"Gearing" up for dynamin-catalyzed membrane fission.

Endocytic dynamins self-assemble into helical scaffolds and utilize energy from GTP hydrolysis to constrict and sever tubular membranous necks of budded endocytic intermediates. They bind the membrane using a pleckstrin-homology domain (PHD). The PHD is characterized by four unstructured loops, two of which partially insert into the membrane. Recent studies reveal that loop insertion lowers the bending rigidity of the membrane and that mutations in these two loops produce separable and opposite effects on the efficiency of dynamin-catalyzed membrane fission. Here, we review the current understanding of dynamin-catalyzed membrane fission and attempt to reconcile contrasting notions that have emerged from biochemical and cellular studies evaluating the role of the PHD in this process. We propose that two membrane-inserting loops act as "gears" that define the catalytic efficiency of the dynamin helical scaffold in membrane fission.

Mechanistic analysis of a novel membrane-interacting variable loop in the pleckstrin-homology domain critical for dynamin function.

Classical dynamins are best understood for their ability to generate vesicles by membrane fission. During clathrin-mediated endocytosis (CME), dynamin is recruited to the membrane through multivalent protein and lipid interactions between its proline-rich domain (PRD) with SRC Homology 3 (SH3) domains in endocytic proteins and its pleckstrin-homology domain (PHD) with membrane lipids. Variable loops (VL) in the PHD bind lipids and partially insert into the membrane thereby anchoring the PHD to the membrane. Recent molecular dynamics (MD) simulations reveal a novel VL4 that interacts with the membrane. Importantly, a missense mutation that reduces VL4 hydrophobicity is linked to an autosomal dominant form of Charcot-Marie-Tooth (CMT) neuropathy. We analyzed the orientation and function of the VL4 to mechanistically link data from simulations with the CMT neuropathy. Structural modeling of PHDs in the cryo-electron microscopy (cryo-EM) cryoEM map of the membrane-bound dynamin polymer confirms VL4 as a membrane-interacting loop. In assays that rely solely on lipid-based membrane recruitment, VL4 mutants with reduced hydrophobicity showed an acute membrane curvature-dependent binding and a catalytic defect in fission. Remarkably, in assays that mimic a physiological multivalent lipid- and protein-based recruitment, VL4 mutants were completely defective in fission across a range of membrane curvatures. Importantly, expression of these mutants in cells inhibited CME, consistent with the autosomal dominant phenotype associated with the CMT neuropathy. Together, our results emphasize the significance of finely tuned lipid and protein interactions for efficient dynamin function.

Taxonomic and genomic characterization of Sporosarcina cyprini sp. nov., moderately tolerant of Cr+6 and Cd+2 isolated from the gut of invasive fish Cyprinus carpio var. communis (Linn., 1758).

A Gram-stain-positive, motile, and rod-shaped bacterium, designated as strain MB25T, was isolated from the gut of Cyprinus carpio from the highly polluted river Yamuna, India. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain MB25T belonged to the genus Sporosarcina, sharing the highest sequence similarity with S. luteola Y1T (98.98%) and S. koreensis S-K12T (98.91%). Digital DNA-DNA hybridization and average nucleotide identity values of strain MB25T with strain Y1T and S-K12T were 18.9, 77.69, and 18.2, 76.80 respectively. Genome analysis of strain MB25T revealed its biotechnological properties such as tolerance to potent heavy metals, genes for the production of carbohydrate-active enzymes, antimicrobial compounds, and also degradation of aromatic compounds. The G + C content of strain MB25T genome was 45%. Growth observed at 10-40 °C (optimum, 28-30 °C), pH 6.0-8.5 (optimum pH 7.5-8.0); NaCl concentrations up to 6.0% (w/v). The dominant respiratory quinone was MK-7, cell wall peptidoglycan is of the A-4 type containing amino acids Lys-Glu and the major fatty acids are anteiso-C11:0 and iso-C15: 0. The major polar lipids of strain MB25T are diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. On the basis of phenotypic, chemotaxonomic, phylogenetic, and phylogenomic data, strain MB25T represents a novel species of the genus Sporosarcina, for which the name Sporosarcina cyprini sp. nov. is proposed. The type strain is MB25T (= MCC 4366 T = JCM 34521 T = CCM 9113 T).

