Diversity and Succession of Microbiota during Fermentation of the Traditional Indian Food Idli.

Idli, a naturally fermented Indian food, is prepared from a mixture of rice and black gram (lentil). To understand its microbial community during fermentation, detailed analysis of the structural and functional dynamics of the idli microbiome was performed by culture-dependent and -independent approaches. The bacterial diversity and microbial succession were assessed at different times of fermentation by 16S rRNA amplicon sequencing. Results highlighted that most microbiota belonged to phylum Firmicutes (70%) and Proteobacteria (22%). Denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) analysis confirmed the diversity and succession involved therein. A culture-dependent approach revealed that the microbially diverse populations were conserved across different geographical locations. The fermentation was primarily driven by lactic acid bacteria as they constitute 86% of the total bacterial population, and genus Weissella emerged as the most important organism in fermentation. The natural microbiota of the grains mainly drives the fermentation, as surface sterilized grains did not show any fermentation. Growth kinetics of idli microbiota and physicochemical parameters corroborated the changes in microbial dynamics, acid production, and leavening occurring during fermentation. Using a metagenomic prediction tool, we found that the major metabolic activities of these microbial fermenters were augmented during the important phase of fermentation. The involvement of the heterofermentative hexose monophosphate (HMP) pathway in batter leavening was substantiated by radiolabeled carbon dioxide generated from d-[1-14C]-glucose. Hydrolases degrading starch and phytins and the production of B vitamins were reported. Moreover, culturable isolates showing beneficial attributes, such as acid and bile tolerance, hydrophobicity, antibiotic sensitivity, and antimicrobial activity, suggest idli to be a potential dietary supplement.IMPORTANCE This is a comprehensive analysis of idli fermentation employing modern molecular tools which provided valuable information about the bacterial diversity enabling its fermentation. The study has demonstrated the relationship between the bacterial population and its functional role in the process. The nature of idli fermentation was found to be more complex than other food fermentations due to the succession of the bacterial population. Further studies using metatranscriptomics and metabolomics may enhance the understanding of this complex fermentation process. Moreover, the presence of microorganisms with beneficial properties plausibly makes idli a suitable functional food.

Immune signal transduction in leishmaniasis from natural to artificial systems: role of feedback loop insertion.

BACKGROUND: Modulated immune signal (CD14-TLR and TNF) in leishmaniasis can be linked to EGFR pathway involved in wound healing, through crosstalk points. This signaling network can be further linked to a synthetic gene circuit acting as a positive feedback loop to elicit a synchronized intercellular communication among the immune cells which may contribute to a better understanding of signaling dynamics in leishmaniasis. METHODS: Network reconstruction with positive feedback loop, simulation (ODE 15s solver) and sensitivity analysis of CD14-TLR, TNF and EGFR was done in SimBiology (MATLAB 7.11.1). Cytoscape and adjacency matrix were used to calculate network topology. PCA was extracted by using sensitivity coefficient in MATLAB. Model reduction was done using time, flux and sensitivity score. RESULTS: Network has five crosstalk points: NIK, IκB-NFκB and MKK (4/7, 3/6, 1/2) which show high flux and sensitivity. PI3K in EGFR pathway shows high flux and sensitivity. PCA score was high for cytoplasmic ERK1/2, PI3K, Atk, STAT1/3 and nuclear JNK. Of the 125 parameters, 20% are crucial as deduced by model reduction. CONCLUSIONS: EGFR can be linked to CD14-TLR and TNF through the MAPK crosstalk points. These pathways may be controlled through Ras and Raf that lie upstream of signaling components ERK ½ (c) and JNK (n) that have a high PCA score via a synthetic gene circuit for activating cell-cell communication to elicit an inflammatory response. Also a disease resolving effect may be achieved through PI3K in the EGFR pathway. GENERAL SIGNIFICANCE: The reconstructed signaling network can be linked to a gene circuit with a positive feedback loop, for cell-cell communication resulting in synchronized response in the immune cell population, for disease resolving effect in leishmaniasis.

The crux and crust of ebolavirus: Analysis of genome sequences and glycoprotein gene.

