Vasvi Chaudhry

Genomic Resource of Rice Seed Associated Bacteria

Plants are host to diverse microbiome that might have co-evolved since millions of years. This resident microbiota can act as extended genome by contributing in plant growth, development and protection from biotic and abiotic stresses. Rice (Oryza Sativa) is a staple food consumed by more than 50% of the worlds population. Herein we targeted the bacterial community associated with the healthy rice seeds. In this direction, we isolated and carried out whole genome sequencing of 100 bacterial isolates. These isolates belong to three major bacterial phyla Proteobacteria, Firmicutes, and Actinobacteria that spread over 15 distinct genus and 29 species. A phylogenetic tree based on a broad set of phylogenomic marker genes confirmed the evolutionary relationship amongst the strains and their phylogenetic grouping. Average Nucleotide Identity was also used to establish species identity of isolates that form a particular phylogenetic and taxonomic grouping. The data generated from the present study is one of the first major genomic resources in the field of phytobiome research. Whole genome sequence of the members will be invaluable in this era of big data driven research. Moreover, the majority of genus and species identified in this study are already known for plant probiotic properties. This genomic data with annotation will aid in comparative, evolutionary and ecological studies of bacteria associated with plants or multi-kingdom bacteria associated with nosocomial infections.

Genomic investigation reveals evolution and lifestyle adaptation of endophytic Staphylococcus epidermidis.

Staphylococcus epidermidis is a major human associated bacterium and also an emerging nosocomial pathogen. There are reports of its association to rodents, sheep and plants. However, comparative and evolutionary studies of ecologically diverse strains of S. epidermidis are lacking. Here, we report the whole genome sequences of four S. epidermidis strains isolated from surface sterilized rice seeds along with genome sequence of type strain. Phylogenomic analysis of rice endophytic S. epidermidis (RESE) with “type strain” unequivocally established their species identity. Whole genome based tree of 93 strains of S. epidermidis revealed RESE as distinct sub-lineage which is more related to rodent sub-lineage than to majority of human lineage strains. Furthermore, comparative genomics revealed 20% variable gene-pool in S. epidermidis, suggesting that genomes of ecologically diverse strains are under flux. Interestingly, we were also able to map several genomic regions that are under flux and gave rise to RESE strains. The largest of these genomic regions encodes a cluster of genes unique to RESE that are known to be required for survival and stress tolerance, apart from those required for adaptation to plant habitat. The genomes and genes of RESE represent distinct ecological resource/sequences and provided first evolutionary insights into adaptation of S. epidermidis to plants.

Characterization of the Antimicrobial Peptide Penisin, a Class Ia Novel Lantibiotic from Paenibacillus sp. Strain A3

ABSTRACT Attempts to isolate novel antimicrobial peptides from microbial sources have been on the rise recently, despite their low efficacy in therapeutic applications. Here, we report identification and characterization of a new efficient antimicrobial peptide from a bacterial strain designated A3 that exhibited highest identity with Paenibacillus ehimensis. Upon purification and subsequent molecular characterization of the antimicrobial peptide, referred to as penisin, we found the peptide to be a bacteriocin-like peptide. Consistent with these results, RAST analysis of the entire genome sequence revealed the presence of a lantibiotic gene cluster containing genes necessary for synthesis and maturation of a lantibiotic. While circular dichroism and one-dimension nuclear magnetic resonance experiments confirmed a random coil structure of the peptide, similar to other known lantibiotics, additional biochemical evidence suggests posttranslational modifications of the core peptide yield six thioether cross-links. The deduced amino acid sequence of the putative biosynthetic gene penA showed approximately 74% similarity with elgicin A and 50% similarity with the lantibiotic paenicidin A. Penisin effectively killed methicillin-resistant Staphylococcus aureus (MRSA) and did not exhibit hemolysis activity. Unlike other lantibiotics, it effectively inhibited the growth of Gram-negative bacteria. Furthermore, 80 mg/kg of body weight of penisin significantly reduced bacterial burden in a mouse thigh infection model and protected BALB/c mice in a bacteremia model entailing infection with Staphylococcus aureus MTCC 96, suggesting that it could be a promising new antimicrobial peptide.

