SEARCH · Search bioRxiv

Results for “Microbiology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37Linked to original sources

bioRxiv2017

FplA From Fusobacterium nucleatum Is A Type Vd Autotransporter Phospholipase With A Proposed Role In Altered Host Signaling And Evasion Of Autophagy

Fusobacterium nucleatum is a pathogenic oral bacterium that is linked to multiple human infections and colorectal cancer. While most Gram-negative pathogens utilize secretion systems for cellular invasion and infection, F. nucleatum lacks Type I, II, III, IV, and VI secretion. By contrast, F. nucleatum strains are enriched in Type V secreted autotransporters, which are Gram-negative bacterial virulence factors critical for binding and entry into host cells. Here we present the first biochemical characterization of a F. nucleatum Type Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby rename Fusobacterium phospholipase autotransporter (FplA). FplA is expressed as a full-length 85 kDa outer membrane embedded protein, or as a truncated phospholipase domain that remains associated with the outer membrane. Using multiple FplA constructs we characterized lipid substrate specificity, potent inhibitors, and chemical probes to detect and track this enzyme family. While the role of FplA is undetermined in F. nucleatum virulence, homologous phospholipases from intracellular pathogens are critical for vacuole escape, altered host signaling, and intracellular survival. We hypothesize that upon intracellular invasion of the host, FplA could play a role in phagosomal escape, subversion of autophagy, or eicosanoid-mediated inflammatory signaling, as we show that FplA binds with high affinity to host phosphoinositide signaling lipids critical to these processes. Our identification of substrates, inhibitors, and chemical probes for FplA, in combination with an fplA gene deletion strain, encompass a powerful set of tools for the future analysis of FplA in vivo. In addition, these studies will guide the biochemical characterization of additional Type Vd autotransporter phospholipases.\n\nIMPORTANCEF. nucleatum is an emerging pathogen that is linked to the pathogenesis of colorectal cancer, yet there is a critical knowledge gap in the mechanisms used by this bacterium to elicit changes in the host for intracellular entry and survival. As phospholipases are critical virulence factors for intracellular bacteria to initiate vacuole lysis, cell-to-cell spread, and evasion of autophagy, we set out to characterize a unique Type Vd secreted phospholipase A1 enzyme from F. nucleatum. Our results show a potential role for modulating host signaling pathways through cleavage of phosphoinositide dependent signaling lipids. These studies open the door for further characterization of this unique enzyme family in bacterial virulence, host-pathogen interactions, and for F. nucleatum, in colorectal carcinogenesis.

microbiology↗

bioRxiv2017

Global Phylogenetic Analysis Of Escherichia coli And Plasmids Carrying The mcr-1 Gene Indicates Bacterial Diversity But Plasmid Restriction

To understand the dynamics behind the worldwide spread of the mcr-1 gene, we determined the population structure of Escherichia coli and of mobile genetic elements (MGEs) carrying the mcr-1 gene. After a systematic review of the literature we included 65 E. coli whole genome sequences (WGS), adding 6 recently sequenced travel related isolates, and 312 MLST profiles. We included 219 MGEs described in 7 Enterobacteriaceae species isolated from human, animal and environmental samples. Despite a high overall diversity, 2 lineages were observed in the E. coli population that may function as reservoirs of the mcr-1 gene, the largest of which was linked to ST10, a sequence type known for its ubiquity in human faecal samples and in food samples. No genotypic clustering by geographical origin or isolation source was observed. Amongst a total of 13 plasmid incompatibility types, the Incl2, lncX4 and IncHI2 plasmids accounted for more than 90% of MGEs carrying the mcr-1 gene. We observed significant geographical clustering with regional spread of lncHI2 plasmids in Europe and Incl2 in Asia. These findings point towards promiscuous spread of the mcr-1 gene by efficient horizontal gene transfer dominated by a limited number of plasmid incompatibility types.

microbiology↗

bioRxiv2017

Modifying And Reacting To The Environmental pH Drives Bacterial Interactions

Microbes usually exist in communities consisting of myriad different but interacting species. These interactions are typically mediated through environmental modifications; microbes change the environment by taking up resources and excreting metabolites, which affects the growth of both themselves and also other microbes. A very common environmental modification is a change of the environmental pH. Here we show that changing and reacting to the pH leads to a feedback loop that can determine the fate of bacterial populations. We find experimentally that these pH changes can decide the fate of bacterial populations; they can either facilitate or inhibit growth, and in extreme cases will cause extinction of the bacterial population. Moreover, modifying the pH can determine the interactions between different species. Understanding how single species change the pH and react to it allowed us to estimate their pairwise interaction outcomes. Those interactions lead to a set of generic interaction motifs--bistability, succession, murder suicide, and mutualism--that may be independent of which environmental parameter is modified and thus reoccur in different microbial systems.