The recent 2013-15 epidemic of Ebola virus disease (EVD) has initiated extensive sequencing and analysis of ebolavirus genomes. All ebolavirus genomes available until December 2014 have been collated and analyzed in this study to obtain phylogenetic relationship and uncover the variations amongst them. The terminal 'leader' and 'trailer' nucleotide sequences of the genomes were omitted and analysis of the intermediate region accommodating the sole seven genes (hepta-CDS region) of the virus showed relative stability of the genome, including the ones isolated from the current epidemic. The genome information was scrutinized to detect the variation in the surface glycoprotein gene and annotate its three protein products, resulting from its atypical transcription. This study will make an easy understanding of the genomes for those who desire to exploit the genome sequences for different investigations in EVD.

A bioinformatics approach to reanalyze the genome annotation of kinetoplastid protozoan parasite Leishmania donovani.

Leishmania donovani is a kinetoplastid protozoan parasite which causes the fatal disease visceral leishmaniasis in humans. Genome sequencing of L. donovani revealed information about the arrangement of genes and genome architecture. After curation of the genome sequence, many genes in L. donovani were assigned as truncated or "partial" genes by the genome sequencing group. In the present study, we have carried out an extensive analysis and attempted to improve the gene models of these partial genes. Our analysis resulted in the identification of 308 partial genes in L. donovani, which were further categorized as C-terminal extensions, joining of genes, tandemly repeated paralogs and wrong chromosomal assignments. We have analyzed each of these genes from these categories and have improved the annotation of existing gene models in L. donovani. Some of these corrections have been confirmed by mass spectrometry derived peptide data from our previous comparative proteogenomics study in L. donovani.

Rifapentine-proliposomes for inhalation: in vitro and in vivo toxicity.

BACKGROUND: Oral therapy for pulmonary tuberculosis (TB) treatment suffers from the limitation of hepatic metabolism leading insufficient concentration of antitubercular (anti-TB) drugs in alveolar macrophage which harbors Mycobacterium tuberculosis (MTB). Targeted aerosol delivery of antituberculous drug to lung is efficient for treating local lung TB infection. OBJECTIVE: The present study was aimed to evaluate rifapentine (RPT) loaded proliposomal dry powder for inhalation (RLDPI) for anti-TBactivity and cytotoxicity in vitro. In vivo toxicity study was also undertaken in Wistar rats to determine safe concentration of RLDPI for administration. MATERIALS AND METHODS: Anti-TB activity of developed RLDPI was assessed using drug susceptibility testing (DST) on Mycobacteria growth indicator tube (MGIT) method. In vitro cytotoxicity was performed in A549 cell lines and IC50 values were used to compare the cytotoxicity of formulation with pure RPT. In vivo repeated dose toxicity study was undertaken using Wistar rats at three different doses for 28-days by intratracheal insufflations method. RESULTS: The results of DST study revealed sensitivity of tubercle bacteria to RLDPI at concentration equivalent to 10 µg/mL of RPT. This study confirmed anti-TB potential of RPT in spray-dried RLDPI, though the spray drying method is reported to reduce activity of drugs. Cytotoxicity study in A549 cells demonstrated that RPT when encapsulated in liposomes as RLDPI was safe to cells as compared to pure RPT. In vivo toxicity study revealed that RPT in the form of RLDPI was safe at 1 and 5 mg/kg dose. However, mortality was seen at higher dose (10 mg/kg), possibly because of liver and kidney damage. CONCLUSION: Thus, these studies demonstrated safety of RLDPI for the treatment of pulmonary TB.

Comparative analysis of midgut bacterial communities of Aedes aegypti mosquito strains varying in vector competence to dengue virus.

