Abundance of sulphur-oxidizing bacteria in coastal aquaculture using soxB gene analyses Kishore K Krishnani, Gopalapillay Gopikrishna, Subramanian M Pillai & Baijnath P Gupta Central Institute of Brackishwater Aquaculture, R. A. Puram, Chennai, India Correspondence: K K Krishnani, Central Institute of Brackishwater Aquaculture, 75, Santhome High Road, R. A. Puram, Chennai 600028, India. E-mail: krishnanik@hotmail.com Abstract Molecular techniques based on sequencing of metage- nomic clone libraries provide an insight into the diver- sity of microbial populations. Using nucleic acid-based methods, the diversity of soxB genes was examined to detect and characterize sulphur-oxidizing bacteria in Indian coastal aquaculture environments. Gene-spe- ci¢c degenerate primers were used to amplify various fragments (710, 753, 483^503, 280 and 239 bp) of soxB genes. Metagenomic clone libraries were constructed for 753, 483^503 and 239 bp fragments of soxB genes. The abundance of soxB revealed the presence of sul- phur-oxidizing organisms. Amino acids in parts of the soxB-encoded proteins were aligned to known conserved amino acid residues. The level of conserva- tion ranged from 23% to 30%. A phylogenetic tree constructed from aligned amino acid sequences of SoxB revealed di¡erent clusters associated with the branches of phototrophic a- and g-proteobacteria. In general, soxB is widespread among the various phylo- genetic groups, although this does not necessarily mean that the organism can use sulphur compounds. Our results suggest that the chemolithoautotrophy based on sulphur oxidation in coastal aquaculture is primarily sustained by the presence of sulphur oxidi- zers, which involve the soxB gene. This study aids identi¢cation of the phylogenetic characteristics re- lated to sulphur bioremediation in poorly character- ized coastal aquaculture environments. Keywords: sulphur-oxidizing bacteria, diversity, soxB genes, sequence analysis, coastal aquaculture Introduction Aerobic chemolithoautotrophic members of domain archaea and bacteria are important for the oxidation of reduced inorganic sulphur compounds, which act as electron donors for their energy-generating sys- tems in aquatic environments. There is a need to analyse these sulphur-oxidizing organisms for their ecological functions in nature, because these organ- isms utilize all types of reduced sulphur compounds such as sulphide, elemental sulphur, polythionates and thiosulphate as energy sources and mostly oxi- dize them to sulphate (Lane, Harrison Jr, Stahl, Pace, Giovannoni, Olsen & Pace 1992; Robertson & Kue- nen 1992; Kelly, Shergill, Lu & Wood 1997). Current e¡orts to assess and monitor sulphide-contami- nated sites rely on nucleic acid-based approaches targeting 16S rRNA. A major limitation of the 16S rRNA gene approach lies in the selective analysis of phylogenetically diverse sulphur-oxidizing bacteria (SOB) (Imho¡ 1992; Friedrich 1998). Alternatively, molecular approaches based on functional genes of the sulphur oxidation pathway are much more sui- ted to analyse the functional group in nature, which use speci¢c genes and allows a greater resolution for the study of genetic di¡erences in natural popula- tions of SOB. Di¡erent enzymes mediate conversion of various reduced sulphur compounds. Their phy- siological function in sulphur oxidation is consid- ered mostly from the biochemical characterization of the enzymatic reaction (Friedrich 1998). The cur- rent model of the Sox enzyme system comprises the four periplasmic complexes SoxXA, SoxYZ, SoxB and Sox (CD)2 that catalyse oxidation of reduced sulphur compound, thiosulphate. There are two groups of thiosulphate-oxidizing organisms that use the Sox enzyme system: the ¢rst group forms sulphur globules as intermediates ( Allochromatium vinosum), which does not have Sox (CD)(2) (Welte, Hafner, Kratzer, Quentmeier, Friedrich & Dahl 2009). In the second group ( Paracoccus pantotro- Aquaculture Research, 2010, 41 , 1290^1301 doi: 10.1111/j.1365-2109.2009.02415.x r 2010 The Authors 1290 Journal Compilation r 2010 Blackwell Publishing Ltd