microbiology↗

bioRxiv2017

Systematic Genetic Dissection Of Chitin Degradation And Uptake In Vibrio cholerae

Vibrio cholerae is a natural resident of the aquatic environment, where a common nutrient is the chitinous exoskeletons of microscopic crustaceans. Chitin utilization requires chitinases, which degrade this insoluble polymer into soluble chitin oligosaccharides. These oligosaccharides also serve as an inducing cue for natural transformation in Vibrio species. There are 7 predicted endochitinase-like genes in the V. cholerae genome. Here, we systematically dissect the contribution of each gene to growth on chitin as well as induction of natural transformation. Specifically, we created a strain that lacks all 7 putative chitinases and from this strain, generated a panel of strains where each expresses a single chitinase. We also generated expression plasmids to ectopically express all 7 chitinases in our chitinase deficient strain. Through this analysis, we found that low levels of chitinase activity are sufficient for natural transformation, while growth on insoluble chitin as a sole carbon source requires more robust and concerted chitinase activity. We also assessed the role that the three uptake systems for the chitin degradation products GlcNAc, (GlcNAc)2, and (GlcN)2, play in chitin utilization and competence induction. Cumulatively, this study provides mechanistic details for how this pathogen utilizes chitin to thrive and evolve in its environmental reservoir.\n\nORIGINALITY-SIGNIFICANCE STATEMENTVibrio cholerae, the causative agent of the diarrheal disease cholera, interacts with the chitinous shells of crustacean zooplankton in the aquatic environment, which serves as an environmental reservoir for this pathogen. It degrades and utilizes chitin-derived products as a source of carbon and nitrogen. Also, chitin serves as an inducing cue for natural transformation - an important mechanism of horizontal gene transfer in this species. Here, we systematically dissect the genes required for chitin degradation and uptake, and characterize the role of these genes for growth on chitin as a nutrient and during chitininduced natural transformation. Thus, this study provides mechanistic details for how this pathogen utilizes chitin to thrive and evolve in its environmental reservoir.

microbiology↗

bioRxiv2017

Priming In A Permissive Type I-C CRISPR-Cas System Reveals Distinct Dynamics Of Spacer Acquisition And Loss

CRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR-Cas system in Legionella pneumophila that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3' to 5' translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe greatly increased interference efficiency coupled with a dramatic shift away from spacer acquisition towards spacer loss. Based on these observations, we propose that the Legionella type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation.

microbiology↗

bioRxiv2017

The CRISPR Spacer Space Is Dominated By Sequences From The Species-Specific Mobilome

The CRISPR-Cas is the prokaryotic adaptive immunity system that stores memory of past encounters with foreign DNA in spacers that are inserted between direct repeats in CRISPR arrays 1,2. Only for a small fraction of the spacers, homologous sequences, termed protospacers, are detectable in viral, plasmid or microbial genomes 3,4. The rest of the spacers remain the CRISPR \"dark matter\". We performed a comprehensive analysis of the spacers from all CRISPR-cas loci identified in bacterial and archaeal genomes, and found that, depending on the CRISPR-Cas subtype and the prokaryotic phylum, protospacers were detectable for 1 to about 19% of the spacers ([~]7% global average). Among the detected protospacers, the majority, typically, 80 to 90%, originate from viral genomes, and among the rest, the most common source are genes integrated in microbial chromosomes but involved in plasmid conjugation or replication. Thus, almost all spacers with identifiable protospacers target mobile genetic elements (MGE). The GC-content, as well as dinucleotide and tetranucleotide compositions, of microbial genomes, their spacer complements, and the cognate viral genomes show a nearly perfect correlation and are almost identical. Given the near absence of self-targeting spacers, these findings are best compatible with the possibility that the spacers, including the dark matter, are derived almost completely from the species-specific microbial mobilomes.

microbiology↗

bioRxiv2017

A Tunable Protein Oscillator Controls Directional Movements In Myxococcus xanthus

