Neelam M. Nathani

Mining of Ruminant Microbial Phytase (RPHY1) from Metagenomic Data of Mehsani Buffalo Breed: Identification, Gene Cloning, and Characterization

Phytases have been widely used as animal feed supplements to increase the availability of digestible phosphorus, especially in monogastric animals fed cereal grains. The present study describes the identification of a full-length phytase gene of Prevotella species present in Mehsani buffalo rumen. The gene, designated as RPHY1, consists of 1,251 bp and is expressed into protein with 417 amino acids. A homology search of the deduced amino acid sequence of the RPHY1 phytase gene in a nonredundant protein database showed that it shares 92% similarity with the histidine acid phosphatase domain. Subsequently, the RPHY1 gene was expressed using a pET32a expression vector in Escherichia coli BL21 and purified using a His60 Ni-NTA gravity column. The mass of the purified RPHY1 was estimated to be approximately 63 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal RPHY1 enzyme activity was observed at 55°C (pH 5) and exhibited good stability at 5°C and within the acidic pH range. Significant inhibition of RPHY1 activity was observed for Mg2+ and K+ metal ions, while Ca2+, Mn2+, and Na+ slightly inhibited enzyme activity. The RPHY1 phytase was susceptible to SDS, and it was highly stimulated in the presence of EDTA. Overall, the observed comparatively high enzyme activity levels and characteristics of the RPHY1 gene mined from rumen prove its promising candidature as a feed supplement enzyme in animal farming.

Functional Characterization Reveals Novel Putative Coding Sequences in Prevotella ruminicola Genome Extracted from Rumen Metagenomic Studies

Aim: To reassemble Prevotella ruminicola genome from rumen metagenomic data of cattle and buffalo and compare with the published reference genome. Method: Rumen microbial communities from Mehsani buffaloes (n = 8) and Kankrej cattle (n = 8), each adapted to different proportions of a dry or green roughage diet, were subjected to metagenomic sequencing by Ion Torrent PGM, and subsequent reads were analyzed by MG-RAST. Using reference-guided assembly of the sequences against the published P. ruminicola strain 23, draft genomes of 2.56 and 2.46 Mb were reconstructed from Mehsani buffalo and Kankrej cows, respectively. The genomes were annotated using the RAST Server and carbohydrate active enzyme (CAZyme) analysis. Results: Taxonomic analysis by MG-RAST revealed P. ruminicola to be the most abundant species present among the rumen microflora. Functional annotation of reconstructed genomes using the RAST Server depicted the maximum assignment of coding sequences involved in the subsystems amino acid and derivatives and carbohydrate metabolism. CAZyme profiling revealed the glycoside hydrolases (GH) family to be the most abundant. GH family subclassification revealed that the extracted genomes had more sequence hits for GH2, GH3, GH92 and GH97 as compared to the reference. Conclusion: The results reflect the metabolic significance of rumen-adapted P. ruminicola in utilizing a coarse diet for animals based on acquisition of novel genetic elements.

Isolation of chitinolytic Clostridium sp. NCR from Mehsani buffalo rumen, its genomic analysis and potential role in rumen.

Genomic analysis of Clostridium sp. NCR, an anaerobic Gram positive bacterium which was isolated from rumen fluid of Mehsani breed of buffalo revealed presence of various environmental gene tags (EGTs) involved in pathways for utilizing a wide range of substrates. Here we report the sequence of this rumen isolate, its whole genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession number JQHY00000000. The genome comprises of a 3.62-Mb draft genome with a G + C content of 28.10%, which encodes a total of 3126 proteins. Functional analysis provides information about the microbes role in maintaining host homeostasis and its fiber degradation potential.

Draft Genome Sequence of Commercial Textile Dye-Decolorizing and -Degrading Bacillus subtilis Strain C3 Isolated in India

ABSTRACT Bacillus subtilis C3, a commercial textile dye-decolorizing and -degrading bacterium, was isolated from the common effluent treatment plant (CEPT) of the Jetpur textile dyeing and printing industrial sector situated in the district of Rajkot, Gujarat, India. Here, we present the annotated 4.18-Mb draft genome sequence of B. subtilis C3, providing information about the metabolic pathways involved in decolorization and degradation of several commercial textile azo dyes. Thus, we confirm B. subtilis C3 as a potential candidate for bioremediation of textile effluents.

Chapter 11 – Comprehensive Exploration of the Rumen Microbial Ecosystem With Advancements in Metagenomics

Abstract Ruminant farming and its environmental impact has long remained an economic concern. Metagenomics unravel the vast structural and functional diversity of the rumen microbial community that plays a major role in animal nutrition. Hereby, we summarize rumen metagenomic studies that have enhanced the knowledge of rumen microbe dynamics subsequently leading to development of better feed strategies to improve livestock production and reduce methane emissions.

Advancements in Molecular Techniques and Bioinformatics for Understanding the Rumen Microbiome

Microbial diversity in the forestomach is of major importance to the ruminants. Rumen microbiome diversity and gene tag predictions are important areas of scientific interests due to their global impacts including methane emission control and ruminant health. Earlier the knowledge of rumen biodiversity was entirely dependent on the anaerobic culturing approach. However, advancements in molecular techniques have revealed hitherto uncovered features of the rumen microbiome. Next-generation sequencing in combination with conventional techniques has provided significant opportunities to understand animal performance in context to rumen ecology and subsequent nutrient manipulation. Metagenomic sequencing generates enormous data making its informatics handling and analysis intricate due to the immense complexity of the data. Bioinformatic resources are rapidly emerging to outwit the intricacies. In this book chapter, we highlight the advances in the metagenomic analysis that help to improve the genomic aspect of the tree of life by getting an in-depth understanding of the Candidatus uncultured population of the rumen microbiome focusing on correlation between the microbial functioning and animal efficiency. It also describes the strategies and specific bioinformatic approaches that can be applied to metagenomic sequences for accurate and in-depth metagenomic analysis, data binning, and genome reconstruction of individual microbes.