Toms C. Joseph

Differential gene expression profile of the hepatopancreas of white spot syndrome virus infected Fenneropenaeus indicus by suppression subtractive hybridization

Suppression Subtractive Hybridization was employed in order to identify the differentially expressed genes in the hepatopancreas of white spot syndrome virus infected Fenneropenaeus indicus. A forward subtracted cDNA library generated 356 clones following a white spot syndrome virus infection. A total of 345 clones with more than 100 nucleotides were selected for further analysis using bioinformatics tools after vector screening. Twenty-three contigs and 111 singletons were generated from a total of 134 consensuses. The consensuses, on a sequence homology search using BLASTX (NCBI), revealed that 74 (55%) of them had no significant match to reported sequences in the database, suggesting that they were found for the first time and are probably associated with shrimp immune function. Out of the remaining 60 (45%) consensuses, 43 had significant homology to known protein sequences in the database while 17 consensuses are homologous to unknown proteins in the database which are considered novel. The most abundant genes in the subtracted library were antimicrobial peptides accounting for 56 clones; among which one is a member of SNF2 family of proteins and another belonged to PfP1 family of proteins on analysis using Antimicrobial peptide predictor software. The other predicted genes in the subtracted library include signal transduction molecules (GTPase, Serine threonine kinase, Armadillo repeats etc), antioxidant enzymes (Cytochrome oxidase, Monomeric sarcosine oxidase and Catalase), active transporters (Nuclear Localization Signal [NLS], calcium ATPase, sodium glutamate symporter, Store-Operated Calcium Entry [SOCE] and ribonucleoprotein [RNP]) contributing to 19, 14 and 5 clones respectively. Three clones are homologous to reverse transcriptase; a first time report in shrimp and one each belong to cell adhesion molecule and Proteinase. InterProScan at EMBL, when used for an integrated search at PROSITE predicted; signal sequences and transmembrane regions for 13 clones. This is the first report on the differential gene expression in WSSV-infected F. indicus. The high expression of immune related genes in response to virus infection in shrimp will provide a new insight into the crustacean innate immunity. Further work on the functionality of the unknown genes in shrimps will give an overview on the role of the differentially expressed genes during viral infection and increase our understanding for developing antiviral measures by making use of the shrimp defense mechanism.

Functional and molecular characterization of a lipopeptide surfactant from the marine sponge-associated eubacteria Bacillus licheniformis NIOT-AMKV06 of Andaman and Nicobar Islands, India

The production of a lipopeptide surfactant from the sponge-associated eubacteria Bacillus licheniformis NIOT-AMKV06 from the Andaman and Nicobar Islands was investigated. The highest production was attained with glucose and yeast extracts as the carbon and nitrogen sources (1.789 mg mL(-1)), respectively. The surfactant was highly stable over a pH range of 5.0-10 and a temperature range of 20-70°C with high NaCl concentrations. Excellent emulsification activity was exhibited by the purified surfactant with crude oil, kerosene, and diesel. A two-fold increase in surfactant production (3.0 mg mL(-1)) was observed using the newly formulated medium in this study. The surfactant biosynthesis gene cluster (sfp, sfpO, and srfA) from B. licheniformis NIOT-AMKV06 was heterologously expressed in Escherichia coli, and the production was increased three-fold (11.78 g L(-1)) over the original strain. The results confirm the potential of the surfactant for use in bioremediation of hydrocarbons in a marine environment and for enhanced oil recovery. To our knowledge, this is the first report on the ability of a hydrocarbon degrading B. licheniformis from marine sponges for the biosynthesis of a potent lipopeptide surfactant possessing characteristics of maximum stability, outstanding surfactant activity, and exceptional emulsifying capability.

Molecular Screening, Isolation, and Characterization of Enterohemorrhagic Escherichia coli O157:H7 from Retail Shrimp

Foodborne outbreaks attributed to the contamination of foods with enterohemorrhagic Escherichia coli (EHEC) O157:H7 are a growing global concern. Fish and shrimp samples obtained from different retail fish markets in Cochin, India, were screened by direct PCR assays targeting three important virulence markers of EHEC, the intimin protein (eaeA gene), enterohemolysin (hlyA gene), and Shiga toxin (stx gene). One shrimp (Fenneropenaeus indicus) sample was positive for all these virulence markers, and seven typical E. coli O157:H7 isolates were recovered from the marker-positive shrimp sample. This is the first report of recovery of typical E. coli O157:H7 from fish or shellfish in India. All the typical EHEC isolates had a characteristic reaction in eosin methylene blue agar and belonged to IMViC (indole, methyl red, Voges Proskauer, Simmons citrate reactions) biotype I. These isolates also were negative for sorbitol and methylumbelliferyl-beta-glucuronide and exhibited beta-hemolytic activity. One isolate showed self-agglutination for E. coli O157 antisera and produced a false-positive reaction with CHROMagar O157. These typical EHEC isolates belonged to a restricted biotype group and had a very low multiple antibiotic resistance index. Isolation of E. coli O157:H7 in fish and shellfish indicates that strict adherence to hygienic handling methods and proper cooking or processing is needed before consumption of these products.

