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bioRxiv2018

Mobile genetic element-encoded hypertolerance to copper protects Staphylococcus aureus from killing by host phagocytes

Pathogens are exposed to toxic levels of copper during infection and copper tolerance may be a general virulence mechanism used by bacteria to resist host defences. In support of this, inactivation of copper-exporter genes has been found to reduce the virulence of bacterial pathogens in vivo. Here we investigate the role of copper-hypertolerance in methicillin resistant Staphylococcus aureus. We show that a copper-hypertolerance locus (copB-mco), carried on a mobile genetic element, is prevalent in a collection of invasive S. aureus strains and more widely among clonal complex 22, 30 and 398 strains. The copB and mco genes encode a copper efflux pump and a multicopper oxidase, respectively. Isogenic mutants lacking copB or mco had impaired growth in subinhibitory concentrations of copper. Transfer of a copB-mco encoding plasmid to a naive clinical isolate resulted in a gain of copper hypertolerance and enhanced bacterial survival inside primed macrophages. The copB and mco genes were upregulated within infected macrophages and their expression was dependent on the copper sensitive operon repressor CsoR. Isogenic copB and mco mutants were impaired in their ability to persist intracellularly in macrophages and were less resistant to phagocytic killing in human blood than the parent strain. The importance of copper-regulated genes in resistance to phagocytic killing was further elaborated using mutants expressing a copper-insensitive variant of CsoR. Our findings suggest that the gain of mobile genetic elements carrying copper-hypertolerance genes contributes to the evolution of virulent strains of S. aureus, better equipped to resist killing by host immune cells.

microbiology↗

bioRxiv2018

Anti-CRISPR phages cooperate to overcome CRISPR-Cas immunity

Some phages encode anti-CRISPR (acr) genes, which antagonize bacterial CRISPR-Cas immune systems by binding components of its machinery, but it is less clear how deployment of these acr genes impacts phage replication and epidemiology. Here we demonstrate that bacteria with CRISPR-Cas resistance are still partially immune to Acr-encoding phage. As a consequence, Acr-phages often need to cooperate in order to overcome CRISPR resistance, with a first phage taking down the host CRISPR-Cas immune system to allow a second Acr- phage to successfully replicate. This cooperation leads to epidemiological tipping points in which the initial density of Acr-phage tips the balance from phage extinction to a phage epidemic. Furthermore, both higher levels of CRISPR-Cas immunity and weaker Acr activities shift the tipping points towards higher phage densities. Collectively these data help to understand how interactions between phage-encoded immune suppressors and the CRISPR systems they target shape bacteria-phage population dynamics.\n\nHighlightsO_LIBacteria with CRISPR immunity remain partially resistant to Acr-phage\nC_LIO_LISequentially infecting Acr phages cooperate to overcome CRISPR resistance\nC_LIO_LIAcr-phage epidemiology depends on the initial phage density\nC_LIO_LICRISPR resistant bacteria can drive Acr phages extinct\nC_LI\n\neTOC blurbSome phages encode Acr proteins that block bacterial CRISPR-Cas immune systems. Although CRISPR-Cas can clear the first infection, this Acr-phage still suppresses the host immune system, which can be exploited by other Acr-phages. This is critical for Acr-phage amplification, but this \"cooperation\" only works beyond a critical Acr-phage density threshold.

microbiology↗

bioRxiv2018

Bacteriophage cooperation suppresses CRISPR-Cas3 and Cas9 immunity

>Bacteria utilize CRISPR-Cas adaptive immune systems for protection from bacteriophages (phages), and some phages produce anti-CRISPR (Acr) proteins that inhibit immune function. Despite thorough mechanistic and structural information for some Acr proteins, how they are deployed and utilized by a phage during infection is unknown. Here, we show that Acr production does not guarantee phage replication, but instead, infections fail when phage population numbers fall below a critical threshold. Failing infections can be rescued by related phages that act as Acr donors, demonstrating that infections succeed if a sufficient Acr dose is contributed to a single cell by multiple phage genomes. The production of Acr proteins by phage genomes that fail to replicate leave the cell immunosuppressed, which predisposes the cell for successful infection by other phages in the population. This \"cooperative\" phage mechanism for CRISPR-Cas inhibition demonstrates inter-virus cooperation that may also manifest in other host-parasite interactions.