Differences in midgut bacterial communities of Aedes aegypti, the primary mosquito vector of dengue viruses (DENV), might influence the susceptibility of these mosquitoes to infection by DENV. As a first step toward addressing this hypothesis, comparative analysis of bacterial communities from midguts of mosquito strains with differential genetic susceptibility to DENV was performed. 16S rRNA gene libraries and real-time PCR approaches were used to characterize midgut bacterial community composition and abundance in three Aedes aegypti strains: MOYO, MOYO-R, and MOYO-S. Although Pseudomonas spp.-related clones were predominant across all libraries, some interesting and potentially significant differences were found in midgut bacterial communities among the three strains. Pedobacter sp.- and Janthinobacterium sp.-related phylotypes were identified only in the MOYO-R strain libraries, while Bacillus sp. was detected only in the MOYO-S strain. Rahnella sp. was found in MOYO-R and MOYO strains libraries but was absent in MOYO-S libraries. Both 16S rRNA gene library and real-time PCR approaches confirmed the presence of Pedobacter sp. only in the MOYO-R strain. Further, real-time PCR-based quantification of 16S rRNA gene copies showed bacterial abundance in midguts of the MOYO-R strain mosquitoes to be at least 10-100-folds higher than in the MOYO-S and MOYO strain mosquitoes. Our study identified some putative bacteria with characteristic physiological properties that could affect the infectivity of dengue virus. This analysis represents the first report of comparisons of midgut bacterial communities with respect to refractoriness and susceptibility of Aedes aegypti mosquitoes to DENV and will guide future efforts to address the potential interactive role of midgut bacteria of Aedes aegypti mosquitoes in determining vectorial capacity for DENV.

A proteogenomic approach to map the proteome of an unsequenced pathogen - Leishmania donovani.

Visceral leishmaniasis or kala azar is the most severe form of leishmaniasis and is caused by the protozoan parasite Leishmania donovani. There is no published report on L. donovani genome sequence available till date, although the genome sequences of three related Leishmania species are already available. Thus, we took a proteogenomic approach to identify proteins from two different life stages of L. donovani. From our analysis of the promastigote (insect) and amastigote (human) stages of L. donovani, we identified a total of 22,322 unique peptides from a homology-based search against proteins from three Leishmania species. These peptides were assigned to 3711 proteins in L. infantum, 3287 proteins in L. major, and 2433 proteins in L. braziliensis. Of the 3711 L. donovani proteins that were identified, the expression of 1387 proteins was detectable in both life stages of the parasite, while 901 and 1423 proteins were identified only in promastigotes and amastigotes life stages, respectively. In addition, we also identified 13 N-terminally and one C-terminally extended proteins based on the proteomic data search against the six-frame translated genome of the three related Leishmania species. Here, we report results from proteomic profiling of L. donovani, an organism with an unsequenced genome.

Determination of Wolbachia diversity in butterflies from Western Ghats, India, by a multigene approach.

Members of the genus Wolbachia are intracellular bacteria that are widespread in arthropods and establish diverse symbiotic associations with their hosts, ranging from mutualism to parasitism. Here we present the first detailed analyses of Wolbachia in butterflies from India with screening of 56 species. Twenty-nine species (52%) representing five families were positive for Wolbachia. This is the first report of Wolbachia infection in 27 of the 29 species; the other two were reported previously. This study also provides the first evidence of infection in the family Papilionidae. A striking diversity was observed among Wolbachia strains in butterfly hosts based on five multilocus sequence typing (MLST) genes, with 15 different sequence types (STs). Thirteen STs are new to the MLST database, whereas ST41 and ST125 were reported earlier. Some of the same host species from this study carried distinctly different Wolbachia strains, whereas the same or different butterfly hosts also harbored closely related Wolbachia strains. Butterfly-associated STs in the Indian sample originated by recombination and point mutation, further supporting the role of both processes in generating Wolbachia diversity. Recombination was detected only among the STs in this study and not in those from the MLST database. Most of the strains were remarkably similar in their wsp genotype, despite divergence in MLST. Only two wsp alleles were found among 25 individuals with complete hypervariable region (HVR) peptide profiles. Although both wsp and MLST show variability, MLST gives better separation between the strains. Completely different STs were characterized for the individuals sharing the same wsp alleles.

Lactobacillus plantarum (VR1) isolated from an ayurvedic medicine (Kutajarista) ameliorates in vitro cellular damage caused by Aeromonas veronii.

