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bioRxiv2017

A dynamic microbial community with high functional redundancy inhabits the cold, oxic subseafloor aquifer

The rock-hosted subseafloor crustal aquifer harbors a reservoir of microbial life that may influence global marine biogeochemical cycles. Here we utilized genomic reconstruction of crustal fluid samples from North Pond, located on the flanks of the Mid-Atlantic Ridge, a site with cold, oxic subseafloor fluid circulation within the upper basement. Twenty-one samples were collected during a two-year period at three different depths and two locations with the basaltic aquifer to examine potential microbial metabolism and community dynamics. We observed minor changes in the geochemical signatures over the two years, yet a dynamic microbial community was present in the crustal fluids that underwent large shifts in the dominant taxonomic groups. An analysis of 195 metagenome-assembled genomes (MAGs) were generated from the dataset and revealed a connection between litho- and autotrophic processes, linking carbon fixation to the oxidation of sulfide, sulfur, thiosulfate, hydrogen, and ferrous iron in a diverse group of microorganisms. Despite oxic conditions, analysis of the MAGs indicated that members of the microbial community were poised to exploit hypoxic or anoxic conditions through the use of microaerobic cytochromes and alternative electron acceptors. Temporal and spatial trends from the MAGs revealed a high degree of functional redundancy that did not correlate with the shifting microbial community membership, suggesting functional stability in mediating subseafloor biogeochemical cycles.

microbiology↗

bioRxiv2017

First Draft Genome Sequence of the Pathogenic Fungus Lomentospora prolificans (formerly Scedosporium prolificans)

Here we describe the sequencing and assembly of the pathogenic fungus Lomentospora prolificans using a combination of short, highly accurate Illumina reads and additional coverage in very long Oxford Nanopore reads. The resulting assembly is highly contiguous, containing a total of 37,630,066 bp with over 98% of the sequence in just 26 scaffolds. Annotation identified 8,656 protein-coding genes. Pulsed-field gel analysis suggests that this organism contains at least 7 and possibly 11 chromosomes, the two longest of which have sizes corresponding closely to the sizes of the longest scaffolds, at 6.6 and 5.7 Mb.

microbiology↗

bioRxiv2017

Anti-infective potential of a quorum modulatory polyherbal extract (Panchvalkal) against certain pathogenic bacteria

Anti-infective potential of a polyherbal ayurvedic formulation namely panchvalkal was assayed against three pathogenic bacteria. This formulation was found to exert quorum-modulatory effect on Chromobacterium violaceum, Serratia marcescens, and Staphylococcus aureus at 250-750 g/mL. Besides altering production of the quorum sensing-regulated pigments in these bacteria, the test formulation also had in vitro effect on antibiotic susceptibility, catalase activity and hemolytic potential of the pathogens. In vivo assay confirmed the protective effect of this panchvalkal formulation on Caenorhabditis elegans, when challenged with the pathogenic bacteria. Repeated exposure of S. aureus to panchvalkal did not induce resistance in this bacterium. To the best of our awareness, this the first report on quorum-modulatory potential of panchvalkal formulation, validating the anti-infective potential and moderate prebiotic property of this polyherbal preparation.

microbiology↗

bioRxiv2017

dRNA-seq implicates sulfide as master regulator of S(0) metabolism in Chlorobaculum tepidum and other green sulfur bacteria