microbiology↗

bioRxiv2018

Identification of cyclic hexapeptides natural products with inhibitory potency against Mycobacterium tuberculosis

A set of [~]500 purified natural product compounds was screened for inhibition against the human pathogen Mycobacterium tuberculosis. A series of cyclic hexapeptides with anti-tubercular activity was identified. Five analogs from a set of sixteen closely related compounds were active, with minimum inhibitory concentrations ranging from 2.3-8.9 M. Eleven structural analogs had no significant activity (MIC > 20 M) demonstrating structure activity relationship. Sequencing of resistant mutant isolates failed to identify changes accounting for the resistance phenotype.

microbiology↗

bioRxiv2018

Evaluation of Caulobacter crescentus strain fitness at the genomic scale during growth in natural freshwater

Bacterial genomes evolve in complex ecosystems and are best understood in this natural context, but replicating such conditions in the lab is challenging. We used transposon sequencing to define the fitness consequences of gene disruption in the bacterium Caulobacter crescentus grown in natural freshwater, compared to axenic growth in common laboratory media. Gene disruptions in amino acid and nucleotide biosynthesis pathways and in metabolic substrate transport machinery impaired fitness in both lake water and defined minimal medium relative to complex peptone broth. Fitness in lake water was enhanced by insertions in genes required for flagellum biosynthesis and reduced by insertions in genes involved in biosynthesis of the holdfast surface adhesin. We further uncovered numerous hypothetical and uncharacterized genes for which disruption impaired fitness in lake water, defined minimal medium, or both. At the genome scale, the fitness profile of mutants cultivated in lake water was more similar to that in complex peptone broth than in defined minimal medium. Microfiltration of lake water did not significantly affect the terminal cell density or the fitness profile of the transposon mutant pool, suggesting that Caulobacter does not strongly interact with other microbes in this ecosystem on the measured timescale. Fitness of select mutants with defects in cell surface biosynthesis and environmental sensing were significantly more variable in lake water than in defined medium, presumably owing to day-to-day heterogeneity in the lake environment. This study reveals genetic interactions between Caulobacter and a natural freshwater environment, and provides a new avenue to study gene function in complex ecosystems.

microbiology↗

bioRxiv2018

Assessment of the petroleum hydrocarbon biodegradation potential of the sediment microbial community from an urban fringing tidal marsh of Northern New England.

We assessed the impact of dodecane, n- hexane and gasoline on the microbial diversity of chronically polluted fringing tidal marsh sediment from the Great Bay Estuary of New Hampshire. Dilution cultures containing saturated alkane concentrations were sampled at zero, one and 10 days, and alkB and cyp153A1 alkane hydroxylase gene libraries and 16S rRNA sequences were analyzed. The initial sediment had the most diverse alkane hydroxylase sequences and phylogenetic composition whereas treated sediments became less functionally and phylogenetically diverse with alkane substrates apparently enriching a few dominant taxa. All 1-and 10-day samples were dominated by Pseudomonas- type alkane hydroxylase sequences except in dodecane treatments where primarily Rhodococcus--type alkane hydroxylases were detected. 16S rRNA profiling revealed that the Gammaproteobacteria, particularly Pseudomonas, dominated all one day samples, especially the n- hexane and gasoline treatments (63.2 and 47.2% respectively) and the 10-day n- hexane treatment (which contained 60.8% Pseudomonas and 18.6%Marinobacter). In contrast, the 10 days of dodecane treatment enriched for Actinobacteria (26.2% Rhodococcus and 32.4%Mycobacterium) and gasoline treatment enriched for Firmicutes (29.7%; mainly Bacillus, Lysinibacillus and Rumelibacillus). Our data indicate that fringing tidal marshes contain microbial communities with alkane-degrading abilities similar to larger meadow marshes, and support the hypothesis that alkane exposure reduces the functional and phylogenetic diversity of microbial communities in an alkane-specific manner. Further research to evaluate the ability of such fringing marsh communities to rebound to pre-pollutant diversity levels should be conducted to better assess the threat of petroleum to these habitats.

