Exploring bacterial diversity in Arctic fjord sediments: a 16S rRNA-based metabarcoding portrait.

The frosty polar environment houses diverse habitats mostly driven by psychrophilic and psychrotolerant microbes. Along with traditional cultivation methods, next-generation sequencing technologies have become common for exploring microbial communities from various extreme environments. Investigations on glaciers, ice sheets, ponds, lakes, etc. have revealed the existence of numerous microorganisms while details of microbial communities in the Arctic fjords remain incomplete. The current study focuses on understanding the bacterial diversity in two Arctic fjord sediments employing the 16S rRNA gene metabarcoding and its comparison with previous studies from various Arctic habitats. The study revealed that Proteobacteria was the dominant phylum from both the fjord samples followed by Bacteroidetes, Planctomycetes, Firmicutes, Actinobacteria, Cyanobacteria, Chloroflexi and Chlamydiae. A significant proportion of unclassified reads derived from bacteria was also detected. Psychrobacter, Pseudomonas, Acinetobacter, Aeromonas, Photobacterium, Flavobacterium, Gramella and Shewanella were the major genera in both the fjord sediments. The above findings were confirmed by the comparative analysis of fjord metadata with the previously reported (secondary metadata) Arctic samples. This study demonstrated the potential of 16S rRNA gene metabarcoding in resolving bacterial composition and diversity thereby providing new in situ insights into Arctic fjord systems.

Marine actinomycete Streptomyces variabilis S26 as a biocontrol agent for vibriosis in shrimp larval rearing systems.

Indiscriminate use of antibiotics has led to the emergence of antibiotic-resistant microbes and the loss of natural flora in aquaculture systems necessitating the ban of many of these chemotherapeutants in aquaculture. Actinobacteria play a profound role in the biogeochemical cycling in the marine environment and represent the principal source of secondary metabolites with antimicrobial property. In the present study, 98 marine-derived actinomycete isolates were screened for antimicrobial activity against the common aquatic pathogens. A potent actinomycete isolate S26, identified as Streptomyces variabilis based on 16 S ribosomal RNA (rRNA) gene sequencing was then checked for the production of antibiotic in five different fermentation media and the one which showed maximum production was chosen for further study. Optimization of the fermentation medium for secondary metabolite production was carried out by response surface methodology (RSM) using DESIGN EXPERT. The analysis of variance (ANOVA) of the quadratic regression model demonstrated that the model was highly significant for the response concerned that is, antimicrobial activity as evident from the Fisher's F- test with a very low probability value [(P model>F) = 0.0001]. Of the 10 different solutions suggested by the software, the most suitable composition was found to be starch, 1.38%; soy powder, 0.88%; ammonium sulfate, 0.16% and salinity, 27.76‰. S. variabilis S26 cultured in the optimized production medium was applied in the Penaeus monodon larval rearing system and the total Vibrio count and survival rate were estimated. S. variabilis S26 treatment showed a significant reduction in vibrios and conferred better protection to P. monodon in culture system compared with control.

Microbial machinery dealing diverse aromatic compounds: Decoded from pelagic sediment ecogenomics in the gulfs of Kathiawar Peninsula and Arabian Sea.

Aromatic hydrocarbons are persistent pollutants in aquatic systems as endocrine disruptors, significantly impacting natural ecosystems and human health. Microbes perform as natural bioremediators to remove and regulate aromatic hydrocarbons in the marine ecosystem. The present study focuses upon the comparative diversity and abundance of various hydrocarbon-degrading enzymes and their pathways from deep sediments along the Gulf of Kathiawar Peninsula and Arabian Sea, India. The elucidation of large number of degradation pathways in the study area under the presence of a wide range of pollutants whose fate needs to be addressed. Sediment core samples were collected, and the whole microbiome was sequenced. Analysis of the predicted ORFs (open reading frames) against the AromaDeg database revealed 2946 aromatic hydrocarbon-degrading enzyme sequences. Statistical analysis portrayed that the Gulfs were more diverse in degradation pathways compared to the open sea, with the Gulf of Kutch being more prosperous and more diverse than the Gulf of Cambay. The vast majority of the annotated ORFs belonged to groups of dioxygenases that included catechol, gentisate, and benzene dioxygenases, along with Rieske (2Fe-2S) and vicinal oxygen chelate (VOC) family proteins. From the sampling sites, only 960 of the total predicted genes were given taxonomic annotations, which mention the presence of many under-explored marine microorganism-derived hydrocarbon degrading genes and pathways. Through the present study, we tried to unveil the array of catabolic pathways of aromatic hydrocarbon degradation and genes from a marine ecosystem that upholds economic and ecological significance in India. Thus, this study provides vast opportunities and strategies for microbial resource recovery in marine ecosystems, which can be investigated to explore aromatic hydrocarbon degradation and their potential mechanisms under various oxic or anoxic environments. Future studies should focus on aromatic hydrocarbon degradation by considering degradation pathways, biochemical analysis, enzymatic, metabolic, and genetic systems, and regulations.