Metagenomic insights to understand the role of polluted river Yamuna in shaping the gut microbial communities of two invasive fish species.

The gastrointestinal microbial community plays a crucial role in host health, immunity, protection, development and provides nutrients to the host. The rising human-induced pollution and heavy metal contamination in all aquatic systems globally has led us to explore the gut microbial diversity of two exotic invasive fish Cyprinus carpio (Linnaeus, 1858) and Oreochromis niloticus (Linnaeus,1857) from river Yamuna, India. These fishes are aquatic bioindicators with high demographic resilience. Exploring these associations would pave the way for addressing problems that inhabitant fishes are facing due to the increasing pollution load in the River Yamuna. Based on 16S rRNA gene amplicon sequencing, our results deliver comparative information on the gut microbiome of these fishes and highlight connotations between the microbiome of gut and water samples. The gut of C. carpio and O. niloticus was dominated by phyla Proteobacteria whereas Bacteroidetes dominated the water sample. Microbial communities showed predicted roles such as pathogenicity (Escherichia-Shigella, Aeromonas veronii, Vibrio cholerae, Streptococcus iniae, Flavobacterium columnare, Klebsiella pneumoniae, Mycobacterium sp.), probiotic applications (Bacillus velezensis, Lactobacillus plantarum, Enterococcus faecalis, Bifidobacterium longum, Lactococcus lactis, Leuconostoc falkenbergense) and involvement in sewage and organic matter decomposition (Nitrosomonas sp., Methanosaeta harundinacea, Dechloromonas agitata, Thauera humireducens, Zoogloea ramigera). Heavy metal degrading members (Leucobacter chromiireducens, Pseudomonas fluorescens, P. aeruginosa, Klebsiella pneumoniae, and Micrococcus luteus) were detected in gut microbiome samples thus supporting the notion that fish shapes its gut microbiota with changing ecology. Functional profiling showed that microbial communities are specialized in metabolic functions thus reflecting the dietary profile of these invasive fishes.

Protein-Protein Interactions on Membrane Surfaces Analysed Using Pull-Downs with Supported Bilayers on Silica Beads.

Discovery-based proteomics workflows that identify novel interactors rely on immunoprecipitations or pull-downs with genetically tagged bait proteins immobilized on appropriate matrices. But strategies to analyse protein interactions on a diffusible-membrane surface combined with the practical ease of pull-downs remain unavailable. Such strategies are important to analyse protein complexes that mature in composition and stability because of diffusion-based encounter between participant proteins. Here, we describe a generic pull-down strategy to analyse such complexes using chelating lipid-containing supported bilayers formed on silica beads. These templates can display desired His-tagged bait proteins on a diffusible-membrane surface. Using clathrin-mediated endocytosis as a paradigm, we find that the clathrin-binding adaptor protein epsin1 displayed as bait on these templates pulls down significantly higher amounts of clathrin from brain lysates than when immobilized on conventional matrices. Together, our results establish the potential of such templates as superior matrices for analysing protein-protein interactions and resultant complexes formed on membrane surfaces.

Gut milieu shapes the bacterial communities of invasive silver carp.

Silver carp is an invasive fish present in the Gobindsagar reservoir, India and has a profound impact on aquaculture. Understanding taxonomic diversity and functional attributes of gut microbiota will provide insights into the important role of bacteria in metabolism of silver carp that facilitated invasion of this exotic species. Microbial composition in foregut, midgut, hindgut and water samples was analysed using 16S rRNA gene amplicon sequencing. The bacterial communities of water samples were distinct from gut microbiota, and unique microbial assemblages were present in different regions of gut depicting profound impact of gut environment on microflora. Proteobacteria was the most abundant phyla across all samples. Ecological network analysis showed dominance of competitive interactions within posteriors region of the gut, promoting niche specialization. Predictive functional profiling revealed the microbiota specialized in digestive functions in different regions of the gut, which also reflects the dietary profile of silver carp.

The genus Sphingopyxis: Systematics, ecology, and bioremediation potential - A review.