microbiology↗

bioRxiv2018

Prolonged lag time results in small colony variants and reflects a sub-population of persisters in vivo

Treatment failure and recurrent infections can occur even when patients are treated with antibiotics to which bacteria are susceptible. These treatment failures could be due to a sub-population of bacteria persisting through the treatment. In this study, we tested the hypothesis that such bacterial persisters manifest in clinical samples as small colony variants (SCVs). We worked with the bacterial pathogen Staphylococcus aureus, where SCVs are frequently observed in clinics. The small size of SCV-colonies is often considered to be the result of mutations that reduce the bacterial growth rate. Alternatively, it is possible that the small colony size could result from a long lag time of bacteria in plated samples. We used automated plate imaging and single-cell microscopy to precisely quantify growth kinetics of bacteria sampled from patient and mice abscesses, and from low-pH in vitro cultures mimicking the host. Under these conditions, the small colony size was the consequence of a long lag time and was associated with tolerance towards antibiotics. We found that bacteria with a long lag time emerged de novo during the growth phase in the murine abscess as well as in the low-pH in vitro cultures, and that their proportion increased during stationary phase. Antibiotic exposure further increased the proportion of bacteria with a long lag time. Thus, the persisters found in low-pH host compartments could be a cause of treatment failure, and antibiotic treatment potentially aggravates the problem. These insights call for antibiotic treatment strategies that explicitly address the problem of persister formation.\n\nSignificance StatementBacterial pathogens often contain subpopulations that are multidrug tolerant and metabolically inactive. These subpopulations are known as persisters and are hypothesised to be linked to treatment failure and recurrent infections even in bacteria that are not genetically resistant to antibiotics. Identifying persisters in clinical samples remains a challenge due to their metabolicaly active state. Here, we used time-lapse plate imaging and single cell microscopy to link the presence of persisters to the nonstable small colony variant (SCV) phenotype that is observed in some Staphylococcus aureus infections. These findings may help to identify treatment regimens that prevent or address the emergence of persistence and thus to reduce relapses.

microbiology↗

bioRxiv2018

Mercury-methylating bacteria are associated with zooplankton: a proof-of-principle survey in the Baltic Sea

Methylmercury (MeHg) is a potent neurotoxin that biomagnifies in marine food-webs. Inorganic mercury (Hg) methylation is generally considered to be conducted by bacteria associated with sediment or detritus, but endogenous methylation by the gut microbiome of animals in the lower food webs is another possible source. We examined the occurrence of the bacterial gene (hgcA), required for Hg methylation, in the guts of dominant Baltic zooplankters. A qPCR assay targeting the hgcA sequence in three main clades (Deltaproteobacteria, Firmicutes and Archaea) was used in the field-collected specimens of copepods (Acartia bifilosa, Eurytemora affinis, Pseudocalanus acuspes and Limnocalanus macrurus) and cladocerans (Bosmina coregoni maritima and Cercopagis pengoi). All copepods were found to carry hgcA genes in their gut microbiome, whereas no positive amplification was recorded in the cladocerans. In the copepods, hgcA genes belonging to only Deltaproteobacteria and Firmicutes were detected. These findings suggest that endogenous Hg methylation can occur in zooplankton and may contribute to seasonal, spatial and vertical MeHg variability in water column and food webs. Additional molecular and metagenomics studies are needed to identify bacteria carrying hgcA genes and improve their quantification in microbiota.

microbiology↗

bioRxiv2018

Prevalence of Antibiotic Resistance in Commensal Escherichia Coli among the Children in Rural Hill Communities of North East India