The green sulfur bacteria (Chlorobiaceae) are anaerobes that use electrons from reduced sulfur compounds (sulfide, S(0), and thiosulfate) as electron donors for photoautotrophic growth. Chlorobaculum tepidum, the model system for the Chlorobiaceae, both produces and consumes extracellular S(0) globules depending on the availability of sulfide in the environment. These physiological changes imply significant changes in gene regulation, which has been observed when sulfide is added to Cba. tepidum growing on thiosulfate. However, the underlying mechanisms driving these gene expression changes, i.e. specific regulators and promoter elements involved, have not yet been defined. Here, differential RNA-seq (dRNA-seq) was used to globally identify transcript start sites (TSS) that were present during growth on sulfide, biogenic S(0), and thiosulfate as sole electron donors. TSS positions were used in combination with RNA-seq data from cultures growing on these same electron donors to identify both basal promoter elements and motifs associated with electron donor dependent transcriptional regulation. These motifs were conserved across homologous Chlorobiaceae promoters. Two lines of evidence suggest that sulfide mediated repression is the dominant regulatory mode in Cba. tepidum. First, motifs associated with genes regulated by sulfide overlap key basal promoter elements. Second, deletion of the gene CT1277, encoding a putative regulatory protein, leads to constitutive over-expression of the sulfide:quinone oxidoreductase CT1087 in the absence of sulfide. The results suggest that sulfide is the master regulator of sulfur metabolism in Cba. tepidum and the Chlorobiaceae. Finally, the identification of basal promoter elements with differing strengths will further the development of synthetic biology in Cba. tepidum and perhaps other Chlorobiaceae.\n\nImportanceElemental sulfur is a key intermediate in biogeochemical sulfur cycling. The photoautotrophic green sulfur bacterium Chlorobaculum tepidum both produces and consumes elemental sulfur depending on the availability of sulfide in the environment. Our results reveal transcriptional dynamics of Chlorobaculum tepidum on elemental sulfur, and increase our understanding of the mechanisms of transcriptional regulation governing growth on different reduced sulfur compounds. This study identifies new genes and sequence motifs that likely play significant roles in the production and consumption of elemental sulfur. Beyond this focused impact, this study paves the way for the development of synthetic biology in Chlorobaculum tepidum and other Chlorobiaceae by providing a comprehensive identification of promoter elements to control gene expression, a key element of strain engineering.

microbiology↗

bioRxiv2017

Multi-clonal evolution of MDR/XDR M. tuberculosis in a high prevalence setting in Papua New Guinea over three decades

An outbreak of multi-drug resistant tuberculosis has been reported on Daru Island, Papua New Guinea. The Mycobacterium tuberculosis strains driving this outbreak and the temporal accrual of drug resistance mutations have not been described. We analyzed 100 isolates using whole genome sequencing and found 95 belonged to a single modern Beijing strain cluster. Molecular dating suggested acquisition of streptomycin and isoniazid resistance in the 1960s, with virulence potentially enhanced by a mycP1 mutation. The outbreak cluster demonstrated a high degree of co-resistance between isoniazid and ethionamide (80/95; 84.2%) attributed to an inhA promoter mutation combined with inhA and ndh coding mutations. Multidrug resistance (MDR), observed in 78/95 samples, emerged with the acquisition of a typical rpoB mutation together with a compensatory rpoC mutation in the 1980s. There was independent acquisition of fluoroquinolone and aminoglycoside resistance; with evidence of local transmission of extensively-drug resistant (XDR) strains from 2009. These findings underscore the importance of whole-genome sequencing in informing an effective public health response to MDR/XDR M. tuberculosis.

microbiology↗

bioRxiv2017

Geobacter sulfurreducens extracellular multiheme cytochrome PgcA facilitates respiration to Fe(III) oxides but not electrodes

Extracellular cytochromes are hypothesized to facilitate the final steps of electron transfer between the outer membrane of the metal-reducing bacterium Geobacter sulfurreducens and solid-phase electron acceptors such as metal oxides and electrode surfaces during the course of respiration. The triheme c-type cytochrome PgcA exists in the extracellular space of G. sulfurreducens, and is one of many multiheme c-type cytochromes known to be loosely bound to the bacterial outer surface. Deletion of pgcA using a markerless method resulted in mutants unable to transfer electrons to Fe(III) and Mn(IV) oxides; yet the same mutants maintained the ability to respire electrode surfaces and soluble Fe(III) citrate. When expressed and purified from Shewanella oneidensis, PgcA demonstrated a primarily alpha helical structure, three bound hemes, and was processed into a shorter 41 kDa form lacking the lipodomain. Purified PgcA bound Fe(III) oxides, but not magnetite, and when PgcA was added to cell suspensions of G. sulfurreducens, PgcA accelerated Fe(III) reduction similar to addition of FMN. Addition of soluble PgcA to {triangleup}pgcA mutants also restored Fe(III) reduction. This report highlights a distinction between proteins involved in extracellular electron transfer to metal oxides and poised electrodes, and suggests a specific role for PgcA in facilitating electron transfer at mineral surfaces.