BACKGROUND: Lactobacillus plantarum is considered as a safe and effective probiotic microorganism. Among various sources of isolation, traditionally fermented foods are considered to be rich in Lactobacillus spp., which can be exploited for their probiotic attribute. Antibacterial property of L. plantarum has been demonstrated against various enteric pathogens in both in vitro and in vivo systems. This study was aimed at characterizing L. plantarum isolated from Kutajarista, an ayurvedic fermented biomedicine, and assessing its antagonistic property against a common enteropathogen Aeromonas veronii. RESULTS: We report the isolation of L. plantarum (VR1) from Kutajarista, and efficacy of its cell free supernatant (CFS) in amelioration of cytotoxicity caused by Aeromonas veronii. On the part of probiotic attributes, VR1 was tolerant to pH 2, 0.3% bile salts and simulated gastric juice. Additionally, VR1 also exhibited adhesive property to human intestinal HT-29 cell line. Furthermore, CFS of VR1 was antibacterial to enteric pathogens like Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Aeromonas veronii and clinical isolates of P. aeruginosa and E. coli. Detailed study regarding the effect of VR1 CFS on A. veronii cytotoxicity showed a significant decrease in vacuole formation and detrimental cellular changes in Vero cells. On the other hand, A. veronii CFS caused disruption of tight junction proteins ZO-1 and actin in MDCK cell line, which was prevented by pre-incubation with CFS of VR1. CONCLUSIONS: This is the first study to report isolation of L. plantarum (VR1) from Kutajarista and characterisation for its probiotic attributes. Our study demonstrates the antagonistic property of VR1 to A. veronii and effect of VR1 CFS in reduction of cellular damage caused by A. veronii in both Vero and MDCK cell lines.

Chitosan-reduced gold nanoparticles: a novel carrier for the preparation of spray-dried liposomes for topical delivery.

Exposure of skin to various chemical and physical agents results in excessive stress to the outermost cell layer of the skin, causing different degenerative effects that can be minimized by using antioxidant formulations. The major challenge, in this regard, is to develop a formulation, which can prevent photodegradation of the actives, thus allowing a significant amount to be deposited at the site. In recent decades, liposomal formulations have been extensively employed to overcome the barrier properties of the skin and photodegradation of actives. In the present study, chitosan-reduced gold nanoparticles were investigated for its potential as a carrier to prepare liposomes by a spray-drying method. Liposomes so obtained were characterized for phospholipid recovery, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, particle size, zeta potential, encapsulation efficiency, and deposition of drug and gold nanoparticles in the rat skin. Further, a liposomal gel formulation was prepared using Carbopol® 980 NF (Noveon Systems, Kochi, India) and evaluated for drug deposition in the skin. Antioxidant activity of vitamin C encapsulated in gold liposomes was determined on a human leukemia (HL-60) cell line. The use of gold nanoparticles as a carrier showed improved phospholipid recovery and thus overcomes the liposome scalability problem. DRIFT spectra confirmed the presence of phospholipid in the formulation. Liposomal gel showed improved drug deposition, as compared to control and marketed preparations. A more interesting contribution of the chitosan-reduced gold nanoparticles was an enhanced antioxidant activity seen in case of the vitamin C-loaded gold liposomal formulation. Liposomal formulation was found to be stable for 3 months at 30°C and 65% relative humidity.

Structural aspects of lesional and non-lesional skin microbiota reveal key community changes in leprosy patients from India.

Although skin is the primary affected organ in Leprosy, the role of the skin microbiome in its pathogenesis is not well understood. Recent reports have shown that skin of leprosy patients (LP) harbours perturbed microbiota which grants inflammation and disease progression. Herein, we present the results of nested Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) which was initially performed for investigating the diversity of bacterial communities from lesional skin (LS) and non-lesional skin (NLS) sites of LP (n = 11). Further, we performed comprehensive analysis of 16S rRNA profiles corresponding to skin samples from participants (n = 90) located in two geographical locations i.e. Hyderabad and Miraj in India. The genus Staphylococcus was observed to be one of the representative bacteria characterizing healthy controls (HC; n = 30), which in contrast was underrepresented in skin microbiota of LP. Taxa affiliated to phyla Firmicutes and Proteobacteria were found to be signatures of HC and LS, respectively. Observed diversity level changes, shifts in core microbiota, and community network structure support the evident dysbiosis in normal skin microbiota due to leprosy. Insights obtained indicate the need for exploring skin microbiota modulation as a potential therapeutic option for leprosy.

