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bioRxiv2018

Identification of novel mutations associated with clofazimine resistance in Mycobacterium abscessus

Mycobacterium abscessus (Mab) is a major non-tuberculous mycobacterial (NTM) pathogen responsible for about 80% of all pulmonary infections caused by rapidly growing mycobacteria. Clofazimine is an effective drug active against Mab and shows synergistic activity when given with amikacin, but the mechanism of resistance to clofazimine in Mab is unknown.\n\nObjectiveTo investigate the molecular basis of clofazimine resistance in Mab.\n\nMethodsWe isolated 29 Mab mutants resistant to clofazimine, and subjected them to whole genome sequencing and Sanger sequencing to identify possible mutations associated with clofazimine resistance.\n\nResultsMutations in MAB_2299c gene which encodes possible transcriptional regulatory protein were identified in 23 of the 29 clofazimine-resistant mutants. In addition, 6 mutations in MAB_1483 were found in 21 of the 29 mutants, and one mutation in MAB_0540 was found in 16 of the 29 mutants. Mutations in MAB_0416c, MAB_4099c, MAB_2613, MAB_0409, MAB_1426 were also associated with clofazimine resistance in less frequency. Two identical mutations which are likely to be polymorphisms unrelated to clofazimine resistance were found in MAB_4605c and MAB_4323 in 13 mutants.\n\nConclusionMutations in MAB_2299c, MAB_1483, and MAB_0540 are the major mechanisms of clofazimine resistance in Mab. Future studies are needed to address the role of the identified mutations in clofazimine resistance in Mab, and our findings have implications for developing a rapid molecular test for detecting clofazimine resistance in this organism.

microbiology↗

bioRxiv2018

Working together: cross-priming in two Legionella pneumophila type I-F CRISPR-Cas systems.

In bacteria and archaea, several distinct types of CRISPR-Cas systems provide adaptive immunity through broadly similar mechanisms: short nucleic acid sequences derived from foreign DNA, known as spacers, engage in complementary base pairing with invasive genetic elements setting the stage for nucleases to degrade the target DNA. A hallmark of type I CRISPR-Cas systems is their ability to acquire spacers in response to both new and previously encountered invaders (naive and primed acquisition, respectively). Our phylogenetic analyses of 47 L. pneumophila type I-F CRISPR-Cas systems and their resident genomes suggest that many of these systems have been horizontally acquired. These systems are frequently encoded on plasmids and can co-occur with nearly identical chromosomal loci. We show that two such co-occurring systems are highly protective and undergo efficient primed acquisition in the lab. Furthermore, we observe that targeting by one systems array can prime spacer acquisition in the other. Lastly, we provide experimental and genomic evidence for a model in which primed acquisition can efficiently replenish a depleted type I CRISPR array following a mass spacer deletion event.

microbiology↗

bioRxiv2018

Ecological selection for small microbial genomes along a temperate-to-thermal soil gradient

Small bacterial and archaeal genomes provide insights into the minimal requirements for life1 and seem to be widespread on the microbial phylogenetic tree2. We know that evolutionary processes, mainly selection and drift, can result in microbial genome reduction 3,4. However, we do not know the precise environmental pressures that constrain genome size in free-living microorganisms. A study including isolates 5 has shown that bacteria with high optimum growth temperatures, including thermophiles, often have small genomes 6. It is unclear how well this relationship may extend generally to microorganisms in nature 7,8, and in particular to those microbes inhabiting complex and highly variable environments like soil 3,6,9. To understand the genomic traits of thermally-adapted microorganisms, here we investigated bacterial and archaeal metagenomes from a 45{degrees}C gradient of temperate-to-thermal soils overlying the ongoing Centralia, Pennsylvania (USA) coal seam fire. There was a strong relationship between average genome size and temperature: hot soils had small genomes relative to ambient soils (Pearsons r = -0.910, p < 0.001). There was also an inverse relationship between soil temperature and cell size (Pearsons r = -0.65, p = 0.021), providing evidence that cell and genome size in the wild are together constrained by temperature. Notably, hot soils had different community structures than ambient soils, implicating ecological selection for thermo-tolerant cells that had small genomes, rather than contemporary genome streamlining within the local populations. Hot soils notably lacked genes for described two-component regulatory systems and antimicrobial production and resistance. Our work provides field evidence for the inverse relationship between microbial genome size and temperature requirements in a diverse, free-living community over a wide range of temperatures that support microbial life. Our findings demonstrate that ecological selection for thermophiles and thermo-tolerant microorganisms can result in smaller average genome sizes in situ, possibly because they have small genomes reminiscent of a more ancestral state.