microbiology↗

bioRxiv2017

Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome

Proper chromosome segregation is essential in all living organisms. In Caulobacter crescentus, the ParA-ParB-parS system is required for proper chromosome segregation and cell viability. The bacterial centromere-like parS DNA locus is the first to be segregated following chromosome replication. parS is bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. Here, we investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of in vivo chromatin immunoprecipitation (ChIP-seq) and in vitro DNA affinity purification with deep sequencing (IDAP-seq). We confirmed two previously identified parS sites and discovered at least three more sites that cluster ~8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites and associates non-specifically with ~10 kb flanking DNA to form a high-order nucleoprotein complex on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a ~500 kb region surrounding the native parS cluster that is tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of parS sites is highly restricted and is crucial for chromosome segregation in Caulobacter.

microbiology↗

bioRxiv2017

A genetic screen suggests an alternative mechanism for inhibition of SecA by azide

Sodium azide prevents bacterial growth by inhibiting the activity of SecA, which is required for translocation of proteins across the cytoplasmic membrane. Azide inhibits ATP turnover in vitro, but its mechanism of action in vivo is unclear. To investigate how azide inhibits SecA in cells, we used transposon directed insertion-site sequencing (TraDIS) to screen a library of transposon insertion mutants for mutations that affect the susceptibility of E. coli to azide. Insertions disrupting components of the Sec machinery generally increased susceptibility to azide, but insertions truncating the C-terminal tail (CTT) of SecA decreased susceptibility of E. coli to azide. Treatment of cells with azide caused increased aggregation of the CTT, suggesting that azide disrupts its structure. Analysis of the metal-ion content of the CTT indicated that SecA binds to iron and the azide disrupts the interaction of the CTT with iron. Azide also disrupted binding of SecA to membrane phospholipids, as did alanine substitutions in the metal-coordinating amino acids. Furthermore, treating purified phospholipid-bound SecA with azide in the absence of added nucleotide disrupted binding of SecA to phospholipids. Our results suggest that azide does not inhibit SecA by inhibiting the rate of ATP turnover in vivo. Rather, azide inhibits SecA by causing it to \"backtrack\" from the ADP-bound to the ATP-bound conformation, which disrupts the interaction of SecA with the cytoplasmic membrane.\n\nSignificance statementSecA is a bacterial ATPase that is required for the translocation of a subset of secreted proteins across the cytoplasmic membrane. Sodium azide is a well-known inhibitor of SecA, but its mechanism of action in vivo is poorly understood. To investigate this mechanism, we examined the effect of azide on the growth of a library of [~]1 million transposon insertion mutations. Our results suggest that azide causes SecA to backtrack in its ATPase cycle, which disrupts binding of SecA to the membrane and to its metal cofactor, which is iron. Our results provide insight into the molecular mechanism by which SecA drives protein translocation and how this essential biological process can be disrupted.

microbiology↗

bioRxiv2017

Signatures of selection at drug resistance loci in Mycobacterium tuberculosis

Tuberculosis (TB) is the leading cause of death by an infectious disease, and global TB control efforts are increasingly threatened by drug resistance in Mycobacterium tuberculosis (M. tb). Unlike most bacteria, where lateral gene transfer is an important mechanism of resistance acquisition, resistant M. tb arises solely by de novo chromosomal mutation. Using whole genome sequencing data from two natural populations of M. tb, we characterized the population genetics of known drug resistance loci using measures of diversity, population differentiation, and convergent evolution. We found resistant sub-populations to be less diverse than susceptible sub-populations, consistent with ongoing transmission of resistant M. tb. A subset of resistance genes (\"sloppy targets\") were characterized by high diversity and multiple rare variants; we posit that a large genetic target for resistance and relaxation of purifying selection contribute to high diversity at these loci. For \"tight targets\" of selection, the path to resistance appeared narrower, evidenced by single favored mutations that arose numerous times on the phylogeny and segregated at markedly different frequencies in resistant and susceptible sub-populations. These results suggest that diverse genetic architectures underlie drug resistance in M. tb, and combined approaches are needed to identify causal mutations. Extrapolating from patterns observed in well-characterized genes, we identified novel candidate variants involved in resistance. The approach outlined here can be extended to identify resistance variants for new drugs, to investigate the genetic architecture of resistance, and, when phenotypic data are available, to find candidate genetic loci underlying other positively selected traits in clonal bacteria.\n\nImportanceMycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), is a significant burden on global health. Antibiotic treatment imposes strong selective pressure on M. tb populations. Identifying the mutations that cause drug resistance in M. tb is important for guiding TB treatment and halting the spread of drug resistance. Whole genome sequencing (WGS) of M. tb isolates can be used to identify novel mutations mediating drug resistance and to predict resistance patterns faster than traditional methods of drug susceptibility testing. We have used WGS from natural populations of drug resistant M. tb to characterize effects of selection for advantageous mutations on patterns of diversity at genes involved in drug resistance. The methods developed here can be used to identify novel advantageous mutations, including new resistance loci, in M. tb and other clonal pathogens.

