Sathyanarayanan Jayashree

Identification of a Novel Antifungal Peptide with Chitin-Binding Property from Marine Metagenome

A novel antifungal peptide with 36 amino acids was identified by functional screening of a marine metagenomic library. The peptide did not show similarity with any existing antimicrobial peptide sequences in the databank. The108 bp ORF designated as mmgp1 was cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. Mass spectrometry analysis of the purified recombinant peptide revealed a molecular mass of 5026.9 Da. The purified recombinant peptide inhibited the growth of Candida albicans and Aspergillus niger. The peptide was predicted to adopt α- helical conformation with an extended coil containing a ligand binding site for N-acetyl-D-glucosamine. The α- helicity of the peptide was demonstrated by circular dichroism spectroscopy in the presence of chitin or membrane mimicking solvent, trifluoroethanol. The chitin binding property of the peptide was also confirmed by fast performance liquid chromatography.

Direct cell penetration of the antifungal peptide, MMGP1, in Candida albicans

An antifungal peptide, MMGP1, was recently identified from marine metagenome. The mechanism of cellular internalization of this peptide in Candida albicans was studied using fluorescein 5–isothiocynate (Sigma, California, USA) labeling followed by fluorescence microscopy and flow cytometry analyses. The peptide could enter C. albicans cells even at 4 °C, where all energy‐dependent transport mechanisms are blocked. In addition, the peptide internalization was not affected by the endocytic inhibitor, sodium azide. The kinetic study has shown that the peptide was initially localized on cell membrane and subsequently internalized into cytosol. The MMGP1 treatment exhibited time‐dependent cytotoxicity in C. albicans as evidenced by SYTOX Green (Molecular Probes Inc., Eugene, Oreg) uptake. Copyright

Genome Sequence of Lactobacillus fermentum Strain MTCC 8711, a Probiotic Bacterium Isolated from Yogurt

ABSTRACT Lactobacillus fermentum strain MTCC 8711 is a lactic acid bacterium isolated from yogurt. Here, we describe the draft genome sequence and annotation of this strain. The 2,566,297-bp-long genome consisted of a single chromosome and seven plasmids. The genome contains 2,609 protein-coding and 74 RNA genes.

Genome Sequence of Staphylococcus arlettae Strain CVD059, Isolated from the Blood of a Cardiovascular Disease Patient

We have isolated a Staphylococcus arlettae strain, strain CVD059, from the blood of a rheumatic mitral stenosis patient. Here, we report the genome sequence and potential virulence factors of this clinical isolate. The draft genome of S. arlettae CVD059 is 2,565,675 bp long with a G+C content of 33.5%.

Microbial Bioremediation: A Metagenomic Approach

Microbial bioremediation serves as an alternative and effective strategy to remove toxic contaminants from a polluted environment. It could be achieved through the interaction of microbes with the toxic contaminants, which leads to immobilization, compartmentalization, and concentration of pollutants rather than their degradation and elimination from the environment. Bioremediation of the contaminated sites employing indigenous microbes is highly advantageous as it is ideally adapted to the environmental conditions prevailing at the site to be remediated. Traditional culture-based approaches have provided only limited information on the metabolic potential and the functional activity of the indigenous microbial communities living in the contaminated environment. Recent development of metagenomic approaches and advancement of high-throughput DNA sequencing technology provides insight into the total microbial community and in-depth knowledge of the metabolic capabilities of the indigenous microbial community prevailing in contaminated sites. Metagenomic approaches could address environmental issues by exploring the phenomenal resources of the uncultivable microorganisms. The present chapter describes the application of metagenomic strategies for better understanding of the indigenous microbial communities and their functional abilities to clean up toxic contaminants from polluted sites, which will provide new perspectives on environmental management.

Microbial Diversity and Phylogeny Analysis of Buttermilk, a Fermented Milk Product, Employing 16S rRNA-Based Pyrosequencing

Buttermilk is a traditional fermented dairy product popular in India and other Asian countries. In this study, the microbial population in a buttermilk sample was investigated by 16S rRNA gene-based bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) approach. A total of 23,080 high-quality pyrosequencing reads was obtained. Of these, 96.3% of reads represented the well-defined domain bacteria and 3.7% represented unclassified domain; no archaeal sequences were found. Lactobacillus delbrueckii was the most predominant species identified, which constituted 94.86% of total reads. The other minor genera found included Streptococcus, Aeromonas, Methylobacter, Enterococcus, Micrococcus, Ralstonia, Moraxella, and Flavobacter. The buttermilk did not contain any pathogenic species. However, the roles of unclassifiable organisms are yet to be studied.

Evaluation of InSeq to Identify Genes Essential for Pseudomonas aeruginosa PGPR2 Corn Root Colonization

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the TnSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5-to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P. aeruginosa PGPR2 for root colonization.

Anti-adhesion Property of the Potential Probiotic Strain Lactobacillus fermentum 8711 Against Methicillin-Resistant Staphylococcus aureus (MRSA)

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen and one of the leading causes of nosocomial infection worldwide. Probiotic bacteria play a significant role in preventive or therapeutic interventions of gastrointestinal infections in human as well as animals. In this study, we have investigated the adhesion property of the probiotic strain Lactobacillus fermentum MTCC 8711 and its ability to prevent the adhesion of MRSA to human colon adenocarcinoma cells, Caco-2. We have shown that L. fermentum could efficiently adhere to the Caco-2 cells. Also, we have shown that L. fermentum significantly reduced MRSA adhesion to Caco-2 cells. Three types of experiments were performed to assess the anti-adhesion property of L. fermentum against MRSA. Inhibition (Caco-2 cells were pre-treated with L. fermentum, and subsequently MRSA was added), competition (both L. fermentum and MRSA were added to Caco-2 cells simultaneously), and displacement or exclusion (Caco-2 cells were pre-treated with MRSA, and subsequently L. fermentum was added). In all three experiments, adhesion of MRSA was significantly reduced. Interestingly, L. fermentum could efficiently displace the adhered MRSA, and hence this probiotic can be used for therapeutic applications also. In cytotoxicity assay, we found that L. fermentum per se was not cytotoxic, and also significantly reduced the MRSA-induced cytotoxicity. The protective effect occurred without affecting Caco-2 cell morphology and viability.