microbiology↗

bioRxiv2018

Escherichia coli Lrp regulates one-third of the genome via direct, cooperative, and indirect routes

The global regulator Lrp plays a crucial role in regulating metabolism, virulence and motility in response to environmental conditions. Lrp has previously been shown to activate or repress approximately 10% of genes in Escherichia coli. However, the full spectrum of targets, and how Lrp acts to regulate them, has stymied earlier study. We have combined matched ChIP-seq and RNA sequencing under nine physiological conditions to map the binding and regulatory activity of Lrp as it directs responses to nutrient abundance. In addition to identifying hundreds of novel Lrp targets, we observe two new global trends: first, that Lrp will often bind to promoters in a poised position under conditions when it has no regulatory activity, and second, that nutrient levels induce a global shift in the equilibrium between non-specific and sequence-specific DNA binding. The overall regulatory behavior of Lrp, which as we now show regulates 35% of E. coli genes directly or indirectly under at least one condition, thus arises from the interaction between changes in Lrp binding specificity and cooperative action with other regulators.

microbiology↗

bioRxiv2018

Gut microbiota density influences host physiology and is shaped by host and microbial factors

To identify factors that regulate gut microbiota density and the impact of varied microbiota density on health, we assayed this fundamental ecosystem property in fecal samples across mammals, human disease, and therapeutic interventions. Physiologic features of the host (carrying capacity) and the fitness of the gut microbiota shape microbiota density. Therapeutic manipulation of microbiota density in mice altered host metabolic and immune homeostasis. In humans, gut microbiota density was reduced in Crohns disease, ulcerative colitis, and ileal pouch-anal anastomosis. The gut microbiota in recurrent Clostridium difficile infection had lower density and reduced fitness that were restored by fecal microbiota transplantation. Understanding the interplay between microbiota and disease in terms of microbiota density, host carrying capacity, and microbiota fitness provide new insights into microbiome structure and microbiome targeted therapeutics.

microbiology↗

bioRxiv2018

A streamlined method for transposon mutagenesis of Rickettsia parkeri yields numerous mutations that impact infection

The rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria, Rickettsia are highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improved mariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group species Rickettsia parkeri. These mutants targeted genes implicated in a variety of pathways, including bacterial replication and metabolism, hypothetical proteins, the type IV secretion system, as well as factors with previously established roles in host cell interactions and pathogenesis. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis.

microbiology↗

bioRxiv2018

The Staphylococcus aureus Two-Component System AgrAC Displays Four Distinct Genomic Arrangements That Delineate Genomic Virulence Factor Signatures

Two-component systems (TCSs) consist of a histidine kinase and a response regulator. Here, we evaluated the conservation of the AgrAC TCS among 149 completely sequenced S. aureus strains. It is composed of four genes: agrBDCA. We found that: i) AgrAC system (agr) was found in all but one of the 149 strains; ii) The agr positive strains were further classified into four agr types based on AgrD protein sequences, iii) the four agr types not only specified the chromosomal arrangement of the agr genes but also the sequence divergence of AgrC histidine kinase protein, which confers signal specificity, iv) the sequence divergence was reflected in distinct structural properties especially in the transmembrane region and second extracellular binding domain, and v) there was a strong correlation between the agr type and the virulence genomic profile of the organism. Taken together, these results demonstrate that bioinformatic analysis of the agr locus leads to a classification system that correlates with the presence of virulence factors and protein structural properties.

microbiology↗

bioRxiv2018

The human leukemia virus HTLV-1 alters the structure and transcription of host chromatin in cis