microbiology↗

bioRxiv2017

MERS-CoV NSP16 necessary for IFN resistance and viral pathogenesis

Coronaviruses encode a mix of highly conserved and novel genes as well as genetic elements necessary for infection and pathogenesis, raising the possibility for common targets for attenuation and therapeutic design. In this study, we focus on the highly conserved nonstructural protein (NSP) 16, a viral 2O methyl-transferase (MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of MERS NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2O MTase activity had only marginal impact on propagation and replication in Vero cells, the MERS dNSP16 mutant demonstrated significant attenuation relative to control both in primary human airway cultures and in vivo. Further examination indicated the MERS dNSP16 mutant had a type I IFN based attenuation and was partially restored in the absence of IFIT molecules. Importantly, the robust attenuation permitted use of MERS dNSP16 as a live attenuated vaccine platform protecting from challenge with a mouse adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2O MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting.\n\nSignificanceCoronavirus emergence in both human and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of SARS-CoV, MERS-CoV, PEDV and swine delta coronavirus in the 21st century. These studies describe an approach that effectively targets the highly conserved 2O methyl-transferase activity of coronaviruses for attenuation. With clear understanding of the IFN/IFIT based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-coronavirus functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2O MTase function across coronaviruses.

microbiology↗

bioRxiv2017

Epidemiology of paediatric gastrointestinal colonisation by extended spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates in north-west Cambodia

Extended-spectrum cephalosporin resistance (ESC-R) in Escherichia coli and Klebsiella pneumoniae is a healthcare threat; high gastrointestinal carriage rates are reported from South-east Asia. Colonisation prevalence data in Cambodia are lacking. We determined gastrointestinal colonisation prevalence of ESC-resistant E. coli (ESC-R-EC) and K. pneumoniae (ESC-R-KP) in Cambodian children/adolescents and associated risk factors; characterised relevant resistance genes, their genetic contexts, and the genetic relatedness of ESC-R strains using whole genome sequencing (WGS). Faeces and questionnaire data were obtained from individuals <16 years in northwestern Cambodia, 2012. WGS of cultured ESC-R-EC/KP was performed (Illumina). Maximum likelihood phylogenies were used to characterise relatedness of isolates; ESC-R-associated resistance genes and their genetic contexts were identified from de novo assemblies using BLASTn and automated/manual annotation. 82/148 (55%) of children/adolescents were ESC-R-EC/KP colonised; 12/148 (8%) were co-colonised with both species. Independent risk factors for colonisation were hospitalisation (OR: 3.12, 95%, CI [1.52-6.38]) and intestinal parasites (OR: 3.11 [1.29-7.51]); school attendance conferred decreased risk (OR: 0.44 [0.21-0.92]. ESC-R strains were diverse; the commonest ESC-R mechanisms were blaCTX-M 1 and 9 sub-family variants. Structures flanking these genes were highly variable, and for blaCTX-M-15, -55 and -27, frequently involved IS26. Chromosomal blaCTX-M integration was common in E. coli. Gastrointestinal ESC-R-EC/KP colonisation is widespread in Cambodian children/adolescents; hospital admission and intestinal parasites are independent risk factors. The genetic contexts of blaCTX-M are highly mosaic, consistent with rapid horizontal exchange. Chromosomal integration of blaCTX-M may result in stable propagation in these community-associated pathogens.