Commensal bacteria are the representative of the reservoir of antibiotic resistance genes present in a community. Merely a few community-based studies on the prevalence of antibiotic resistance in commensal bacteria have been conducted so far in Southeast Asia and other parts of India. Northeastern India is still untapped regarding the surveillance of antibiotic-resistant genes and prevalence in commensal bacteria. In the present work, the prevalence of antibiotic resistance in commensal Escherichia coli was investigated along with the associated demographic factors in pre-school and school going children in rural areas of Sikkim. A total of 550 fecal E. coli isolates were obtained from children of the age 1-14 years living in different villages at various altitudes of Sikkim from July 2015 to June 2017. Standard antibiotic susceptibility testing of these isolates was performed. A structured questionnaire was designed to study the factors associated with carriage of antibiotic resistance in commensal E. coli isolates among children. Descriptive statistics analysis and a logistic regression model were used to identify the effect of external factors on antibiotic resistance pattern. High prevalence of resistance was found against commonly used antibiotics ampicillin (92%), ceftazidime (90%), cefoxitin (88%), streptomycin (40%) and tetracycline (36%) among the samples examined in our present study. No resistance to chloramphenicol was recorded. Fifty-two percent of the isolates were resistant to the combination of penicillin and quinolone group of antibiotics. Children living in nuclear families showed higher incidence of resistance to ampicillin (63.15%, OR 0.18,95% CI:0.11 - 0.28, p<0.01) while children of mothers having education up to school level displayed higher incidence of ceftazidime (59.27% OR 0.75, 95% CI:0.55 - 1.02, p<0.02). Our study demonstrates a high prevalence of antibiotic-resistant commensal E. coli against the commonly used antibiotics among children in the study area. A close association between different demographic factors and the pattern of carriage of antibiotic-resistant isolates was observed suggesting a concern over misuse of antibiotics and warrants a future threat of emerging multidrug resistant isolates.

microbiology↗

bioRxiv2018

Strain-level diversity drives alternative community types in millimeter scale granular biofilms

Microbial communities are often highly diverse in their composition, both at the level of coarse-grained taxa such as genera as well as at the level of strains within species. This variability can be driven by both extrinsic factors like temperature, pH, etc., as well as by intrinsic ones, such as demographic fluctuations or ecological interactions. The relative contributions of these factors and the taxonomic level at which they influence community structure remain poorly understood, in part because of the difficulty of identifying true community replicates assembled under the same environmental parameters. Here, we address this problem using an activated granular sludge reactor in which millimeter scale biofilm granules represent true community replicates whose differences in composition are expected to be driven primarily by biotic factors. Using 142 shotgun metagenomes of single biofilm granules we found that, at the commonly used genuslevel resolution, community replicates varied much more in their composition than would be expected from neutral assembly processes. This variation, however, did not translate into any clear partitioning into discrete community types, i.e. the equivalent of enterotypes in the human gut. However, a strong partition into community types did emerge at the strain level for the most abundant organism: strains of Candidatus Accumulibacter that coexisted in the metacommunity--i.e. the reactor--excluded each other within community replicates. Single-granule communities maintained a significant lineage structure, whereby the strain phylogeny of Accumulibacter correlated with the overall species composition of the community, indicating high potential for co-diversification among species and communities. Our results suggest that due to the high functional redundancy and competition between close relatives, alternative community types are most likely observed at the level of recently differentiated genotypes but not higher orders of genetic resolution.

microbiology↗

bioRxiv2018

Mosaic patterns of B-vitamin synthesis and utilization in a natural marine microbial community