Chromatin looping controls gene expression by regulating promoter-enhancer contacts, the spread of epigenetic modifications, and the segregation of the genome into transcriptionally active and inactive compartments. We studied the impact on the structure and expression of host chromatin by the human retrovirus HTLV-1. We show that HTLV-1 disrupts host chromatin structure by forming loops between the provirus and the host genome; certain loops depend on the critical chromatin architectural protein CTCF, which we recently showed binds to the HTLV-1 provirus. Finally, we show that the provirus causes two distinct patterns of abnormal transcription of the host genome in cis: bidirectional transcription in the host genome immediately flanking the provirus, and clone-specific transcription in cis at non-contiguous loci up to >300 kb from the integration site. We conclude that HTLV-1 causes insertional mutagenesis up to the megabase range in the host genome in >104 persistently-maintained HTLV-1+ T-cell clones in vivo.

microbiology↗

bioRxiv2018

Comparative Genomics of Staphylococcus Reveals Determinants of Speciation and Diversification of Antimicrobial Defense.

The bacterial genus Staphylococcus comprises diverse species with most being described as colonizers of human and animal skin. A relational analysis of features that discriminate its species and contribute to niche adaptation and survival remains to be fully described. In this study, an interspecies, whole-genome comparative analysis of 21 Staphylococcus species was performed based on their orthologues. Three well-defined multi-species groups were identified: group A (including aureus/epidermidis); group B (including saprophyticus/xylosus) and group C (including pseudintermedius/delphini). The machine learning algorithm Random Forest was applied to identify variable orthologues that drive formation of the Staphylococcus species groups A-C. Orthologues driving staphylococcal infrageneric diversity comprised regulatory, metabolic and antimicrobial resistance proteins. Notably, the BraSR (NsaRS) two-component system (TCS) and its associated BraDE transporters that regulate antimicrobial resistance distinguish group A Staphylococcus species from others in the genus that lack the BraSR TCS. Divergence of BraSR and GraSR antimicrobial peptide survival TCS and their associated transporters was observed across the staphylococci, likely reflecting niche specific evolution of these TCS/transporters and their specificities for AMPs. Experimental evolution, with selection for resistance to the lantibiotic nisin, revealed multiple routes to resistance and differences in the selection outcomes of the BraSR- positive species S. hominis and S. aureus. Selection supported a role for GraSR in nisin survival responses of the BraSR-negative group B species S. saprophyticus. Our study reveals diversification of antimicrobial-sensing TCS across the staphylococci and hints at differential relationships between GraSR and BraSR in those species positive for both TCS.\n\nImportanceThe genus Staphylococcus includes species that are commensals and opportunist pathogens of humans and animals. Identifying the features that discriminate species of staphylococci is relevant to understanding niche selection and the structure of their microbiomes. Moreover, the determinants that structure the community are relevant for strategies to modify the frequency of individual species associated with dysbiosis and disease. In this study, we identify orthologous proteins that discriminate genomes of staphylococci. In particular, species restriction of a major antimicrobial survival system, BraSR (NsaRS), to a group of staphylococci dominated by those that can colonize human skin. The diversity of antimicrobial sensing loci was revealed by comparative analysis and experimental evolution with selection for nisin resistance identified the potential for variation in antimicrobial sensing in BraRS-encoding staphylococci. This study provides insights into staphylococcal species diversity.