The genus Sphingopyxis was first reported in the year 2001. Phylogenetically, Sphingopyxis is well delineated from other genera Sphingobium, Sphingomonas and Novosphingobium of sphingomonads group, family Sphingomonadaceae of Proteobacteria. To date (at the time of writing), the genus Sphingopyxis comprises of twenty validly published species available in List of Prokaryotic Names with Standing in Nomenclature. Sphingopyxis spp. have been isolated from diverse niches including, agricultural soil, marine and fresh water, caves, activated sludge, thermal spring, oil and pesticide contaminated soil, and heavy metal contaminated sites. Sphingopyxis species have drawn considerable attention not only for their ability to survive under extreme environments, but also for their potential to degrade number of xenobiotics and other environmental contaminants that impose serious threat to human health. At present, genome sequence of both cultivable and non-cultivable strains (metagenome assembled genome) are available in the public databases (NCBI) and genome wide studies confirms the presence of mobile genetic elements and plethora of degradation genes and pathways making them a potential candidate for bioremediation. Beside genome wide predictions there are number of experimental evidences confirm the degradation potential of bacteria belonging to genus Sphingopyxis and also the production of different secondary metabolites that help them interact and survive in their ecological niches. This review provides detailed information on ecology, general characteristic and the significant implications of Sphingopyxis species in environmental management along with the bio-synthetic potential.

Gut microbiome of endangered Tor putitora (Ham.) as a reservoir of antibiotic resistance genes and pathogens associated with fish health.

BACKGROUND: Tor putitora, the largest freshwater fish of the Indian subcontinent, is an endangered species. Several factors have been attributed towards its continuous population decrease, but very little is known about the gut microbiome of this fish. Also, the fish gut microbiome serves as a reservoir of virulence factors and antibiotic resistance determinants. Therefore, the shotgun metagenomic approach was employed to investigate the taxonomic composition and functional potential of microbial communities present in the gut of Tor putitora, as well as the detection of virulence and antibiotic resistance genes in the microbiome. RESULTS: The analysis of bacterial diversity showed that Proteobacteria was predominant phylum, followed by Chloroflexi, Bacteroidetes, and Actinobacteria. Within Proteobacteria, Aeromonas and Caulobacter were chiefly present; also, Klebsiella, Escherichia, and plant symbionts were noticeably detected. Functional characterization of gut microbes endowed the virulence determinants, while surveillance of antibiotic resistance genes showed the dominance of ß-lactamase variants. The antibiotic-resistant Klebsiella pneumoniae and Escherichia coli pathovars were also detected. Microbial genome reconstruction and comparative genomics confirmed the presence of Aeromonads, the predominant fish pathogens. CONCLUSIONS: Gut microbiome of endangered Tor putitora consisted of both commensals and opportunistic pathogens, implying that factors adversely affecting the non-pathogenic population would allow colonization and proliferation of pathogens causing diseased state in asymptomatic Tor putitora. The presence of virulence factors and antibiotic resistance genes suggested the potential risk of dissemination to other bacteria due to horizontal gene transfer, thereby posing a threat to fish and human health. The preservation of healthy gut microflora and limited use of antibiotics are some of the prerequisites for the conservation of this imperilled species.

Genomic insights into the phylogeny of Bacillus strains and elucidation of their secondary metabolic potential.

The genus Bacillus constitutes a plethora of species that have medical, environmental, and industrial applications. While genus Bacillus has been the focus of several studies where genomic data have been used to resolve many taxonomic issues, there still exist several ambiguities. Through the use of in-silico genome-based methods, we tried to resolve the taxonomic anomalies of a large set of Bacillus genomes (n = 178). We also proposed species names for uncharacterized strains and reported genome sequence of a novel isolate Bacillus sp. RL. In the hierarchical clustering on genome-to-genome distances, we observed 11 distinct monophyletic clusters and investigated the functional pathways annotated as the property of these clusters and core-gene content of the entire dataset. Thus, we were able to assert the possible outlier strains (n = 17) for this genus. Analyses of secondary metabolite potential of each strain helped us unravel still unexplored diversity for various biosynthetic genes.