Aquatic environments contain diverse microbial communities whose complex interactions mediate the cycling of major and trace nutrients such as vitamins. B-vitamins are essential coenzymes that many organisms cannot synthesize. Thus their exchange among de-novo synthesizers and auxotrophs is expected to play an important role in the microbial consortia and explain some of the temporal and spatial changes observed in diversity. In this study, we analyzed metatranscriptomes of a natural coastal microbial community, diel sampled-quarterly over one year to try to identify the potential major B-vitamin synthesizers and consumers. Our transcriptomic data show that the best-represented taxa dominated the expression of synthesis genes for some B-vitamins but lacked transcripts for others. For instance, Rhodobacterales dominated the expression of vitamin-B12 synthesis, but not of vitamin-B7, whose synthesis transcripts were mainly represented by Flavobacteria.In contrast, bacterial groups that constituted less than 4% of the community (e.g., Verrucomicrobia) accounted for most of the vitamin-B1 synthesis transcripts. Furthermore, ambient vitamin-B1 concentrations were higher in samples collected during the day, and were positively correlated with chlorophyll-a concentration. Our analysis supports the hypothesis that the mosaic of metabolic interdependencies through B-vitamin synthesis and exchange are key processes that contribute to shaping microbial communities in nature.

microbiology↗

bioRxiv2018

From proteins to polysaccharides: lifestyle and genetic evolution of Coprothermobacter proteolyticus.

Microbial communities that degrade lignocellulosic biomass are typified by high levels of species- and strain-level complexity as well as synergistic interactions between both cellulolytic and non-cellulolytic microorganisms. Coprothermobacter proteolyticus frequently dominates thermophilic, lignocellulose-degrading communities with wide geographical distribution, which is in contrast to reports that it ferments proteinaceous substrates and is incapable of polysaccharide hydrolysis. Here we deconvolute a highly efficient cellulose-degrading consortium (SEM1b) that is co-dominated by Clostridium (Ruminiclostridium) thermocellum- and multiple heterogenic strains affiliated to C. proteolyticus. Metagenomic analysis of SEM1b recovered metagenome-assembled genomes (MAGs) for each constituent population, whilst in parallel two novel strains of C. proteolyticus were successfully isolated and sequenced. Annotation of all C. proteolyticus genotypes (two strains and one MAG) revealed their genetic acquisition of various carbohydrate-active enzymes (CAZymes), presumably derived from horizontal gene transfer (HGT) events involving C. thermocellum- or Thermotogae-affiliated populations that are historically co-located. HGT material included whole saccharolytic operons and dockerin-encoding enzymatic subunits that are synonymous with cellulosomes. Finally, temporal genome-resolved metatranscriptomic analysis of SEM1b revealed expression of C. proteolyticus CAZymes at different SEM1b life-stages as well as co-expression of CAZymes from multiple SEM1b populations, inferring deeper microbial interactions that are dedicated towards co-degradation of cellulose and hemicellulose. We show that C. proteolyticus, a ubiquitous keystone population, consists of closely related strains that have adapted via HGT to degrade both oligo- and longer polysaccharides present in decaying plants and microbial cell walls, thus explaining its dominance in thermophilic anaerobic digesters on a global scale.

microbiology↗

bioRxiv2018

Haemoproteus paraortalidum n. sp. in captive Black-fronted Piping-guans Aburria jacutinga (Galliformes, Cracidae): high prevalence in a population reintroduced into the wild

Haemosporidian parasites of the genus Haemoproteus are widespread and can cause disease and even mortality in birds under natural and captive conditions. The Black-fronted Piping-guan (Aburria jacutinga) is an endangered Neotropical bird of the Cracidae (Galliformes) going through a reintroduction program to avoid extinction. We used microscopic examination and partial cytochrome b DNA sequencing to describe a new Haemoproteus species infecting Black-fronted Piping-guans bred and raised in captivity that were reintroduced into the Atlantic rainforest. Haemoproteus (Parahaemoproteus) paraortalidum n. sp. was detected in the blood of 19 out of 29 examined birds. The new species is distinguished from other haemoproteids due to the shape of gametocytes, which have pointed ends in young stages, and due to the presence of vacuole-like unstained spaces in macrogametocytes and numerous volutin granules both in macro- and microgametocytes. Illustrations of the new species are provided. Phylogenetic inference positioned this parasite in the Parahaemoproteus subgenus clade together with the other two Haemoproteus genetic lineages detected in cracids up to date. We discuss possible implications of the reintroduction of birds infected with haemosporidian parasites into de wild. Treatment of Haemoproteus infections remains insufficiently studied, but should be considered for infected birds before reintroduction to improve host reproductive and survival rates after release.