Methylobacterium indicum sp. nov., a facultative methylotrophic bacterium isolated from rice seed ☆

Two pink pigmented, Gram-negative, motile, aerobic, rod shaped endophytic bacteria designated as SE2.11(T) and SE3.6 were isolated in different experiments from surface sterilized rice seeds. Both strains grew optimally at 28°C temperature. They were positive for catalase and nitrate reduction. The 16S rRNA gene sequence of the strains SE2.11(T) and SE3.6 displayed between 98.1% and 97.2% similarities with the validly published species of the genus Methylobacterium. The major cellular fatty acid was C18:1 ω7c in both the strains, a characteristic feature observed for members of the genus Methylobacterium. The predominant polar lipids were phospholipids including phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG). Phylogenetic analysis of 16S rRNA gene sequences resulted in the formation of a coherent cluster of strains SE2.11(T) and SE3.6 with closest relative Methylobacterium platani JCM 14648(T). However, digital DNA-DNA hybridization (dDDH) of strains SE2.11(T) and SE3.6 with the closest type strain M. platani JCM 14648(T) revealed similarity of 35.5% and 35.4%, respectively. Further, the ANI analysis of strains SE2.11(T) and SE3.6 genomes revealed only 87.9% identity with M. platani JCM 14648(T). Based on differences in biochemical, chemotaxonomic characteristics along with low identity at whole genome level we conclude that both strains represent a novel species of the genus Methylobacterium, for which the name Methylobacterium indicum sp. nov., is proposed. The type strain Methylobacterium indicum is SE2.11(T) (=MTCC 12298(T)=JCM 30761(T)) and SE3.6 (=MTCC 12299=JCM 30762) is another strain.

Laterosporulin10: a novel defensin like Class IId bacteriocin from Brevibacillus sp. strain SKDU10 with inhibitory activity against microbial pathogens

Bacteriocins are antimicrobial peptides (AMPs) produced by bacteria to acquire survival benefits during competitive inter- and intra-species interactions in complex ecosystems. In this study, an AMP-producing soil bacterial strain designated SKDU10 was isolated and identified as a member of the genus Brevibacillus. The AMP produced by strain SKDU10 identified as a class IId bacteriocin with 57.6 % homology to laterosporulin, a defensin-like class IId bacteriocin. However, substantial differences were observed in the antimicrobial activity spectrum of this bacteriocin named laterosporulin10 when compared to laterosporulin. Laterosporulin10 effectively inhibited the growth of Staphylococcus aureus and Mycobacterium tuberculosis (Mtb H37Rv) with LD50 values of 4.0 µM and 0.5 µM, respectively. Furthermore, laterosporulin10 inhibited the growth of Mtb H37Rv strain at about 20 times lower MIC value compared to S. aureus MTCC 1430 or M. smegmatis MC2 155 in vitro and ex vivo. Electron micrographs along with membrane permeabilization studies using FACS analysis revealed that laterosporulin10 is a membrane-permeabilizing peptide. Interestingly, laterosporulin10 was able to efficiently kill Mtb H37Rv strain residing inside the macrophages and did not show haemolysis up to 40 µM concentration.

Glimpse into the Genomes of Rice Endophytic Bacteria: Diversity and Distribution of Firmicutes

Endophytic bacteria inhabit within plant tissues without causing any evident damage to the host and play crucial roles in plant growth, development, fitness, and protection (Farrar et al., 2014; Truyens et al., 2015). These endophytic bacteria spend a portion of their life cycle inside plants and normally resides on intercellular spaces and gain carbohydrates, amino acids, and inorganic nutrients from plants (Bacon andHinton, 2007). Despite their beneficial effects on plant growth and development, seed-borne endophytic bacteria are still largely unexplored. Recent developments in high-throughput technologies, such as next-generation sequencing (NGS), permit the investigation of endophytic microbiomes, facilitate sequencing of a larger number of bacteria and encourage in depth analyses of bacterial communities from taxonomy, phylogeny, and evolutionary studies (Kaul et al., 2016). Genomes of endophytic bacteria encode all the information that is required for an organism to grow under a range of both favorable and unfavorable conditions depending on the plant habitats. Along with the house-keeping machinery, these bacterial genomes also encode genes that are required for their endophytic life style and plant beneficial properties (Hardoim et al., 2015; Sheibani-Tezerji et al., 2015). In the present study, whole genome sequencing of 21 rice seed endophytic bacterial species belonging to the phylum Firmicutes was performed to ascertain their phylogenetic position and to get clue of the genomic signatures for their adaptation to endophytic lifestyle. Genomic dataset of endophytic bacterial strains are valuable pool of information that provides insights into the diversity, distribution, and lifestyle associated genes of these endophytes associated with plants.