Dynamic control of cell polarity is of critical importance for many aspects of cellular development and motility. In Myxococcus xanthus, a G-protein and its cognate GTPase-activating protein establish a polarity axis that defines the direction of movement of the cell and which can be rapidly inverted by the Frz chemosensory system. Although vital for collective cell behaviours, how Frz triggers this switch has remained unknown. Here, we use genetics, imaging and mathematical modelling to show that Frz controls polarity reversals via a gated relaxation oscillator. FrzX, which we newly identify as the primary Frz output, provides the gating and thus acts as the trigger for reversals. Slow relocalisation of the polarity protein RomR then creates a refractory period during which another switch cannot be triggered. A secondary Frz output, FrzZ, decreases this delay allowing rapid reversals when required. This architecture thus results in a highly tunable switch that allows a wide range of motility responses.

microbiology↗

bioRxiv2017

Uncovering The Drivers Of Animal-Host Microbiotas With Joint Distribution Modeling

In addition to the processes structuring free-living communities, host-associated microbiota are directly or indirectly shaped by the host. Therefore, microbiota data have a hierarchical structure where samples are nested under one or several variables representing host-specific factors, often spanning multiple levels of biological organization. Current statistical methods do not accommodate this hierarchical data structure, and therefore cannot explicitly account for the effect of the host in structuring the microbiota. We introduce a novel extension of joint species distribution models which can straightforwardly accommodate and discern between effects such as host phylogeny and traits, recorded covariates like diet and collection site, among other ecological processes. Our proposed methodology includes powerful yet familiar outputs seen in community ecology overall, including: (i) model-based ordination to visualize and quantify the main patterns in the data; (ii) variance partitioning to asses how influential the included host-specific factors are in structuring the microbiota; and (iii) co-occurrence networks to visualize microbe-to-microbe associations.

microbiology↗

bioRxiv2017

Yeasts Dominate Soil Fungal Communities In Three Lowland Neotropical Rainforests

Forest soils typically harbour a vast diversity of fungi, but are usually dominated by filamentous (hyphae-forming) taxa. Compared to temperate and boreal forests, though, we have limited knowledge about the fungal diversity in tropical rainforest soils. Here we show, by environmental metabarcoding of soil samples collected in three Neotropical rainforests, that Yeasts dominate the fungal communities in terms of the number of sequencing reads and OTUs. These unicellular forms are commonly found in aquatic environments, and their hyperdiversity may be the result of frequent inundation combined with numerous aquatic microenvironments in these rainforests. Other fungi that are frequent in aquatic environments, such as the abundant Chytridiomycotina, were also detected. While there was low similarity in OTU composition within and between the three rainforests, the fungal communities in Central America were more similar to each other than the communities in South America, reflecting a general biogeographic pattern also seen in animals, plants, and protists.\n\nContactdunthorn@rhrk.uni-kl.de

microbiology↗

bioRxiv2017

Bifunctionality Of A Biofilm Matrix Protein Controlled By Redox State

Biofilms are communities of microbial cells that are encapsulated within a self-produced polymeric matrix. The matrix is critical to the success of biofilms in diverse habitats, but despite this many details of the composition, structure, and function remain enigmatic. Biofilms formed by the Gram-positive bacterium Bacillus subtilis depend on the production of the secreted film-forming protein BslA. Here we show that a gradient of electron acceptor availability through the depth of the biofilm gives rise to two distinct functional roles for BslA and that these can be genetically separated through targeted amino acid substitutions. We establish that monomeric BslA is necessary and sufficient to give rise to complex biofilm architecture, while dimerization of BslA is required to render the community hydrophobic. Dimerization of BslA, mediated by disulfide bond formation, depends on two conserved cysteine residues located in the C-terminal region. Our findings demonstrate that bacteria have evolved multiple uses for limited elements in the matrix, allowing for alternative responses in a complex, changing environment.\n\nSignificanceThe biofilm matrix is a critical target in the hunt for novel strategies to destabilise or stabilise biofilms. Knowledge of the processes controlling matrix assembly is therefore an essential prerequisite to exploitation. Here we highlight that the complexity of the biofilm matrix is even higher than anticipated with one matrix component making two independent functional contributions to the community. The influence the protein exerts is dependent on the local environmental properties, providing another dimension to consider during analysis. These findings add to the evidence that bacteria can evolve multifunctional uses for the extracellular matrix components.

microbiology↗

bioRxiv2017

The effect of treatment on the microbiota of patients diagnosed with colonic lesions