Temporal bacterial diversity and detection of putative methanotrophs in surface mats of Lonar crater lake.

The phylogenetic diversity of bacterial communities in microbial mats of two different seasons from saline and hyperalkaline Lonar Lake was investigated using 16S rRNA gene library analysis. Arthrospira (Cyanobacteria) related clones (>80% of total clones) dominated libraries of both the seasons. Clear differences were found in both the seasons as the operational taxonomic units (OTUs) related to Fusibacter (LAI-1 and LAI-59) and Tindallia magadiensis (LAI-27) found in post-monsoon were not found in the pre-monsoon library. Likewise, OTUs related to Planococcus rifietensis (LAII-67), Bordetella hinzii (LAII-2) and Methylobacterium variabile (LAII-25) found in the pre-monsoon were not found in post-monsoon. The study was extended to identify methanotrophs in the surface mats. Libraries constructed with type I and type II methanotroph specific 16S rRNA gene primers showed the presence of clones (LAMI-99 and LAMII-2) closely related to Methylomicrobium buryaticum and Beijerinckiaceae family members. Denaturing gradient gel electrophoresis (DGGE) fingerprinting based on protein-coding genes (pmoA and mxaF) further confirmed the detection of Methylomicrobium sp. Hence, we report here for the first time the detection of putative methanotrophs in surface mats of Lonar Lake. The finding of clones related to organisms with interesting functional attributes such as assimilation of C(1) compounds (LAII-25, LAMI-39, LAMI-99 and LAMII-2), non-sulfur photosynthetic bacteria (LAMII-43) and clones distantly affiliated to organisms of heavily polluted environments (LAI-59 and LAMII-52), is of significant note. These preliminary results would direct future studies on the functional dynamics of microbial mat associated food web chain in the extreme environment.

Genome-Wide Proteomics and Phosphoproteomics Analysis of Leishmania spp. During Differentiation.

Determining variations in protein abundance and/or posttranslational modification as a function of time or upon induction by a signal in a particular cell type is central to quantitative proteomics. Isobaric labeling methodologies now allow for parallel quantification of proteins at various conditions concurrently or multiplexing in relatively quantitative proteomics workflows. Hence, mapping the protein expression profiles of various developmental stages of Leishmania parasites is possible with high-resolution mass spectrometry. To analyze global changes in protein expression and cellular signaling pathways during Leishmania differentiation and development is possible with a quantitative proteomics approach. The tandem mass tags (TMT) approach provides a chemical labeling method based on the principle of amine reactive tags; the maximum number of conditions that can be multiplexed is 10-plex. We describe herein a detailed method for sample preparation, TMT-labeling, mass spectrometry and data analysis of different developmental stages of Leishmania donovani parasites. This quantitative proteomic approach is useful to study dynamic changes in protein expression levels during L. donovani differentiation, and also allows in-depth analysis of signaling pathways via phosphoproteomics.

Distribution of Pathogenic Yeasts in Different Clinical Samples: Their Identification, Antifungal Susceptibility Pattern, and Cell Invasion Assays.

