Sriprakash Mohanty

RNA-seq analysis of mucosal immune responses reveals signatures of intestinal barrier disruption and pathogen entry following Edwardsiella ictaluri infection in channel catfish, Ictalurus punctatus.

The mucosal surfaces of fish (gill, skin, gastrointestinal tract) are important sites of bacterial exposure and host defense mechanisms. In mammalian systems, the intestinal epithelium is well characterized as both a selectively permeable barrier regulated by junctional proteins and as a primary site of infection for a number of enteric pathogens including viruses, bacteria, and parasites. The causative bacterium of enteric septicemia of catfish, Edwardsiella ictaluri, is believed to gain entry through the intestinal epithelium, with previous research using a rat intestinal epithelial cell line (IEC-6) indicating actin polymerization and receptor-mediated endocytosis as potential mechanisms of uptake. Here, we utilized high-throughput RNA-seq to characterize the role of the intestinal epithelial barrier following E. ictaluri challenge. A total of 197.6 million reads were obtained and assembled into 176,481 contigs with an average length of 893.7 bp and N50 of 1676 bp. The assembled contigs contained 14,457 known unigenes, including 2719 genes not previously identified in other catfish transcriptome studies. Comparison of digital gene expression between challenged and control samples revealed 1633 differentially expressed genes at 3 h, 24 h, and 3 day following exposure. Gene pathway analysis of the differentially expressed gene set indicated the centrality of actin cytoskeletal polymerization/remodelling and junctional regulation in pathogen entry and subsequent inflammatory responses. The expression patterns of fifteen differentially expressed genes related to intestinal epithelial barrier dysfunction were validated by quantitative real-time RT-PCR (average correlation coeff. 0.92, p < 0.001). Our results set a foundation for future studies comparing mechanisms of pathogen entry and mucosal immunity across several important catfish pathogens including E. ictaluri, Edwardsiellatarda, Flavobacterium columnare, and virulent atypical Aeromonas hydrophila. Understanding of molecular mechanisms of pathogen entry during infection will provide insight into strategies for selection of resistant catfish brood stocks against various diseases.

Utilization of fermented silkworm pupae silage in feed for carps.

Fermented silkworm pupae (SWP) silage or untreated fresh SWP pastes were incorporated in carp feed formulations replacing fishmeal. The feed formulations were isonitrogenous (30.2-30.9% protein) and isocaloric (ME = 2905-2935 kcal/kg). Feeding under a polyculture system consisting of 30% each of catla (Catla catla), mrigal (Cirrhinus mrigala) and rohu (Labeo rohita) with 10% silver carps (Hypophthalmychthys molitrix) was carried out in ponds to evaluate the nutritive quality of SWP silage. Survival rate, feed conversion ratio and specific growth rate, respectively, were 84.2%, 2.10 and 2.39 for fermented SWP silage, 65.8%, 2.98 and 2.26 for untreated SWP and 67.5%, 3.16 and 2.20 for fishmeal indicating clearly that the fermented SWP silage was nutritionally superior to untreated SWP or fishmeal. The dietary influence on the proximate composition of whole fish was marginal.

Aerobic and heterotrophic nitrogen removal by Enterobacter cloacae CF-S27 with efficient utilization of hydroxylamine

Heterotrophic bacterium, Enterobacter cloacae CF-S27 exhibited simultaneous nitrification and aerobic denitrification in presence of high concentration of hydroxylamine. With the initial nitrogen concentration of 100mgL-1h-1, ammonium, nitrate and nitrite removal efficiencies were 81%, 99.9% and 92.8%, while the corresponding maximum removal rates reached as high as 11.6, 15.1 and 11.2mgL-1h-1 respectively. Quantitative amplification by real time PCR and enzyme assay demonstrated that hydroxylamine reductase gene (hao) is actively involved in hetrotrophic nitrification and aerobic denitrification process of Enterobacter cloacae CF-S27. PCR primers were designed targeting amplification of hao gene from diversified environmental soil DNA. The strain Enterobacter cloacae CF-S27 significantly maintained the undetectable amount of dissolved nitrogen throughout 60days of zero water exchange fish culture experiment in domestic wastewater.

Draft Genome Sequence of Brevibacillus borstelensis cifa_chp40, a Thermophilic Strain Having Biotechnological Importance.