BackgroundColorectal cancer is a worldwide health problem. Despite growing evidence that members of the gut microbiota can drive tumorigenesis, little is known about what happens to it after treatment for an adenoma or carcinoma. This study tested the hypothesis that treatment for adenoma or carcinoma alters the abundance of bacterial populations associated with disease to those associated with a normal colon. We tested this hypothesis by sequencing the 16S rRNA genes in the feces of 67 individuals before and after treatment for adenoma (N = 22), advanced adenoma (N = 19), and carcinoma (N = 26).\n\nResultsThere were small changes to the bacterial community associated with adenoma or advanced adenoma and large changes associated with carcinoma. The communities from patients with carcinomas changed significantly more than those with adenoma following treatment (P-value < 0.001). Although treatment was associated with intrapersonal changes, the change in the abundance of individual OTUs in response to treatment was not consistent within diagnosis groups (P-value > 0.05). Because the distribution of OTUs across patients and diagnosis groups was irregular, we used the Random Forest machine learning algorithm to identify groups of OTUs that could be used to classify pre and post-treatment samples for each of the diagnosis groups. Although the three models could differentiate between the pre and post-treatment samples, there was little overlap between the OTUs that were indicative of each treatment. To determine whether individuals who underwent treatment were more likely to have OTUs associated with normal colons we used a larger cohort that contained individuals with normal colons and those with adenomas, advanced adenomas, and carcinomas. We again built Random Forest models and measured the change in the positive probability of having one of the three diagnoses to assess whether the post-treatment samples received the same classification as the pre-treatment samples. Samples from patients who had carcinomas changed towards a microbial milieu that resembles the normal colon after treatment (P-value < 0.05). Finally, we were unable to detect any significant differences in the microbiota of individuals treated with surgery alone and those treated with chemotherapy or chemotherapy and radiation (P-value > 0.05).\n\nConclusionsBy better understanding the response of the microbiota to treatment for adenomas and carcinomas, it is likely that biomarkers will eventually be validated that can be used to quantify the risk of recurrence and the likelihood of survival. Although it was difficult to identify significant differences between pre and post-treatment samples from patients with adenoma and advanced adenoma, this was not the case for carcinomas. Not only were there large changes in pre versus post-treatment samples for those with carcinoma, but these changes were towards a more normal microbiota.

microbiology↗

bioRxiv2017

Prediction of fluoroquinolone susceptibility directly from whole genome sequence data using liquid chromatography-tandem mass spectrometry to identify mutant genotypes.

Fluoroquinolone resistance in bacteria is multifactorial, involving target site mutations, reductions in fluoroquinolone entry due to reduced porin production, increased fluoroquinolone efflux, enzymes that modify fluoroquinolones, and Qnr, a DNA mimic that protects the drug target from fluoroquinolone binding. Here we report a comprehensive analysis using transformation and in vitro mutant selection, of the relative importance of each of these mechanisms in fluoroquinolone resistance and non-susceptibility, using Klebsiella pneumoniae, one of the most clinically important multi-drug resistant bacterial species known, as a model system. Our improved biological understanding was then used to generate rules that could be predict fluoroquinolone susceptibility in K. pneumoniae clinical isolates. Key to the success of this predictive process was the use of liquid chromatography tandem mass spectrometry to measure the abundance of proteins in extracts of cultured bacteria, identifying which sequence variants seen in the whole genome sequence data were functionally important in the context of fluoroquinolone susceptibility.

microbiology↗

bioRxiv2017

Correct And Efficient Initiation Of Viral RNA Synthesis By The Influenza A Virus RNA Polymerase

The influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2 and PA. To copy the viral RNA (vRNA) genome segments and the complementary RNA (cRNA) segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two different de novo initiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3 terminus, while on the cRNA promoter the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into prime-realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly.\n\nImportanceInfluenza A viruses cause severe disease in humans and are considered a major threat to our economy and health. The viruses replicate and transcribe their genome using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the RNA polymerase must work correctly. In this report, we provide insight into the mechanism that the RNA polymerase employs to ensure that the viral genome is copied correctly.

microbiology↗

bioRxiv2017

Comparative Genomics Sheds Light On Niche Differentiation And The Evolutionary History Of Comammox Nitrospira

