Molecular characterization of mosquitocidal Bacillus sphaericus isolated from Tamil Nadu, India.

Forty-two Bacillus sphaericus strains were isolated from soil around Tamil Nadu, India. The phylogenetic relationship among the B. sphaericus isolates was analysed by REP-PCR and multiplex PCR was performed for the detection of mosquito larvicidal genes binA, binB, mtx1, mtx2 and mtx3 in B. sphaericus isolates. According to the REP-PCR band pattern, B. sphaericus isolates were divided into group A comprising I-XI clusters and group B comprising cluster XII. Three of the isolates BSTN01, 23 and 24 were gathered under cluster XII showed a high level of larvicidal activity against Culex quinquefasciatus and Anopheles stephensi, the other 39 isolates grouped under I-XI clusters were non-toxic or weak or moderately toxic to mosquito larvae. Even though BSTN23 and 24 were isolated from the same location and both contained all the five mosquito larvicidal genes, their intraspecies difference was clearly elucidated by REP-PCR analysis. Among high toxic isolates, BSTN23 and 24 were observed to contain all the five toxin genes and BSTN01 showed the presence of binary toxin and Mtx1 toxin genes. The isolates BSTN02, 03, 07, 14, 16, 19, 20, 21, 25, 31, 36 and 39 were found to contain mtx1 gene with combination of mtx2 and/or mtx3 showed moderate or low toxicity against mosquito larvae. binA, binB and mtx1 genes were not present in non-toxic isolates. The present study revealed the genetic heterogeneity between both toxic and non-toxic isolates and indicates that there is a good correlation between the presence of toxin genes and toxicity of the strains. These techniques could be developed in screening of novel highly toxic B. sphaericus strains from environment without bioassay on mosquito larvae.

Cellulosic ethanol production by Zymomonas mobilis harboring an endoglucanase gene from Enterobacter cloacae.

Enterobactercloacae was isolated from the gut of the wood feeding termite, Heterotermes indicola, and a 2.25-kb fragment conferring cellulase activity was cloned in Escherichia coli. The cloned fragment contained a 1083-bp ORF which could encode a protein belonging to glycosyl hydrolase family 8. The cellulase gene was introduced into Zymomonas mobilis strain Microbial Type Culture Collection centre (MTCC) on a plasmid and 0.134 filter paper activity unit (FPU)/ml units of cellulase activity was observed with the recombinant bacterium. Using carboxymethyl cellulose and 4% NaOH pretreated bagasse as substrates, the recombinant strain produced 5.5% and 4% (V/V) ethanol respectively, which was threefold higher than the amount obtained with the original E.cloacae isolate. The recombinant Z. mobilis strain could be improved further by simultaneous expression of cellulase cocktails before utilizing it for industrial level ethanol production.

Isolation and characterization of bacteria from the gut of Bombyx mori that degrade cellulose, xylan, pectin and starch and their impact on digestion.

Bombyx mori L. (Lepidoptera: Bombycidae) have been domesticated and widely used for silk production. It feeds on mulberry leaves. Mulberry leaves are mainly composed of pectin, xylan, cellulose and starch. Some of the digestive enzymes that degrade these carbohydrates might be produced by gut bacteria. Eleven isolates were obtained from the digestive tract of B. mori, including the Gram positive Bacillus circulans and Gram negative Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Serratia liquefaciens, Enterobacter sp., Pseudomonas fluorescens, P. aeruginosa, Aeromonas sp., and Erwinia sp.. Three of these isolates, P. vulgaris, K. pneumoniae, C. freundii, were cellulolytic and xylanolytic, P. fluorescens and Erwinia sp., were pectinolytic and K. pneumoniae degraded starch. Aeromonas sp. was able to utilize the CMcellulose and xylan. S. liquefaciens was able to utilize three polysaccharides including CMcellulose, xylan and pectin. B. circulans was able to utilize all four polysaccharides with different efficacy. The gut of B. mori has an alkaline pH and all of the isolated bacterial strains were found to grow and degrade polysaccharides at alkaline pH. The number of cellulolytic bacteria increases with each instar.

Pathogenicity, antibiotic susceptibility and genetic similarity of environmental and clinical isolates of Vibrio cholerae.

Isolates of Vibrio cholerae were obtained from clinical and environmental samples and the pathogenicity of these isolates was confirmed by hemolytic assay. The clinical isolates were more pathogenic than environmental isolates. Antibiotic susceptibility of V. cholerae to a set of antibiotics showed a marked variation. The environmental isolates exhibited more resistance to the antibiotics than clinical isolates. The plasmid curing technique was used to check the encoding of antibiotic resistance gene in genome. In both isolates, the resistance to vancomycin and co-trimaxazole was not mediated by plasmid and it may probably be encoded in genome. RAPD method was adopted to find out the variation in the genome of the clinical isolates and environmental isolates of V. cholerae. The genomic similarity pattern revealed that the environmental Ogawa isolates were closely related to clinical Ogawa isolates. This study confirmed the existence of the complex nature of V. cholerae in its pathogenicity, response to a set of antibiotics and genetic similarity.