EVALUATION OF AMPLIFIED RDNA RESTRICTION ANALYSIS (ARDRA) FOR IDENTIFICATION AND CHARACTERIZATION OF ARSENITE RESISTANT BACTERIAL ISOLATES

-The disposal of toxic heavy metals such as arsenic posed high risk to the environment. Arsenite [As(III)], a reduced form of arsenic, is more toxic and mobile than arsenate [As(V)]. The aim of this work was to isolate arsenic-tolerant bacteria from contaminated soil collected from various industrial landfills of Gujarat, India. This study explores the potential of the amplified ribosomal DNA restriction analysis (ARDRA) for intra and interspecies identification of the Bacillus sp. A set of primers was used to amplify part of the 16S rDNA as well as the 16S spacer from isolates belonging to different bacillus species. Restriction analysis was carried out with nine different restriction enzymes, namely Alu I, Hae III, Hpa II, Sau 3AI , Taq I, RsaI, Cac8I, Bst NI and Bsa JI. We present a phylogenetic analysis of three strains of arsenite resistant bacterial isolates of Gujarat, India. Sequencing and BLAST search was done for the partial 16S rDNA of the isolates for submitting query to the NCBI-BLAST. Homologous sequence obtained by standard nucleotidenucleotide BLAST (BLASTn) were aligned with the isolate sequence using CLUSTAL program. The phylogenetic tree showed that the isolates falls under evolutionary clustal comprising members of bacillus group. This PCR-RFLP method provides a rapid tool for the identification of arsenite resistant bacterial isolates and the detection of new taxa. INTRODUCTION Arsenic is the 20th most abundant element in the Earth`s crust 14th in the seawater and 12th in the human body (Woolson, 1975) and is widely distributed throughout nature as a result of weathering, dissolution, fire, volcanic activity and anthropogenic input (Cullen and Reimer, 1989) . Arsenic is a semi-metalic element,which may be found in a variety of form,viz.-3, 0, +3, +5. The most common arsenic species observed in the environment are the trivalent form arsenite [As(III)] and pentavalent form of arsenate[As(V)]. (Pattamaporn Aksornchu et al., 2008). The toxicity of different forms of arsenic decrease in the order: arsine>inorganic arsenite>organic arsenite> inorganic arsenate>organic arsenate>free arsenic (Mandal and Suzuki, 2002). Many bacteria are known for their ability to transform inorganic arsenic species by oxidation or reduction (Duquesne, K et al., 2003, Erhlich; H.L. et al., 2001; Gihring et al., 2001; Macy et al., 1996; Weeger et al., 1993). The bacterial oxidation of As [III] to As [V] represents a potential detoxification mechanism and has applications in bioremediation because it generates less toxic and less mobile forms of arsenic. Nevertheless, the roles of the bacteria in the arsenic cycle are yet to be fully elucidated. (Diliana D. Simeonova et al., 2004) During the past decade, the development of molecular techniques using nucleic acids has led to many new findings in studies of microbial ecology (Amann et al., 1995). The PCR, a molecular technique, is used to amplify a specific gene or DNA fragment present in a species within the genome (Almeida et al., 2000). A molecular approach based on 16S rDNA is useful in detecting bacterial