification using integron- specific primers HS286 and HS287. Recovery of differ- ent integron-linked genes from one of the isolates indicated the usefulness of this approach for gene mining in place of traditional gene recovery methodologies. Keywords Acidophilic moderate thermophiles Æ Gene cassettes Æ Integrons Æ 16S rDNA analysis Æ Uranium rich environments Introduction The interest in the study of microbial diversity has been enhanced by the fact that only a small proportion of the microbial community is culturable. This has stimulated the development and use of new molecular methods for detecting taxon-specific genes without the need for cul- ture (Lane 1991). The microbial genomic era has revolutionized our understanding of the composition of bacterial gene pools and how different genes have been transferred vertically as well as horizontally. It has been clearly demonstrated that a substantial part of the bacterial gene pool is extrachromosomal and can vary between members of the same species. In addition, a large proportion of bacterial genes has been acquired by horizontal gene transfer (Ochman et al. 2000). Horizontal gene transfer in bac- teria is facilitated by a number of genetic elements including phages, plasmids, transposons, and integrons. Previously, most attention has been focused on plasmids and transposons. This is particularly true for environ- mental microorganisms (Smalla et al. 2000). Therefore, one possible way to recover a proportion of the envi- ronmental gene pool is to target conserved genetic se- quences associated with mobile genetic elements. An integron is defined as a genetic element that possesses a site, attI, at which additional DNA, in the form of gene cassettes, can be integrated by site-specific recombination, and which encodes an enzyme, integrase, that mediates these site-specific recombination events (Bennett 1999). Gene cassettes are discrete genetic ele- ments that may exist as free, circular, non-replicating DNA molecules when moving from one genetic site to another (Collis and Hall 1992), but which are normally found as linear sequences that constitute part of a larger DNA molecule, such as a plasmid or chromo- some. Gene cassettes normally contain only a single gene and an additional short sequence, called a 59-base element, that functions as a specific recombination site (Hall et al. 1991). The genes carried on gene cassettes usually lack a promoter and are expressed from a promoter on the integron (Hall and Collis 1995). The range of genera now known to host integrons is very broad and includes Vibrio, Pseudomonas, Shewanella, Geobacter, Xanthomonas, Listonella, Photobacterium, Extremophiles (2003) 7:341–345 DOI 10.1007/s00792-003-0354-3 Muhammad A. Ghauri Æ Ahmad M. Khalid Æ Susan Grant Shaun Heaphy Æ William D. Grant Communicated by K. Horikoshi S. Grant Æ S. Heaphy Æ W. D. Grant (&) Department of Microbiology and Immunology, Maurice Shock Medical Sciences Building, University of Leicester, University Road, Leicester, LE1 9HN, UK E-mail: wdg1@le.ac.uk Tel.: +44-116-2522948 Fax: +44-116-2525030 M. A. Ghauri Æ A. M. Khalid National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan Present address: M. A. Ghauri Department of Microbiology and Immunology, University of Leicester, Leicester, UK