Viral footprints across Gulfs of Kathiawar Peninsula and Arabian Sea: Unraveled from pelagic sediment metagenomic data.

Marine biosphere is one of the largest, diverse and dynamic system hosting numerous of microorganisms. Viruses being the most abundant under explored lifeforms in ocean, represent a reservoir of great genetic diversity. We report the metagenomic insights on the viral communities in the deep sediments of the two Gulfs of Gujarat i.e. Gulf of Khambhat and Gulf of Kutch, with one sample from Arabian Sea, treated as open sea control. The viral reads were filtered from the whole dataset, assembled and studied for viral diversity, which was visualized by Pavian. The sequences were checked for the viral abundance, diversity and functionality. The resulting viral taxonomic classification contained 6 orders, 8 families and 47 genera. The results revealed that the phages infecting Cyanobacterium, Bacillus and Vibrio dominated the sediments. Further, it was observed that majority of viral sequences belonged to double-stranded DNA phages. The present study attempts to provide a primary insight of the viral signals and potential genetic content in the Gulfs of Kathiawar.

A novel solvent tolerant esterase of GDSGG motif subfamily from solar saltern through metagenomic approach: Recombinant expression and characterization.

A novel esterase, designated as EstSP was identified by function based screening from a soil metagenomic fosmid library of solar saltern of Goa. EstSP gene of 1065 bp encoding a putative esterase of 354 amino acids showing 55% identity to esterase from gamma proteobacterium HIMB55 was identified. The enzyme EstSP belongs to family IV hormone sensitive lipase with novel sequence characteristics and a unique motif GDSGG. EstSP expressed as a His-tag fusion protein of mass 58 kDa was visualized on SDS PAGE and confirmed by Western blot analysis. The enzyme is an alkaline esterase that exhibited highest catalytic activity towards p-nitrophenyl acetate with optimum temperature 40 °C and pH 8.0. The catalytic efficiency and specific activity of EstSP for p-nitrophenyl acetate was 7407.4 min-1 mM-1 and 915.23 U mg-1 respectively. EstSP showed remarkable stability in the presence of polar and non-polar solvents, retaining >80% of its activity after 72 h. Furthermore, the enzyme is halotolerant with optimum activity at 1 M NaCl and maintained 60% residual activity after 24 h exposure to 5 M NaCl. This novel enzyme with remarkable properties could be a promising candidate for industrial bioprocesses in non-aqueous media as well as pharmaceutical, food and biotechnological applications.

Marine actinomycetes as bioremediators in Penaeus monodon rearing system.