INTRODUCTION: Species of genus Candida are part of the common microbiota of humans; however, some of the Candida species are known opportunistic pathogens. Formation of biofilms, resistance to antifungal drugs, and increase in asymptomatic infections demands more studies on isolation, identification and characterization of Candida from clinical samples. METHODS: The present manuscript deals with assessment of authentic yeast identification by three methods viz., DNA sequencing of 28S rRNA gene, protein profiles using MALDI-TOF MS, and colony coloration on chromogenic media. Antifungal susceptibility and in vitro cell invasion assays were performed to further characterize these isolates. RESULTS: Comparison of three methods showed that DNA sequence analysis correctly identified more than 99.4% of the isolates up to species level as compared to 89% by MALDI-TOF MS. In this study, we isolated a total of 176 yeasts from clinical samples and preliminary morphological characters indicated that these yeast isolates belong to the genus Candida. The species distribution of isolates was as follows: 75 isolates of Candida albicans (42.61%), 50 of C. tropicalis (28.40%), 22 of C. glabrata (12.5%), 14 of C. parapsilosis (7.95%) and 4 of Clavispora lusitaniae (2.27%). Other species like Cyberlindnera fabianii, Issatchenkia orientalis, Kluyveromyces marxianus, Kodamaea ohmeri, Lodderomyces sp., and Trichosporon asahii were less than 2%. Antifungal susceptibility assay performed with 157 isolates showed that most of the isolates were resistant to the four azoles viz., clotrimazole, fluconazole, itraconazole, and ketoconazole, and the frequency of resistance was more in non-albicans Candida isolates. The susceptibility to azole drugs ranged from 7% to 48%, while 75% of the tested yeasts were susceptible to nystatin. Moreover, 88 isolates were also tested for their capacity to invade human cells using HeLa cells. In vitro invasion assay showed that most of the C. albicans isolates showed epithelial cell invasion as compared to isolates belonging to C. glabrata, C. parapsilosis and C. tropicalis. DISCUSSION: The identification of yeasts of clinical origin by sequencing of 28S rRNA gene performed better than MALDI-TOF MS. The present study reiterates the world scenario wherein there is a shift from Candida strains to emerging opportunistic pathogens which were earlier regarded as environmental strains. The present study enlightens the current understanding of identification methods for clinical yeast isolates, increased antifungal drug resistance, epithelial cell invasion as a virulence factor, and diversity of yeasts in Indian clinical samples.

Generation, annotation, and analysis of ESTs from midgut tissue of adult female Anopheles stephensi mosquitoes.

BACKGROUND: Malaria is a tropical disease caused by protozoan parasite, Plasmodium, which is transmitted to humans by various species of female anopheline mosquitoes. Anopheles stephensi is one such major malaria vector in urban parts of the Indian subcontinent. Unlike Anopheles gambiae, an African malaria vector, transcriptome of A. stephensi midgut tissue is less explored. We have therefore carried out generation, annotation, and analysis of expressed sequence tags from sugar-fed and Plasmodium yoelii infected blood-fed (post 24 h) adult female A. stephensi midgut tissue. RESULTS: We obtained 7061 and 8306 ESTs from the sugar-fed and P. yoelii infected mosquito midgut tissue libraries, respectively. ESTs from the combined dataset formed 1319 contigs and 2627 singlets, totaling to 3946 unique transcripts. Putative functions were assigned to 1615 (40.9%) transcripts using BLASTX against UniProtKB database. Amongst unannotated transcripts, we identified 1513 putative novel transcripts and 818 potential untranslated regions (UTRs). Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes. Out of 3946 unique transcripts, only 1387 transcripts were mapped on the A. gambiae genome. These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome. The EST data is available as ESTDB at http://mycompdb.bioinfo-portal.cdac.in/cgi-bin/est/index.cgi. CONCLUSION: 3946 unique transcripts were successfully identified from the adult female A. stephensi midgut tissue. These data can be used for microarray development for better understanding of vector-parasite relationship and to study differences or similarities with other malaria vectors. Mapping of putative novel transcripts from A. stephensi on the A. gambiae genome proved fruitful in identification and annotation of several genes. Failure of some novel transcripts to map on the A. gambiae genome indicates existence of substantial genomic dissimilarities between these two potent malaria vectors.

16S rDNA based skin microbiome data of healthy individuals and leprosy patients from India.

Leprosy is an infectious disease that has predilection in skin and peripheral nerves. Skin has its own microbiome, however it is not extensively studied in Indian leprosy patients. Here, by using next-generation 16S rDNA sequencing, we have attempted to assess the skin associated microbial diversity pertaining to affected and unaffected skin of Indian leprosy patients. A total of 90 skin swab samples were collected from 60 individuals (30 healthy controls, 30 patients) residing in Hyderabad and Miraj, two distinct geographical locations in India to assess the homo/heterogeneity of skin microbial signatures. While a large increase in genus Methylobacterium and Pseudomonas was seen in patients from Miraj and Hyderabad respectively, a considerable decrease in genus Staphylococcus in the leprosy patients (as compared to controls) from both geographical locations was also observed. We expect that, these datasets can not-only provide further interesting insights, but will also help to observe dynamics of microbiome in the diseased state and generate hypotheses to test for skin microbiome transplantation studies in leprosy.