microbiology↗

bioRxiv2018

MetaWRAP - a flexible pipeline for genome-resolved metagenomic data analysis

BackgroundThe study of microbiomes using whole-metagenome shotgun sequencing enables the analysis of uncultivated microbial populations that may have important roles in their environments. Extracting individual draft genomes (bins) facilitates metagenomic analysis at the single genome level. Software and pipelines for such analysis have become diverse and sophisticated, resulting in a significant burden for biologists to access and use them. Furthermore, while bin extraction algorithms are rapidly improving, there is still a lack of tools for their evaluation and visualization.\n\nResultsTo address these challenges, we present metaWRAP, a modular pipeline software for shotgun metagenomic data analysis. MetaWRAP deploys state-of-the-art software to handle metagenomic data processing starting from raw sequencing reads and ending in metagenomic bins and their analysis. MetaWRAP is flexible enough to give investigators control over the analysis, while still being easy-to-install and easy-to-use. It includes hybrid algorithms that leverage the strengths of a variety of software to extract and refine high-quality bins from metagenomic data through bin consolidation and reassembly. MetaWRAPs hybrid bin extraction algorithm outperforms individual binning approaches and other bin consolidation programs in both synthetic and real datasets. Finally, metaWRAP comes with numerous modules for the analysis of metagenomic bins, including taxonomy assignment, abundance estimation, functional annotation, and visualization.\n\nConclusionsMetaWRAP is an easy-to-use modular pipeline that automates the core tasks in metagenomic analysis, while contributing significant improvements to the extraction and interpretation of high-quality metagenomic bins. The bin refinement and reassembly modules of metaWRAP consistently outperform other binning approaches. Each module of metaWRAP is also a standalone component, making it a flexible and versatile tool for tackling metagenomic shotgun sequencing data. MetaWRAP is open-source software available at https://github.com/bxlab/metaWRAP.

microbiology↗

bioRxiv2018

The effect of strain level diversity on robust inference of virus-induced mortality

Infection and lysis of phytoplankton by viruses affects population dynamics and nutrient cycles within oceanic microbial communities. However, estimating the quantitative rates of viral-induced lysis remains challenging in situ. The modified dilution method is the most commonly utilised empirical approach to estimate virus-induced killing rates of phytoplankton. The lysis rate estimates of the modified dilution method are based on models of virus-host interactions involving only a single virus and a single host population. Here, using modelling approaches, we examine the robustness of the modified dilution method in multi-strain, complex communities. We assume that strains differ in their life history traits, including growth rates (of hosts) and lysis rates (by viruses). We show that trait differences affect resulting experimental dynamics such that lysis rates measured using the modified dilution method may be driven by the fastest replicating strains; which are not necessarily the most abundant in situ. We discuss the implications of using the modified dilution method and alternative dilution-based approaches for estimating viral-induced lysis rates in marine microbial communities.

microbiology↗

bioRxiv2018

Dynamic virulence-related regions of the fungal plant pathogen Verticillium dahliae display remarkably enhanced sequence conservation

Selection pressure impacts genomes unevenly, as different genes adapt with differential speed to establish an organisms optimal fitness. Plant pathogens co-evolve with their hosts, which implies continuously adaption to evade host immunity. Effectors are secreted proteins that mediate immunity evasion, but may also typically become recognized by host immune receptors. To facilitate effector repertoire alterations, in many pathogens, effector genes reside in dynamic genomic regions that are thought to display accelerated evolution, a phenomenon that is captured by the two-speed genome hypothesis. The genome of the vascular wilt pathogen Verticillium dahliae has been proposed to obey to a similar two-speed regime with dynamic, lineage-specific regions that are characterized by genomic rearrangements, increased transposable element activity and enrichment in in planta-induced effector genes. However, little is known of the origin of, and sequence diversification within, these lineage-specific regions. Based on comparative genomics among Verticillium spp. we now show differential sequence divergence between core and lineage-specific genomic regions of V. dahliae. Surprisingly, we observed that lineage-specific regions display markedly increased sequence conservation. Since single nucleotide diversity is reduced in these regions, host adaptation seems to be merely achieved through presence/absence polymorphisms. Increased sequence conservation of genomic regions important for pathogenicity is an unprecedented finding for filamentous plant pathogens and signifies the diversity of genomic dynamics in host-pathogen co-evolution.

microbiology↗

bioRxiv2018

Comprehensive analysis of small RNA profile by massive parallel sequencing in HTLV-1 asymptomatic subjects with monoclonal and polyclonal rearrangement of the T-cell antigen receptor γ-chain