microbiology↗

bioRxiv2018

Effects of Temperatures and High Pressures on the Growth and Survivability of Methanogens and Stable Carbon Isotope Fractionation: Implications for Deep Subsurface Life on Mars

In order to examine the potential survivability of life in the Martian deep subsurface, we have investigated the effects of temperature (45{degrees}C, 55{degrees}C, and 65{degrees}C) and pressure (1 atm, 400 atm, 800 atm, and 1200 atm) on the growth, carbon isotopic data, and morphology of chemolithoautotrophic anaerobic methanogenic archaea, Methanothermobacter wolfeii. The growth and survivability of this methanogen were determined by measuring the methane concentration in headspace gas samples after the cells were returned to their conventional growth conditions. Interestingly, this methanogen survived at all the temperatures and pressures tested. M. wolfeii demonstrated the highest methane concentration following exposure to pressure of 800 atm and a temperature of 65{degrees}C. We found that the stable carbon isotopic fractionation of methane, {delta}13C(CH4), was slightly more enriched in 12C at 1 atm and 55{degrees}C than the carbon isotopic data obtained in other temperature and pressure conditions. A comparison of the images of the cells before and after the exposure to different temperatures and pressures did not show any obvious alteration in the morphology of M. wolfeii. The research reported here suggests that at least one methanogen, M. wolfeii, may be able to survive under hypothetical Martian subsurface conditions with respect to temperature and pressure.

microbiology↗

bioRxiv2018

Avian influenza viruses in wild birds: virus evolution in a multi-host ecosystem

Wild ducks and gulls are the major reservoirs for avian influenza A viruses (AIVs). The mechanisms that drive AIV evolution are complex at sites where various duck and gull species from multiple flyways breed, winter or stage. The Republic of Georgia is located at the intersection of three migratory flyways: Central Asian Flyway, East Asian/East African Flyway and Black Sea/Mediterranean Flyway. For six consecutive years (2010-2016), we collected AIV samples from various duck and gull species that breed, migrate and overwinter in Georgia. We found substantial subtype diversity of viruses that varied in prevalence from year to year. Low pathogenic (LP)AIV subtypes included H1N1, H2N3, H2N5, H2N7, H3N8, H4N2, H6N2, H7N3, H7N7, H9N1, H9N3, H10N4, H10N7, H11N1, H13N2, H13N6, H13N8, H16N3, plus two H5N5 and H5N8 highly pathogenic (HP)AIVs belonging to clade 2.3.4.4. Whole genome phylogenetic trees showed significant host species lineage restriction for nearly all gene segments and significant differences for LPAIVs among different host species in observed reassortment rates, as defined by quantification of phylogenetic incongruence, and in nucleotide diversity. Hemagglutinin clade 2.3.4.4 H5N8 viruses, circulated in Eurasia during 2014-2015 did not reassort, but analysis after its subsequent dissemination during 2016-2017 revealed reassortment in all gene segments except NP and NS. Some virus lineages appeared to be unrelated to AIVs in wild bird populations in other regions with maintenance of local AIV viruses in Georgia, whereas other lineages showed considerable genetic inter-relationship with viruses circulating in other parts of Eurasia and Africa, despite relative under-sampling in the area.\n\nImportanceWaterbirds (e.g., gulls/ducks) are natural reservoirs of avian influenza viruses (AIVs) and have been shown to mediate dispersal of AIV at inter-continental scales during seasonal migration. The segmented genome of influenza viruses enables viral RNA from different lineages to mix or re-assort when two viruses infect the same host. Such reassortant viruses have been identified in most major human influenza pandemics and several poultry outbreaks. Despite their importance, we have only recently begun to understand AIV evolution and reassortment in their natural host reservoirs. This comprehensive study illustrates of AIV evolutionary dynamics within a multi-host ecosystem at a stop-over site where three major migratory flyways intersect. Our analysis of this ecosystem over a six-year period provides a snapshot of how these viruses are linked to global AIV populations. Understanding the evolution of AIVs in the natural host is imperative to both mitigating the risk of incursion into domestic poultry and potential risk to mammalian hosts including humans.