The description of comammox Nitrospira spp., performing complete ammonium-to-nitrate oxidation, and their co-occurrence with canonical betaproteobacterial ammonium oxidizing bacteria ({beta}-AOB) in the environment, call into question the metabolic potential of comammox Nitrospira and the evolutionary history of their ammonium oxidation pathway. We report four new comammox Nitrospira genomes, constituting two novel species, and the first comparative genomic analysis on comammox Nitrospira.\n\nComammox Nitrospira has lost the potential to use external nitrite as energy and nitrogen source: compared to strictly nitrite oxidizing Nitrospira; they lack genes for assimilative nitrite reduction and reverse electron transport from nitrite. By contrast, compared to other Nitrospira, their ammonium oxidizer physiology is exemplified by genes for ammonium and urea transporters and copper homeostasis and the lack of cyanate hydratase genes. Two comammox clades are different in their ammonium uptake systems. Contrary to {beta}-AOB, comammox Nitrospira genomes have single copies of the two central ammonium oxidation pathway genes, lack genes involved in nitric oxide reduction, and encode genes that would allow efficient growth at low oxygen concentrations. Hence, comammox Nitrospira seems attuned to oligotrophy and hypoxia compared to {beta}-AOB.\n\n{beta}-AOBs are the clear origin of the ammonium oxidation pathway in comammox Nitrospira: reconciliation analysis indicates two separate early amoA gene transfer events from {beta}-AOB to an ancestor of comammox Nitrospira, followed by clade specific losses. For haoA, one early transfer from {beta}-AOB to comammox Nitrospira is predicted - followed by intra-clade transfers. We postulate that the absence of comammox genes in most Nitrospira genomes is the result of subsequent loss.\n\nSignificanceThe recent discovery of comammox bacteria - members of the Nitrospira genus able to fully oxidize ammonia to nitrate - upset the long-held conviction that nitrification is a two-step process. It also opened key questions on the ecological and evolutionary relations of these bacteria with other nitrifying prokaryotes. Here, we report the first comparative genomic analysis of comammox Nitrospira and related nitrifiers. Ammonium oxidation genes in comammox Nitrospira had a surprisingly complex evolution, originating from ancient transfer from the phylogenetically distantly related ammonia-oxidizing betaproteobacteria, followed by within-lineage transfers and losses. The resulting comammox genomes are uniquely adapted to ammonia oxidation in nutrient-limited and low-oxygen environments and appear to have lost the genetic potential to grow by nitrite oxidation alone.

microbiology↗

bioRxiv2017

Prediction Of Cephalosporin And Carbapenem Susceptibility In Multi-Drug Resistant Gram-Negative Bacteria Using Liquid Chromatography-Tandem Mass Spectrometry

In vitro antibacterial susceptibility testing informs clinical decision making concerning antibacterial therapeutics. Predicting, in a timely manner, which bacterial infection will respond to treatment by a given antibacterial drug reduces morbidity, mortality, and healthcare costs. It also allows prudent antibacterial use, because clinicians can focus on the least broad-spectrum agent suitable for each patient. Existing susceptibly testing methodologies rely on growth of bacteria in the presence of an antibacterial drug. There is significant interest in the possibility of predicting antibacterial drug susceptibility directly though the analysis of bacterial DNA or protein, because this may lead to more rapid susceptibility testing directly from clinical samples. Here we report a robust and tractable methodology that allows measurement of the abundance of key proteins responsible for antibacterial drug resistance within samples of 1 {micro}g of total bacterial protein. The method allowed correct prediction of {beta}-lactam susceptibility in clinical isolates from four key bacterial species and added considerable value over and above the information generated by whole genome sequencing, allowing for gene expression, not just gene presence to be considered, which is key when considering the complex interplays of multiple mechanisms of resistance.

microbiology↗

bioRxiv2017

Hydrogen Isotope Composition Of Thermoanaerobacterium saccharolyticum Lipids: Comparing Wild Type To A nfn- Transhydrogenase Mutant

AbstractThe 2H/1H ratio in microbial fatty acids can record information about the energy metabolism of microbes and about the isotopic composition of environmental water. However, the mechanisms involved in the fractionation of hydrogen isotopes between water and lipid are not fully resolved. We provide data aimed at understanding this fractionation in the Gram-positive obligately thermophilic anaerobe, Thermoanaerobacterium saccharolyticum, by comparing a wild-type strain to a deletion mutant in which the nfnAB genes encoding electron-bifurcating transhydrogenase have been removed. The wild-type strain showed faster growth rates and larger overall fractionation [Formula] than the mutant strain [Formula]. The overall trend in growth rate and fractionation, along with the isotopic ordering of individual lipids, is consistent with results reported for the Gram-negative sulfate reducer, Desulfovibrio alaskensis G20.