IntroductionIn this study, we used a massive parallel sequencing technology to investigate the cellular small RNA (sRNA) operating in peripheral blood mononuclear cells (PBMCs) of the Human T-lymphotropic virus type I (HTLV-I) infected asymptomatic subjects with a monoclonal and polyclonal rearrangement of the T-cell antigen receptor {gamma}-chain.\n\nMaterials and MethodsBlood samples from 15 HTLV-1 asymptomatic carriers who were tested for clonal TCR-{gamma} gene (seven and eight subjects presented monoclonal and polyclonal expansion of HTLV-1 infected cells, respectively), and were submitted to Illumina for small RNA library construction. sRNA libraries were prepared from cryoperserved PBMCs using TrueSeq Small RNA Library Preparation Kit (Illumina). The sRNA-Seq reads were aligned, annotated, and profiled by different bioinformatics tools.\n\nResultsThrough bioinformatics analysis, we identified a total of 494 known sRNAs and 120 putative novel sRNAs. Twenty-two known and 15 novel sRNA showed a different expression (>2-fold) between the asymptomatic monoclonal (ASM) and asymptomatic polyclonal carriers (ASP). The hsa-mir-196a-5p was the most abundantly upregulated micro RNA (miRNA) and the hsa-mir-133a followed by hsa-mir-509-3p were significantly downregulated miRNAs with more than a three-fold difference in the ASM than ASP group. The target genes predicted to be regulated by the differentially expressed miRNAs play essential roles in diverse biological processes including cell proliferation, differentiation, and/or apoptosis.\n\nDiscussionOur results provide an opportunity for a further understanding of sRNA regulation and function in HTLV-1 infected subjects with monoclonality evidence.

microbiology↗

bioRxiv2018

American Gut: an Open Platform for Citizen-Science Microbiome Research

Although much work has linked the human microbiome to specific phenotypes and lifestyle variables, data from different projects have been challenging to integrate and the extent of microbial and molecular diversity in human stool remains unknown. Using standardized protocols from the Earth Microbiome Project and sample contributions from over 10,000 citizen-scientists, together with an open research network, we compare human microbiome specimens primarily from the USA, UK, and Australia to one another and to environmental samples. Our results show an unexpected range of beta-diversity in human stool microbiomes as compared to environmental samples, demonstrate the utility of procedures for removing the effects of overgrowth during room-temperature shipping for revealing phenotype correlations, uncover new molecules and kinds of molecular communities in the human stool metabolome, and examine emergent associations among the microbiome, metabolome, and the diversity of plants that are consumed (rather than relying on reductive categorical variables such as veganism, which have little or no explanatory power). We also demonstrate the utility of the living data resource and cross-cohort comparison to confirm existing associations between the microbiome and psychiatric illness, and to reveal the extent of microbiome change within one individual during surgery, providing a paradigm for open microbiome research and education.\n\nImportanceWe show that a citizen-science, self-selected cohort shipping samples through the mail at room temperature recaptures many known microbiome results from clinically collected cohorts and reveals new ones. Of particular interest is integrating n=1 study data with the population data, showing that the extent of microbiome change after events such as surgery can exceed differences between distinct environmental biomes, and the effect of diverse plants in the diet which we confirm with untargeted metabolomics on hundreds of samples.

microbiology↗

bioRxiv2018

EukRef: phylogenetic curation of ribosomal RNA to enhance understanding of eukaryotic diversity and distribution

Environmental sequencing has greatly expanded our knowledge of micro-eukaryotic diversity and ecology by revealing previously unknown lineages and their distribution. However, the value of these data is critically dependent on the quality of the reference databases used to assign an identity to environmental sequences. Existing databases contain errors, and struggle to keep pace with rapidly changing eukaryotic taxonomy, the influx of novel diversity, and computational challenges related to assembling the high-quality alignments and trees needed for accurate characterization of lineage diversity. EukRef (eukref.org) is a community driven initiative that addresses these challenges by bringing together taxonomists with expertise spanning the complete eukaryotic tree of life and microbial ecologists that actively use environmental sequencing data for the purpose of developing reliable reference databases across the diversity of microbial eukaryotes. EukRef organizes and facilitates rigorous sequence data mining and annotation by providing protocols, guidelines and tools to do so.

microbiology↗

bioRxiv2018

A dual sRNA in Staphylococcus aureus induces a metabolic switch responding to glucose consumption