Heterologous expression, purification, and phylogenetic analysis of oil-degrading biosurfactant biosynthesis genes from the marine sponge-associated Bacillus licheniformis NIOT-06

Surfactin is a lipopeptide, composed of one β-hydroxy fatty acid, a long fatty acid moiety, and seven amino acids. In this study, the biosurfactant biosynthesis genes; 4′-pantetheinyl transferase (sfp), phosphopantetheinyl transferase (sfpO), and surfactin synthetase (srfA) have been characterized from the marine sponge-associated Bacillus licheniformis NIOT-06 from the Andaman and Nicobar Islands. The purified recombinant biosurfactant revealed excellent emulsification activity with crude oil and kerosene. Reverse-phase high-performance liquid chromatography resolved the purified recombinant biosurfactant into several fractions and one of which had significant surface tension reducing property. Fourier transform infrared spectroscopy spectrum also revealed the presence of C–N–N, alkenes, and N–H as the functional groups, and a similar overlapping pattern was observed with that of standard lipopeptide surfactin. The diversity and phylogeny of sfp, sfpO, and srfA gene sequences were compared with other eubacteria. The sfp, sfpO, and srfA gene sequences obtained from Bacillus licheniformis NIOT-06 were diverse and appeared to be partially conserved when compared with the GenBank reported sequences of several eubacteria.

Characterization and phylogenetic analysis of ectoine biosynthesis genes from Bacillus halodurans

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), is a natural compound, which serves as a protective substance in many bacterial cells. In this study, the putative ectABC gene cluster from Bacillus halodurans was heterologously expressed in E. coli and the production of ectoine was confirmed by HPLC analysis. The activity of the enzymes coded by the ectA, B and C genes were found to be higher in induced transgenic cells compared to the uninduced cells. Phylogenetic analysis revealed sequence identities ranging from 36–73% for ectA gene, 55–81% for ectB gene and 55–80% for ectC gene indicating that the enzymes are evolutionarily well conserved.

White spot syndrome virus infection: Threat to crustacean biodiversity in Vembanad Lake, India

Highlights • The prevalence of white spot syndrome virus (WSSV) among crustaceans from farmed, estuarine and marine environments surrounding the Vembanad Lake, India was detected using PCR.• A total of 308 samples from aquaculture ponds consisting of six species of crustaceans collected from five different farms were tested for the presence of WSSV.• WSSV was not detected from three species of deep sea crustaceans tested. The black tiger shrimp,• P. monodon had the highest incidence of WSSV among the species screened in farmed, estuarine and marine environments.

First draft genome sequence of a member of the genus mangrovibacter, isolated from an aquaculture farm in India.

ABSTRACT Mangrovibacter sp. MFB070, a Gram-negative, facultatively anaerobic, nitrogen-fixing bacterium, was isolated from an aquaculture farm in Cochin, India. Here, we report the first draft genome sequence of a member of the genus Mangrovibacter, which may help us to elucidate the evolutionary status of this genus. The draft genome sequence of the Mangrovibacter sp. consists of 5,361,682 bp, encoding 4,971 predicted coding sequences in 57 contigs.

Draft Genome Sequence of the Halophilic and Highly Halotolerant Gammaproteobacteria Strain MFB021.

ABSTRACT We report the 4.25-Mbp first draft sequence of Gammaproteobacteria strain MFB021, a moderate halophile isolated from petroleum-contaminated soil in Cochin, India. The genome of the strain MFB021 was sequenced to understand the mechanism of hydrocarbon degradation and the halophilicity of the bacterium.

Variations of structural protein sequences among geographical isolates of white spot syndrome virus

White spot syndrome virus (WSSV) is the causative agent of a disease that causes severe mortalities in cultured shrimp worldwide. The sequence of five structural genes of an Indian isolate of WSSV was compared with sequences from other WSSV isolates deposited in GenBank. Among the structural genes analyzed, the sequences of vp28 and vp19 had maximum divergence with nucleotide changes at ten different positions from twenty eight submissions for vp28 and six changes from seventeen submissions for vp19. The vp68, vp26, and vp281 genes were found to be highly conserved between isolates.

Occurrence of viral pathogens in Penaeus monodon post-larvae from aquaculture hatcheries

Viral pathogens appear to exert the most significant constraints on the growth and survival of crustaceans under culture conditions. The prevalence of viral pathogens White Spot Syndrome Virus (WSSV), Hepatopancreatic Parvo Virus (HPV), Monodon Baculo Virus (MBV) and Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) in Penaeus monodon post-larvae was studied. Samples collected from different hatcheries and also samples submitted by farmers from Kerala were analyzed. Out of 104 samples collected, WSSV was detected in 12.5% of the post-larvae samples. Prevalence of concurrent infections by HPV, MBV and WSSV (either dual or triple infection) was present in 60.6% of the total post-larvae tested. Out of the 51 double positives, 98% showed either HPV or IHHNV infection. HPV or IHHNV was detected in 11 post-larval samples showing triple viral infection. This is the first report of IHHNV from India. Result of this study reveals the lack of efficient screening strategies to eradicate viruses in hatchery reared post-larvae.