Actinomycetes (277 Nos) isolated from marine environment and shrimp culture pond sediments were tested for hydrolytic enzyme production and biogranulation property. Potential isolates were screened for their efficacy in bioremediation of shrimp culture system. Based on the BOD reduction efficiency and water quality parameters, five actinomycete isolates viz., Streptomyces coelicoflavus (A6), Streptomyces diastaticus (A44), Nocardiopsis alba (A55), Streptomyces parvus (A56) and Streptomyces champavatii (R32) were subjected for tertiary screening in Penaeus monodon larval rearing system and the animals were challenged with white spot syndrome virus (WSSV). The bioremediating effect of actinomycete treatments were assessed by analysing the expression profile of five antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-2, crustin-3, penaeidin-3 and penaeidin-5 and eight immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, glutathione-S-transferase, haemocyanin, peroxinectin, pmCathepsinC, prophenol oxidase (proPO) and Rab-7. Expression of eight WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, ribonucleotide reductase, thymidine kinase and VP28 were also analyzed to detect the presence and intensity of viral infection in the experimental animals post-challenge. Theapplication of consortia (1 g/5 L water) yields better results in terms of significant reduction in BOD of shrimp rearing system showing the bioremediation potential of the marine actinomycete strains. The application of marine actinomycetes viz., Streptomyces coelicoflavus (A6), Streptomyces diastaticus (A44), Nocardiopsis alba (A55), Streptomyces parvus (A56) and Streptomyces champavatii (R32) in granulated form were found to be potential bioremediators in shrimp rearing system.

CTAB influenced differential elution of metagenomic DNA from saltpan and marine sediments.

A simple, reliable method for genomic DNA extraction from sediments with minimum contaminants was developed to address the risk of poor quality DNA in metagenomic studies. Nine DNA extraction methods using 20% cetyl-trimethyl-ammonium bromide (CTAB) were performed and compared to develop an extraction protocol that can offer humic acid-free metagenomic DNA from marine and saltpan sediments. Community DNA extraction was executed via., Zhou et al. modified protocol using 20% CTAB treatment at different steps to compare the efficacy of humic acid removal. Out of nine DNA extraction methods, method 6 significantly improved the quality of DNA with efficient removal of humic substances. 16S rRNA gene amplification and spectrophotometric analysis confirmed the efficiency of method 6 to remove DNA inhibitors from marine sediments as well as saltpan samples. Inhibitors extracted along with metagenomic DNA outcome increased DNA yield and PCR inhibition in method 1 and 3. However, repeated 20% CTAB wash in method 6 ensured 16S amplification and least yield and concentration. Current study explains a detailed protocol based on 20% CTAB wash for the extraction of humic acid-free DNA from diverse sediment samples.

Amplicon sequencing based profiling of bacterial diversity from Krossfjorden, Arctic.

In this study, Illumina Miseq sequencing of 16S rRNA gene amplicon was performed on sediments collected from Krossfjorden, Arctic for analyzing the bacterial community structure. Metagenome contained 15,936 sequences with 5,809,491 bp size and 53% G+C content. Metagenome sequence information are now available at NCBI under the Sequence Read Archive (SRA) database with accession no. SRP159159. Taxonomic hits distribution from MG-RAST analysis revealed the dominance of Alpha- and Gamma-subdivisions of Proteobacteria (88.89%) along with Bacteriodetes (8.89%) and Firmicutes (2.22%). Predominant species were Alteromonadales bacterium TW-7 (24%), Pseudoalteromonas haloplanktis (20%) and Pseudoalteromonas spp. SM9913 (18%). MG-RAST assisted analysis also detected the presence of a variety of marine taxa like Bacteriodes, Pseudovibrio, Marinobacter, Idiomarina, Teredinibacter, etc. which take part in key ecological functions and biogeochemical activities of Arctic fjord ecosystems.

High-quality metagenomic DNA from marine sediment samples for genomic studies through a preprocessing approach.

Recent advances in culture-independent studies of microbes had proved to be more reliable and efficient than the conventional ones. The isolation of good quality and quantity of total community DNA are one of the major hurdles in this endeavour. Shearing of DNA during the extraction process and the co-extraction of inhibitory compounds reduce the quality of the isolated nucleic acids making it unsuitable for the construction of large insert metagenomic libraries. In the present study, a multi-level filtration step was brought in which efficiently isolated total bacterial DNA from three different environment samples. The preprocessing method could efficiently improve the 260/230 ratio of the isolated DNA by 2.3-45 % and decreased the protein contamination by 22.5-34.5 % on saltpan and arctic sediment samples, respectively. The more significant part of the experiment was that the DNA obtained was of high quality with minimal shearing making it most suitable for the construction of large insert genomic libraries. PCR amplification of 16S rRNA gene confirmed that the filtration method was effective in the isolation of high-quality DNA.