Pathogenic bacteria must rapidly adapt to ever-changing environmental signals or nutrient availability resulting in metabolism remodeling. The carbon catabolite repression represents a global regulatory system, allowing the bacteria to express genes involved in carbon utilization and metabolization of the preferred carbon source. In Staphylococcus aureus, regulation of catabolite repressing genes is mediated by the carbon catabolite protein A (CcpA). Here, we have identified a CcpA-dependent small non-coding RNA, RsaI that is inhibited by high glucose concentrations. RsaI represses the translation of mRNAs encoding a major permease of glucose uptake, the FN3K enzyme that protects proteins against damages caused by high glucose concentrations, and IcaR, the transcriptional repressor of exopolysaccharide production. Besides, RsaI regulates the activities of other sRNAs responding to the uptake of glucose-6 phosphate or NO. Finally, RsaI inhibits the expression of several enzymes involved in carbon catabolism pathway, and activates genes involved in energy production, fermentation and NO detoxification when the glucose concentration decreases. This multifunctional RNA provides a signature for a metabolic switch when glucose is scarce and growth is arrested.

microbiology↗

bioRxiv2018

Physiology and lipid accumulation capacity of different Yarrowia lipolytica and Rhodosporidium toruloides strains on glycerol

ObjectiveTo compare physiological and process parameters, as well as lipid accumulation capacity, of six strains of Yarrowia lipolytica and two strains of Rhodosporidium toruloides in media containing glycerol as the main carbon and energy source.\n\nResultsThe strains Y. lipolytica IMUFRJ 50678, Po1g, W29 and CCT 5443 displayed very similar physiological parameters, with {micro}max, [Formula] and [Formula] average values of 0.31 h-1, 0.53 (g DW/g S), 0.60 (g S/g DW.h) and 0.46 (g DW/g S), respectively. The two strains of R. toruloides presented physiological and process parameters with no significant difference, with average values of 0.084 h-1, 0.53 (g DW/g S), 0.17 (g S/g DW.h) and 0.44 (g DW/g S). Among all Y. lipolytica strains, Y. lipolytica CCT 5443 strain presented the highest YLip/S, 0.054 (g Lip/g S), and PLipof 0.040 (g Lip/l.h). Among all investigated strains (Y. lipolytica and R. toruloides), the yeast R. toruloides CCT 7815 displayed the highest lipid accumulation capacity, with YLip/Sequal to 0.11 (g Lip/g S) and PLipequal to 0.10 (g Lip/l.h).\n\nConclusionAmong all strains investigated in our study, the yeast strain R. toruloides CCT 7815 presents the most promising characteristics for industrial single cell oil production.\n\nList of abbreviations

microbiology↗

bioRxiv2018

Species- and C-terminal linker-dependent variations in the dynamic behavior of FtsZ on membranes in vitro

Bacterial cell division requires the assembly of FtsZ protofilaments into a dynamic structure called the Z-ring. The Z-ring recruits the division machinery and directs local cell wall remodeling for constriction. The organization and dynamics of protofilaments within the Z-ring coordinate local cell wall synthesis during cell constriction, but their regulation is largely unknown. The disordered C-terminal linker (CTL) region of Caulobacter crescentus FtsZ (CcFtsZ) regulates polymer structure and turnover in solution in vitro, and regulates Z-ring structure and activity of cell wall enzymes in vivo. To investigate the contributions of the CTL to the polymerization properties of FtsZ on its physiological platform, the cell membrane, we reconstituted CcFtsZ polymerization on supported lipid bilayers (SLB) and visualized polymer dynamics and structure using total internal reflection fluorescence microscopy. Unlike E. coli FtsZ protofilaments that organized into large, bundled patterns, CcFtsZ protofilaments assembled into small, dynamic clusters on SLBs. Moreover, CcFtsZ lacking its CTL formed large networks of straight filament bundles that underwent slower turnover than the dynamic clusters of wildtype FtsZ. Our in vitro characterization provides novel insights into species- and CTL-dependent differences between FtsZ assembly properties that are relevant to Z-ring assembly and function on membranes in vivo.

microbiology↗