microbiology↗

bioRxiv2017

Indole-3-Acetaldehyde Dehydrogenase-dependent Auxin Synthesis Contributes to Virulence of Pseudomonas syringae Strain DC3000

The bacterial pathogen Pseudomonas syringae modulates plant hormone signaling to promote infection and disease development. P. syringae uses several strategies to manipulate auxin physiology in Arabidopsis thaliana to promote pathogenesis, including synthesis of indole-3-acetic acid (IAA), the predominant form of auxin in plants, and production of virulence factors that alter auxin responses in the host; however, the role of pathogen-derived auxin in P. syringae pathogenesis is not well understood. Here we demonstrate that P. syringae strain DC3000 produces IAA via a previously uncharacterized pathway and identify a novel indole-3-acetaldehyde dehydrogenase, AldA, that functions in IAA biosynthesis by catalyzing the NAD-dependent formation of IAA from indole-3-acetaldehyde (IAAld). Biochemical analysis and solving of the 1.9 [A] resolution x-ray crystal structure reveal key features of AldA for IAA synthesis, including the molecular basis of substrate specificity. Disruption of aldA and a close homolog, aldB, lead to reduced IAA production in culture and reduced virulence on A. thaliana. We use these mutants to explore the mechanism by which pathogen-derived auxin contributes to virulence and show that IAA produced by DC3000 suppresses salicylic acid-mediated defenses in A. thaliana. Thus, auxin is a DC3000 virulence factor that promotes pathogenicity by suppressing host defenses.\n\nAuthor SummaryPathogens have evolved multiple strategies for suppressing host defenses and modulating host physiology to promote colonization and disease development. For example, the plant pathogen Pseudomonas syringae uses several strategies to the manipulate hormone signaling of its hosts, including production of virulence factors that alter hormone responses in and synthesis of plant hormones or hormone mimics. Synthesis of indole-3-acetic acid (IAA), a common form of the plant hormone auxin, by many plant pathogens has been implicated in virulence. However, the role of pathogen-derived IAA during pathogenesis by leaf spotting pathogens such as P. syringae strain DC3000 is not well understood. Here, we demonstrate that P. syringae strain DC3000 uses a previously uncharacterized biochemical pathway to synthesize IAA, catalyzed by a novel aldehyde dehydrogenase, AldA, and carry out biochemical and structural studies of the AldA protein to investigate AldA activity and substrate specificity. We also generate an aldA mutant disrupted in IAA synthesis to show that IAA is a DC3000 virulence factor that promotes pathogenesis by suppressing host defense responses.

microbiology↗

bioRxiv2017

The fallacy of identification by neutralization in the light of cytopathology nonproducing enterovirus strains

We describe the characterization of an enterovirus isolate recovered from untreated raw sewage in Ibadan, southwest Nigeria in 2010. The isolate was neutralized by specific antisera and consequently identified as Echovirus 7 (E7). Subsequent molecular characterization showed the isolate to be a mixture of E7 and Coxsackievirus A24 (CV-A24) thereby suggesting the CV-A24 strain to be non-cytopathology producing. Further molecular analysis suggested that the CV-A24 might have recombined with a Sabin poliovirus 2 (PV2) in its non-structural region. This is the first description of a Nigerian CV-A24 strain.

microbiology↗

bioRxiv2017

Evolution of exploitative interactions during diversification in Bacillus subtilis biofilms

Microbial biofilms are tightly packed, heterogeneous structures that serve as arenas for social interactions. Studies on Gram negative models reveal that during evolution in structured environments like biofilms, isogenic populations commonly diversify into phenotypically and genetically distinct variants. These variants can settle in alternative biofilm niches and develop new types of interactions that greatly influence population productivity. Here, we explore the evolutionary diversification of pellicle biofilms of the Gram positive, spore-forming bacterium Bacillus subtilis. We discover that - similarly to other species - B. subtilis diversifies into distinct colony variants. These variants dramatically differ in biofilm formation abilities and expression of biofilm-related genes. In addition, using a quantitative approach, we reveal striking differences in surface complexity and hydrophobicity of the evolved colony types. Interestingly, one of the morphotypes completely lost the ability of independent biofilm formation and evolved to hitchhike with other morphotypes with improved biofilm forming abilities. Genome comparison suggests that major phenotypic transformations between the morphotypes can be triggered by subtle genetic differences. Our work demonstrates how positive complementarity effects and exploitative interactions intertwine during evolutionary diversification in biofilms.