Extracellular synthesis of crystalline silver nanoparticles and molecular evidence of silver resistance from Morganella sp.: towards understanding biochemical synthesis mechanism.

There has been significant progress in the biological synthesis of nanomaterials. However, the molecular mechanism of synthesis of such bio-nanomaterials remains largely unknown. Here, we report the extracellular synthesis of crystalline silver nanoparticles (AgNPs) by using Morganella sp., and show molecular evidence of silver resistance by elucidating the synthesis mechanism. The AgNPs were 20+/-5 nm in diameter and were highly stable at room temperature. The kinetics of AgNPs formation was investigated. Detectable particles were formed after an hour of reaction, and their production remained exponential up to 18 h, and saturated at 24 h. Morganella sp. was found to be highly resistant to silver cations and was able to grow in the presence of more than 0.5 mM AgNO(3). Three gene homologues viz. silE, silP and silS were identified in silver-resistant Morganella sp. The homologue of silE from Morganella sp. showed 99 % nucleotide sequence similarity with the previously reported gene, silE, which encodes a periplasmic silver-binding protein. The homologues of silP and silS were also highly similar to previously reported sequences. Similar activity was totally absent in closely related Escherichia coli; this suggests that a unique mechanism of extracellular AgNPs synthesis is associated with silver-resistant Morganella sp. The molecular mechanism of silver resistance and its gene products might have a key role to play in the overall synthesis process of AgNPs by Morganella sp. An understanding of such biochemical mechanisms at the molecular level might help in developing an ecologically friendly and cost-effective protocol for microbial AgNPs synthesis.

Isolation of urease-positive Ochrobactrum intermedium in the stomach of a non-ulcer dyspeptic patient from north India.

Ochrobactrum intermedium is an opportunistic human pathogen found in immunocompromised individuals. We report the case of a north Indian patient with non-ulcer dyspepsia whose gastric biopsy revealed the presence of O. intermedium, along with Helicobacter pylori. Further description of O. intermedium was performed with 16S rRNA (1500 nucleotides) and RecA (1065 nucleotides) gene sequencing, and the identity and phylogenetic affiliation of the isolate was confirmed by 100% nucleotide similarity with O. intermedium LMG3301. Further investigation is required in order to evaluate the link between H. pylori and O. intermedium in the gastric niche.

Bacterial synthesis of copper/copper oxide nanoparticles.

A bacterial mediated synthesis of copper/copper oxide nanoparticle composite is reported. A Gram-negative bacterium belonging to the genus Serratia was isolated from the midgut of Stibara sp., an insect of the Cerambycidae family of beetles found in the Northwestern Ghats of India. This is a unique bacterium that is quite specific for the synthesis of copper oxide nanoparticles as several other strains isolated from the same insect and common Indian mosquitoes did not result in nanoparticle formation. By following the reaction systematically, we could delineate that the nanoparticle formation occurs intracellularly. However, the process results in the killing of bacterial cells. Subsequently the nanoparticles leak out as the cell wall disintegrates. The nanoparticles formed are thoroughly characterized by UV-Vis, TEM, XRD, XPS and FTIR studies.

Investigation of methanogen population structure in biogas reactor by molecular characterization of methyl-coenzyme M reductase A (mcrA) genes.

The methanogen community in biogas reactor running on cattle dung was investigated in two different seasons; summer (April, 36 degrees C) and winter (December, 24 degrees C), in the year 2004 by a culture-independent approach. Community structure was determined by phylogenetic analyses of 343 and 278 mcrA clones belonging to summer and winter month libraries, respectively. In summer month's library, 41.7% clones were affiliated to Methanomicrobiales, 30% to Methanosarcinales, 19% to Methanobacteriales, 5% to Methanococcales and a total of 4.3% clones belonged to unclassified euryarchaeotal lineages. In winter month's library, Methanomicrobiales encompassed 98.6% clones, and Methanobacteriales included 1.4% of total clone diversity. Biogas plant performance data collected during the winter month indicated significant reduction in daily biogas produced as compared to summer month because of lowering in ambient temperature and associated shift in microbial community. Results from this molecular study showed the existence of highly diverse and complex methanogens communities present in biogas plant.