microbiology↗

bioRxiv2017

Evaluating The Accuracy Of DNA Stable Isotope Probing

Originality-Significance StatementBy combining DNA Stable Isotope Probing (DNA-SIP) with multiplexed high throughput DNA sequencing (HTS-DNA-SIP), it is now possible to identify patterns of isotope incorporation for thousands of microbial taxa. HTS-DNA-SIP has enormous potential to reveal patterns of carbon and nitrogen exchange within microbial food webs. A current limitation is that, due to the expense of these experiments, it has been impossible to evaluate the accuracy of DNA-SIP methods. We have developed a model that simulates DNA-SIP data, and we use the model to systematically evaluate and validate the accuracy of DNA-SIP analyses. This model can determine the analytical accuracy of DNA-SIP experiments in a range of contexts. Furthermore, the ability to predict experimental outcomes, as a function of experimental design and community characteristics, should be of great use in the design and interpretation DNA-SIP experiments.\n\nSummaryDNA Stable isotope probing (DNA-SIP) is a powerful method that identifies in situ isotope assimilation by microbial taxa. Combining DNA-SIP with multiplexed high throughput DNA sequencing (HTS-DNA-SIP) creates the potential to map in situ assimilation dynamics for thousands of microbial taxonomic units. However, the accuracy of methods for analyzing DNA-SIP data has never been evaluated. We have developed a toolset (SIPSim) for simulating HTS-DNA-SIP datasets and evaluating the accuracy of methods for analyzing HTS-DNA-SIP data. We evaluated two different approaches to analyzing HTS-DNA-SIP data: \"high resolution stable isotope probing\" (HR-SIP) and \"quantitative stable isotope probing\" (q-SIP). HR-SIP was highly specific and moderately sensitive, with very few false positives but potential for false negatives. In contrast, q-SIP had fewer false negatives but many false positives. We also found HR-SIP more robust than q-SIP with respect to experimental variance. Furthermore, we found that the detection sensitivity of HTS-DNA-SIP can be increased without compromising specificity by evaluating evidence of isotope incorporation over multiple windows of buoyant density (MW-HR-SIP). SIPSim provides a platform for determining the accuracy of HTS-DNA-SIP methods across a range of experimental parameters, which will be useful in the design, analysis, and validation of DNA-SIP experiments.

microbiology↗

bioRxiv2017

Niche Partitioning In Sympatric Populations Of Aquatic Stenotrophomonas Uncovered By Evolutionary Species Delimitation

The genus Stenotrophomonas (Gammaproteobacteria) has a broad environmental distribution. S. maltophilia is its best known species because it is a globally emerging, multidrug-resistant (MDR), opportunistic pathogen. Members of this species are known to display high genetic, ecological and phenotypic diversity, forming the so-called S. maltophilia complex (Smc). Heterogeneous resistance and virulence phenotypes have been reported for environmental Smc isolates of diverse ecological origin. We hypothesized that this heterogeneity could be in part due to the potential lumping of several cryptic species in the Smc. Here we used state-of-the-art phylogenetic and population genetics methods to test this hypothesis based on the multilocus dataset available for the genus at pubmlst.org. It was extended with sequences from complete and draft genome sequences to assemble a comprehensive set of reference sequences. This framework was used to analyze 108 environmental isolates obtained in this study from the sediment and water column of four rivers and streams in Central Mexico, affected by contrasting levels of anthropogenic pollution. The aim of the study was to identify species in this collection, defined as genetically cohesive sequence clusters, and to determine the extent of their genetic, ecological and phenotypic differentiation. The multispecies coalescent, coupled with Bayes factor analysis was used to delimit species borders, together with population genetic structure analyses, recombination and gene flow estimates between sequence clusters.\n\nThese analyses consistently revealed that the Smc contains at least 5 significantly differentiated lineages: S. maltophilia and Smc1 to Smc4. Only S. maltophilia was found to be intrinsically MDR, all its members expressing metallo-{beta}-lactamases (MBLs). The other Smc lineages were not MDR and did not express MBLs. We also obtained isolates related to S. acidaminiphila, S. humi and S. terrae. They were significantly more susceptible to antibiotics than S. maltophilia. We demonstrate that the sympatric lineages recovered display significantly differentiated habitat preferences, antibiotic resistance profiles and beta-lactamase expression phenotypes, as shown by diverse multivariate analyses and robust univariate statistical tests. We discuss our data in light of current models of bacterial speciation, which fit these data well, stressing the implications of species delimitation in ecological, evolutionary and clinical research.

microbiology↗