microbiology↗

bioRxiv2017

Unexpected mixed-mode transmission and moderate genetic regulation of Symbiodinium communities in a brooding coral

Determining the extent to which Symbiodinium communities in corals are inherited versus environmentally-acquired is fundamental to understanding coral resilience and to predicting coral responses to stressors like warming oceans that disrupt this critical endosymbiosis. We examined the fidelity with which Symbiodinium communities in the brooding coral Seriatopora hystrix are vertically transmitted and the extent to which communities are genetically regulated, by genotyping 60 larvae and their parents (9 maternal and 45 paternal colonies) using high throughput sequencing of the ITS-2 locus. Unexpectedly, Symbiodinium communities associated with brooded larvae were distinct from those within parent colonies, including the presence of types not detected in adults. Bayesian heritability (h2) analysis revealed that 33% of variability in larval Symbiodinium communities was genetically controlled. Results highlight flexibility in the establishment of larval communities and overturn the paradigm that symbiont transmission is exclusively vertical in brooding corals. Instead, we show that Symbiodinium transmission in S. hystrix involves a mixed-mode strategy, similar to many terrestrial invertebrate symbioses. Also, variation in the abundances of common Symbiodinium types among adult communities suggests that microhabitat differences influence the structure of in hospite Symbiodinium communities. Partial genetic regulation coupled with flexibility in the environmentally-acquired component of larval Symbiodinium communities implies that corals with vertical transmission, like S. hystrix, may be more resilient to environmental change than previously thought.

microbiology↗

bioRxiv2017

N-methyl-bacillithiol, a new metabolite discovered in the Chlorobiaceae, indicates that bacillithiol and derivatives are widely phylogenetically distributed.

Low-molecular weight (LMW) thiols are metabolites that mediate redox homeostasis and the detoxification of chemical stressors in cells. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including the Chlorobiaceae. Fluorescent thiol labeling of metabolite extracts coupled with HPLC showed that Chlorobaculum tepidum contained a novel LMW thiol with a mass of 412 {+/-} 1 Da corresponding to a molecular formula of C14H24N2O10S. These data suggested the new thiol is closely related to bacillithiol (BSH), the major LMW thiol from low G+C% Gram-positive bacteria. By comparing the as-isolated bimane adduct with chemically synthesized candidate structures, the Cba. tepidum thiol structure was identified as N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, a rarely observed modification in metabolism. Orthologs of bacillithiol biosynthetic genes in the Cba. tepidum genome were required for the biosynthesis of N-Me-BSH. Furthermore, the CT1040 gene product was genetically identified as the BSH N-methyltransferase. N-Me-BSH was found in all Chlorobi examined as well as Polaribacter sp. strain MED152, a member of the Bacteroidetes. A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of the Chlorobi, Bacteroidetes, Deinococcus-Thermus, and Firmicutes, but also by Acidobacteria, Chlamydiae, Gemmatimonadetes, and Proteobacteria.\n\nSignificance StatementHere, N-Me-BSH is shown to be a redox-responsive LMW thiol cofactor in Cba. tepidum and the gene, nmbA, encoding the BSH N-methyltransferase responsible for its synthesis is identified. The co-occurrence of orthologs to BSH biosynthesis genes and bacillithiol N-methyltransferase was confirmed to correctly predict LMW thiol biosynthesis in phylogenetically distant genomes. The analysis indicates that BSH/N-Me-BSH are likely the most widely distributed class of LMW thiols in biology. This finding sheds light on the evolution of LMW thiol metabolism, which is central to redox homeostasis, regulation and stress resistance in all cellular life. It also sheds light on a rare chemical modification. N-Me-BSH is the fourth instance of cysteinyl nitrogen methylation in metabolism. Identification of the BSH N-methyltransferase reported here will enable detailed in vivo and in vitro dissection of the functional consequences of this modification. As a standalone N-methyltransferase, NmbA may be useful as a component of constructed biosynthetic pathways for novel product (bio)synthesis.

