A polymerase chain reaction (PCR) assay was developed for discrimination of
Bacillus subtilis from other members of
B. subtilis group as well as rapid
identification from environmental samples. Primers ENIF and EN1R from endoglucanase
gene were used to amplify a1311 bp DNA fragment. The specificity of the primers was
tested with seven reference strains and 28 locally isolated strains of endoglucanase
positive Bacillus species. The PCR product was
only produced from B. subtilis. The results
demonstrated high specificity of two oligonucleotides for B.
subtilis. This species-specific PCR method provides a quick, simple,
powerful and reliable alternative to conventional methods in the detection and
identification of B. subtilis. To our knowledge
this is the first report of a B. subtilis
specific primer set.
Klebsiella pneumoniae being ubiquitous in nature encounters wide differences in environmental condition. The organism's abundance in natural water reservoirs exposed to temperature variation forms the basis of its persistence and spread in the soil and other farm produce. In order to investigate the effect of temperature changes on the survival and adaptation of the bacteria, the transcriptional response of K. pneumoniae subjected to low (20 °C) and high (50 °C) temperature shock were executed using Applied Biosystems SOLiD platform. Approximately, 33 and 34% of protein coding genes expressed in response to 20 and 50 °C, respectively, displayed significant up- or downregulation (p < 0.01). Most of the significantly expressed transcripts mapped to metabolism, membrane transport, and cell motility were downregulated at 50 °C, except for protein folding, sorting, and degradation, suggesting that heat stress causes general downregulation of gene expression together with induction of heat shock proteins. While at 20 °C, the transcripts of carbohydrate, lipid, and amino acid metabolism were highly upregulated. Hypothetical proteins as well as canonical heat and cold shock proteins, viz. grpE, clpX, recA, and deaD were upregulated commonly in response to 20 and 50 °C. Significant upregulation of genes encoding ribosomal proteins at 20 and 50 °C possibly suggest their role in the survival of K. pneumoniae cells under low- and high-temperature stress.
The genus Bacillus comprises of a diverse group with a wide range of nutritional requirements and physiological and metabolic diversity. Their role in nutrient cycle is well documented. 16S rDNA sequences do not always allow the species to be discriminated. In this study 40 Bacillus spp. obtained from fish culture pond and 10 culture type strains were analysed for their genomic diversity by PCR–RFLP of intergenic spacer region of 16S-23S and HSP60 genes. TaqI digestion of PCR products amplified by ITS PCR did not render distinctive RFLP patterns. Numerical analysis of ITS PCR–RFLP pattern differentiated the isolates into 11 clusters. Same species were found to be grouped in different clusters. But PstI digested PCR products amplified from HSP60 gene of the isolates showed distinctive RFLP patterns. The dendrogram constructed from HSP60 PCR–RFLP delineated the isolates into 11 clusters also. All the clusters, except cluster I grouped only one type of species. The results showed that Bacillus spp. could be clearly distinguished by PCR–RFLP of HSP60 gene. Therefore, the HSP60 gene is proposed as an additional molecular marker for discrimination of Bacillus group.
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