Metabolite Profiling Reveals Abiotic Stress Tolerance in Tn5 Mutant of Pseudomonas putida

Pseudomonas is an efficient plant growth–promoting rhizobacteria (PGPR); however, intolerance to drought and high temperature limit its application in agriculture as a bioinoculant. Transposon 5 (Tn5) mutagenesis was used to generate a stress tolerant mutant from a PGPR Pseudomonas putida NBRI1108 isolated from chickpea rhizosphere. A mutant NBRI1108T, selected after screening of nearly 10,000 transconjugants, exhibited significant tolerance towards high temperature and drought. Southern hybridization analysis of EcoRI and XhoI restricted genomic DNA of NBRI1108T confirmed that it had a single Tn5 insertion. The metabolic changes in the polar and non-polar extracts of NBRI1108 and NBRI1108T were examined using 1H, 31P nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Thirty six chemically diverse metabolites consisting of amino acids, fatty acids and phospholipids were identified and quantified. Insertion of Tn5 influenced amino acid and phospholipid metabolism and resulted in significantly higher concentration of aspartic acid, glutamic acid, glycinebetaine, glycerophosphatidylcholine (GPC) and putrescine in NBRI1108T as compared to that in NBRI1108. The concentration of glutamic acid, glycinebetaine and GPC increased by 34%, 95% and 100%, respectively in the NBRI1108T as compared to that in NBRI1108. High concentration of glycerophosphatidylethanolamine (GPE) and undetected GPC in NBRI1108 indicates that biosynthesis of GPE may have taken place via the methylation pathway of phospholipid biosynthesis. However, high GPC and low GPE concentration in NBRI1108T suggest that methylation pathway and phosphatidylcholine synthase (PCS) pathway of phospholipid biosynthesis are being followed in the NBRI1108T. Application of multivariate principal component analysis (PCA) on the quantified metabolites revealed clear variations in NBRI1108 and NBRI1108T in polar and non-polar metabolites. Identification of abiotic stress tolerant metabolites from the NBRI1108T suggest that Tn5 mutagenesis enhanced tolerance towards high temperature and drought. Tolerance to drought was further confirmed in greenhouse experiments with maize as host plant, where NBRI1108T showed relatively high biomass under drought conditions.

Insights from the draft genome of Paenibacillus lentimorbus NRRL B-30488, a promising plant growth promoting bacterium ☆

Paenibacillus lentimorbus NRRL B-30488, a plant growth-promoting bacterium was isolated from Sahiwal cows milk. The strain shows antagonism against phytopathogens, Fusarium oxysporum f. sp. ciceri and Alternaria solani. Its genome contains gene clusters involved in nonribosomal synthesis of secondary metabolites involved in antimicrobial activities. The genome sequence of P. lentimorbus NRRL B-30488 provides the genetic basis for application of this bacterial strain in plant growth promotion, plant protection and degradation of organic pollutants.

Draft Genome Sequence of Pseudomonas putida Strain MTCC5279.

ABSTRACT Here we report the genome sequence of a plant-growth-promoting rhizobacterium, Pseudomonas putida strain MTCC5279. The length of the draft genome sequence is approximately 5.2 Mb, with a GC content of 62.5%. The draft genome sequence reveals a number of genes whose products are possibly involved in plant growth promotion and abiotic stress tolerance.

Chlorella vulgaris and Pseudomonas putida interaction modulates phosphate trafficking for reduced arsenic uptake in rice (Oryza sativa L.)

Rice grown in arsenic (As) contaminated areas contributes to high dietary exposure of As inducing multiple adverse effects on human health. The As contamination and application of phosphate fertilizers during seedling stage creates a high P and As stress condition. The flooded paddy fields are also conducive for algal growth and microbial activity. The present study proposes potential role of microalgae, Chlorella vulgaris (CHL) and bacteria, Pseudomonas putida (RAR) on rice plant grown under excess As and phosphate (P) conditions. The results show synchronized interaction of CHL + RAR which, reduces As uptake through enhanced P:As and reduced As:biomass ratio by modulating P trafficking. Gene expression analysis of different phosphate transporters exhibited correlation with reduced As uptake and other essential metals. The balancing of reactive oxygen species (ROS), proline accumulation, hormone modulation, and As sequestration in microbial biomass were elucidated as possible mechanisms of As detoxification. The study concludes that RAR and CHL combination mitigates the As stress during P-enriched conditions in rice by: (i) reducing As availability, (ii) modulating the As uptake, and (iii) improving detoxification mechanism of the plant. The study will be important in assessing the role and applicability of P solubilizing biofertilizers in these conditions.