microbiology↗

bioRxiv2017

Development of Inflammatory Bowel Disease is Linked to a Longitudinal Restructuring of the Gut Metagenome in Mice

The gut microbiome is linked to inflammatory bowel disease (IBD) severity and altered in late stage disease. However, it is unclear how gut microbial communities change over the course of IBD development, especially in regards to function. To investigate microbiome mediated disease mechanisms and discover early biomarkers of IBD, we conducted a longitudinal metagenomic investigation in an established mouse model of IBD, where dampened TGF-{beta} signaling in T cells leads to peripheral immune activation, weight loss, and severe colitis. IBD development is associated with abnormal gut microbiome temporal dynamics, including dampened acquisition of functional diversity and significant differences in abundance trajectories for KEGG modules such as glycosaminoglycan degradation, cellular chemotaxis, and type III and IV secretion systems. Most differences between sick and control mice emerge when mice begin to lose weight and heightened T cell activation is detected in peripheral blood. However, lipooligosaccharide transporter abundance diverges prior to immune activation, indicating that it could be a pre-disease indicator or microbiome-mediated disease mechanism. Taxonomic structure of the gut microbiome also significantly changes in association with IBD development, and the abundance of particular taxa, including several species of Bacteroides, correlate with immune activation. These discoveries were enabled by our use of generalized linear mixed effects models to test for differences in longitudinal profiles between healthy and diseased mice while accounting for the distributions of taxon and gene counts in metagenomic data. These findings demonstrate that longitudinal metagenomics is useful for discovering potential mechanisms through which the gut microbiome becomes altered in IBD.\n\nImportanceIBD patients harbor distinct microbial communities with different functional capabilities compared to healthy people. But is this cause or effect? Answering this question requires data on changes in gut microbial communities leading up to disease onset. By performing weekly metagenomic sequencing and mixed effects modeling on an established mouse model of IBD, we identified several functional pathways encoded by the gut microbiome that covary with host immune status. These pathways are novel early biomarkers that may either enable microbes to live inside an inflamed gut or contribute to immune activation in IBD mice. Future work will validate the potential roles of these microbial pathways in host-microbe interactions and human disease. This study is novel in its longitudinal design and focus on microbial pathways, which provided new mechanistic insights into the role of gut microbes in IBD development.

microbiology↗

bioRxiv2017

A strain-specific multiplex RT-PCR for Australian rabbit haemorrhagic disease viruses uncovers a new recombinant virus variant in rabbits and hares

Rabbit haemorrhagic disease virus (RHDV, or GI.1), is a calicivirus in the genus Lagovirus that has been widely utilised in Australia as a biological control agent for the management of overabundant wild European rabbit (Oryctolagus cuniculus) populations since 1996. Recently, two exotic incursions of pathogenic lagoviruses have been reported in Australia; GI.1a-Aus, previously called RHDVa-Aus, is a GI.1a virus detected in January 2014, and the novel lagovirus GI.2 (previously known as RHDV2). Furthermore, an additional GI.1a strain, GI.1a-K5 (also known as 08Q712), was released nationwide in March 2017 as a supplementary tool for wild rabbit management. To discriminate between these lagoviruses, a highly sensitive strain-specific multiplex RT-PCR assay was developed, which allows fast, cost-effective, and sensitive detection of the four pathogenic lagoviruses currently known to be circulating in Australia. In addition, we developed a universal qRT-PCR assay to be used in conjunction with the multiplex assay that broadly detects all four viruses and facilitates quantification of viral RNA load in samples. These assays enable rapid detection, identification, and quantification of pathogenic lagoviruses in the Australian context. Using these assays, a novel recombinant lagovirus was detected in rabbit tissues samples, which contained the non-structural genes of GI.1a-Aus and the structural genes of GI.2. This variant was also recovered from the liver of a European brown hare (Lepus europaeus). The impact of this novel recombinant on Australian wild lagomorph populations and its competitiveness in relation to circulating field strains, particularly GI.2, requires further studies.

microbiology↗