microbiology↗

bioRxiv2018

A role for Fibroblast Growth Factor Receptor 1 in the pathogenesis of Neisseria meningitidis

Neisseria meningitidis remains an important cause of human disease. It is highly adapted to the human host - its only known reservoir. Adaptations to the host environment include many specific interactions with human molecules including iron-binding proteins, components of the innate and adaptive immune systems, and cell surface receptors such as the Epidermal Growth Factor Receptor (EGFR). Interaction of the meningococcus with EGFR has been elucidated in some detail and leads to intracellular signalling and cytoskeletal changes contributing to the pathogenesis of the organism. Here, we show that the meningococcus also recruits Fibroblast Growth Factor Receptor 1 (FGFR1) onto the surface of human blood microvascular epithelial cells (HBMECs). Furthermore, meningococci internalised into these cells recruit the activated form of this receptor, and that expression and activation of FGFR1 is necessary for efficient internalisation of meningococci into HBMECs. We show that Neisseria meningitidis interacts specifically with the IIIc isoform of FGFR1.

microbiology↗

bioRxiv2018

In vivo cloning of β 1-4 endoglucanase gene of Serratia liquefaciens using Muduction and its in silico analysis

Cellulose is the major structural component in the plant cell wall. The bio-degradation of cellulose molecules is facilitated by cellulase. In the present study, in vivo cloning of cellulase ({beta}-1, 4 endoglucanase) gene from a cellulolytic bacterium Serratia liquefaciens into E. coli DH5 has been performed using mini-Mu phage transduction. The enzyme activity of cloned endoglucanase was 81.2U/mg at optimum temperature (40{degrees}C) and 80.2U/mg at optimum pH7, while the wildtype has 65.9U/mg and 64.9U/mg respectively. The conserved domain analysis shows that S. liquefaciens endoglucanase belongs to GH8 family. The nucleotide sequence analysis of wildtype and cloned endoglucanase shows that mutations were found at residues 51(Lys - Asn), 203(Trp-Cys), 246(Thr-Iso), 260(Gly-Ala) and 288(Phe-Leu). The structural analysis shows the active site of wildtype endoglucanase is a narrow groove which lies parallel to the central axis, whereas cloned endoglucanase is broad and tilted to [~]70{degrees} from the central axis. The increased enzyme activity in the cloned endoglucanase is due to the structural modification conferred by changes in amino acid resulting in widening of the cleft in the active site.

microbiology↗

bioRxiv2018

Ecological patterns are robust to use of exact sequence variants versus operational taxonomic units

Recent controversy focuses on the best method for delineating microbial taxa, based on either traditional operational taxonomic units (OTUs) or exact sequence variants (ESVs) of marker gene sequences. We sought to test if the binning approach (ESVs versus OTUs) affected the ecological conclusions of a large field study. The dataset included sequences of both bacteria (16S) and fungi (ITS), across multiple environments diverging markedly in abiotic conditions, over three collection times. Despite quantitative differences in microbial richness, we found that all alpha- and beta-diversity metrics were highly positively correlated (r > 0.90) between samples analyzed with both approaches. Moreover, the community composition of the dominant taxa did not vary between approaches. Consequently, statistical inferences were nearly indistinguishable. Thus, we conclude that for typical alpha- and beta-diversity analyses, OTU or ESV methods will likely reveal similar ecological results and determining which method to employ will depend on the question at hand.

microbiology↗