Comparison of DNA Extraction Methods for Optimal Recovery of Metagenomic DNA from Human and Environmental Samples.

Metagenomics is the study of gene pool of an entire community in a particular niche. This provides valuable information about the functionality of host-microbe interaction in a biological ecosystem. Efficient metagenomic DNA extraction is a critical pre-requisite for a successful sequencing run in a metagenomic study. Although isolation of human stool metagenomic DNA is fairly standardized, the same protocol does not work as efficiently in fecal DNA from other organisms. In this study, we report a comparison of manual and commercial DNA extraction methods for diverse samples such as human stool, fish gut and soil. Fishes are known to have variable microbial diversity based on their food habits, so the study included two different varieties of fishes. A modified protocol for effective isolation of metagenomic DNA from human milk samples is also reported, highlighting critical precautions. Recent studies have emphasized the importance of studying functionality of human milk metagenome to understand its influence on infants' health. While manual method works well with most samples and therefore can be a method of choice for testing new samples, broad-range commercial kit offers advantage of high purity and quality. DNA extraction of different samples would go a long way in unraveling the unexplored association between microbes and host in a biological system.

Microbial taxonomy in the era of OMICS: application of DNA sequences, computational tools and techniques.

The current prokaryotic taxonomy classifies phenotypically and genotypically diverse microorganisms using a polyphasic approach. With advances in the next-generation sequencing technologies and computational tools for analysis of genomes, the traditional polyphasic method is complemented with genomic data to delineate and classify bacterial genera and species as an alternative to cumbersome and error-prone laboratory tests. This review discusses the applications of sequence-based tools and techniques for bacterial classification and provides a scheme for more robust and reproducible bacterial classification based on genomic data. The present review highlights promising tools and techniques such as ortho-Average Nucleotide Identity, Genome to Genome Distance Calculator and Multi Locus Sequence Analysis, which can be validly employed for characterizing novel microorganisms and assessing phylogenetic relationships. In addition, the review discusses the possibility of employing metagenomic data to assess the phylogenetic associations of uncultured microorganisms. Through this article, we present a review of genomic approaches that can be included in the scheme of taxonomy of bacteria and archaea based on computational and in silico advances to boost the credibility of taxonomic classification in this genomic era.

Complete Genome Analysis of Thermus parvatiensis and Comparative Genomics of Thermus spp. Provide Insights into Genetic Variability and Evolution of Natural Competence as Strategic Survival Attributes.

Thermophilic environments represent an interesting niche. Among thermophiles, the genus Thermus is among the most studied genera. In this study, we have sequenced the genome of Thermus parvatiensis strain RL, a thermophile isolated from Himalayan hot water springs (temperature >96°C) using PacBio RSII SMRT technique. The small genome (2.01 Mbp) comprises a chromosome (1.87 Mbp) and a plasmid (143 Kbp), designated in this study as pTP143. Annotation revealed a high number of repair genes, a squeezed genome but containing highly plastic plasmid with transposases, integrases, mobile elements and hypothetical proteins (44%). We performed a comparative genomic study of the group Thermus with an aim of analysing the phylogenetic relatedness as well as niche specific attributes prevalent among the group. We compared the reference genome RL with 16 Thermus genomes to assess their phylogenetic relationships based on 16S rRNA gene sequences, average nucleotide identity (ANI), conserved marker genes (31 and 400), pan genome and tetranucleotide frequency. The core genome of the analyzed genomes contained 1,177 core genes and many singleton genes were detected in individual genomes, reflecting a conserved core but adaptive pan repertoire. We demonstrated the presence of metagenomic islands (chromosome:5, plasmid:5) by recruiting raw metagenomic data (from the same niche) against the genomic replicons of T. parvatiensis. We also dissected the CRISPR loci wide all genomes and found widespread presence of this system across Thermus genomes. Additionally, we performed a comparative analysis of competence loci wide Thermus genomes and found evidence for recent horizontal acquisition of the locus and continued dispersal among members reflecting that natural competence is a beneficial survival trait among Thermus members and its acquisition depicts unending evolution in order to accomplish optimal fitness.