Brevibacillus borstelensis cifa_chp40 is a thermophilic, strictly aerobic gram positive motile bacteria isolated from the alkaline hot water spring located in the Eastern Ghats zone of India. It could grow in a wide range of temperature and degrade low-density polythene at 37°C. The strain cifa_chp40 produces essential enzymes like protease, lipase, esterase and amidase at 50°C. Here, we report the draft genome sequence of B. borstelensis cifa_chp40 which will provide further insight into the metabolic capabilities, function and evolution of this important organism.

Assessment of genetic diversity of Bacillus spp. isolated from eutrophic fish culture pond

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.

Survey of the transcriptome of Brevibacillus borstelensis exposed to low temperature shock

Molecular mechanisms underlying the ability of Brevibacillus borstelensis to survive and adapt to various environmentally relevant stresses are poorly understood. To define organisms molecular response to low temperature, gene expression profile of B. borstelensis at 20 °C was carried out by high-throughput sequencing technology. A total of 4579 transcripts with a maximum transcript length of 9919 bp were annotated. Gene expression profiling identified 712 genes that were significantly up- or down-regulated during cold shock. Functional categorization of the differentially expressed genes revealed that response to stress, regulation of transcription, transport, signal transduction and cytoplasm were the differentially regulated processes. The microbial stress responsive genes (hsp90, hslU, grpE, dnaK, dnaJ, hslV) and genes under regulatory adaptive responses (rpoN) were identified. The gene encoding cold shock protein purine nucleoside phosphorylase was found to be remarkably up-regulated. RT-PCR experiments carried out on genes expressed under cold shock independently verified the transcriptome data results. In addition, a large number of genes encoding hypothetical protein were identified. The brief survey of the transcripts obtained in response to cold shock underlines the survival strategy of thermophilic bacteria exposed to low temperature environment, which is further helpful in generating genetic information associated with this bacteria.

Profiling of Brevibacillus borstelensis transcriptome exposed to high temperature shock.

To understand the molecular mechanisms underlying the ability of the bacteria to survive at high temperature, gene expression profile of Brevibacillusborstelensis at 55°C during 5 and 10min heat shock period was carried out by high-throughput sequencing technology. A total of 2555 non-redundant transcripts were annotated. A total of 575 genes at 5min and 400 genes at 10min exhibited significant differential expression in response to temperature upshift from 50 to 55°C. Genes up-regulated under heat shock were associated with metabolism (mtnE), membrane transport, signal transduction, transcriptional regulation (ycxD, codY) and folding and sorting (hsp90). A larger number of genes encoding hypothetical proteins were identified. RT-PCR experimental results carried out on genes expressed under heat shock were found to be consistent with transcriptome data. The results enhance our understanding of adaptation strategy of thermophilic bacteria thereby providing a strong background for in depth research in thermophiles.

Sequence Polymorphism of GroEL Gene in Natural Population of Bacillus and Brevibacillus spp. that Showed Variation in Thermal Tolerance Capacity and mRNA Expression

GroEL, a class I chaperonin, plays an important role in the thermal adaptation of the cell and helps to maintain the viability of the cell under heat shock condition. Function of groEL in vivo depends on the maintenance of proper structure of the protein which in turn depends on the nucleotide and amino acid sequence of the gene. In this study, we investigated the changes in nucleotide and amino acid sequences of the partial groEL gene that may affect the thermotolerance capacity as well as mRNA expression of bacterial isolates. Sequences among the same species having differences in the amino acid level were identified as different alleles. The effect of allelic variation on the groEL gene expression was analyzed by comparison and relative quantification in each allele under thermal shock condition by RT-PCR. Evaluation of Ka/Ks ratio among the strains of same species showed that the groEL gene of all the species had undergone similar functional constrain during evolution. The strains showing similar thermotolerance capacity was found to carry same allele of groEL gene. The isolates carrying allele having amino acid substitution inside the highly ATP/ADP or Mg2+-binding region could not tolerate thermal stress and showed lower expression of the groEL gene. Our results indicate that during evolution of these bacterial species the groEL gene has undergone the process of natural selection, and the isolates have evolved with the groEL allelic sequences that help them to withstand the thermal stress during their interaction with the environment.