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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↗

bioRxiv2017

Revisiting The Ancylostoma caninum Secretome Provides New Information On Hookworm-Host Interactions

Hookworm infection is a major tropical parasitic disease affecting almost 500 million people worldwide. These soil-transmitted helminths can survive for many years in the intestine of the host, where they feed on blood, causing iron deficiency anaemia and other complications. To avoid the hosts immune response the parasite releases excretory/secretory products (ESPs), a complex mixture of glycans, lipids and proteins that represent the major host-parasite interface. Using a combination of separation techniques such as SDS-PAGE and OFFGEL electrophoresis, in combination with state-of-the-art mass spectrometry we have reanalysed the dog hookworm, Ancylostoma caninum, ESPs (AcES). We identified 315 proteins present in the AcES, compared with just 105 identified in previous studies. The most highly represented family of proteins is the SCP/TAPs (90 of the 315 proteins), and the most abundant constituents of AcES are homologues of the tissue inhibitors of metalloproteases (TIMP) family. We identified putative vaccine candidates and proteins that could have immunomodulatory effects for treating inflammatory diseases. This study provides novel information about the proteins involved in host-hookworm interactions, and constitutes a comprehensive dataset for the development of vaccines and the discovery of new immunoregulatory biologics.

microbiology↗

bioRxiv2017

An Aphrodisiac Produced By Vibrio fischeri Stimulates Mating In The Closest Living Relatives Of Animals

We serendipitously discovered that the marine bacterium Vibrio fischeri induces sexual reproduction in one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Although bacteria influence everything from nutrition and metabolism to cell biology and development in eukaryotes, bacterial regulation of eukaryotic mating was unexpected. Here we show that a single V. fischeri protein, the previously uncharacterized EroS, fully recapitulates the aphrodisiac activity of live V. fischeri. EroS is a chondroitin lyase; although its substrate, chondroitin sulfate, was previously thought to be an animal synapomorphy, we demonstrate that S. rosetta produces chondroitin sulfate and thus extend the ancestry of this important glycosaminoglycan to the premetazoan era. Finally, we show that V. fischeri, purified EroS, and other bacterial chondroitin lyases induce S. rosetta mating at environmentally-relevant concentrations suggesting that bacterially-produced aphrodisiacs likely regulate choanoflagellate mating in nature.

microbiology↗

bioRxiv2017

Identifying Parameters Of Host Cell Vulnerability During Salmonella Infection By Quantitative Image Analysis And Modeling

Salmonella target and enter epithelial cells at permissive entry sites: some cells are more likely to be infected than others. However the parameters that lead to host cell heterogeneity are not known. Here, we quantitatively characterized host cell \"vulnerability\" towards Salmonella infection based on imaged parameters. We performed successive infections of the same host cell population followed by automated high-throuput microscopy and observed that infected cells have higher probability of being re-infected. Establishing a predictive model we identified two combined origins of host cell vulnerability: the pathogen-induced cellular vulnerability emerging from Salmonella uptake and persisting at later stage of the infection, and the host cell-inherent vulnerability. We linked the host cell inherent vulnerability with its morphological attributes such as the local cell crowding, and with host cell cholesterol content. This showed that the probability of Salmonella infection success can be forecast from morphological or molecular host cell parameters.

microbiology↗

bioRxiv2017

The industrial anaerobe Clostridium acetobutylicum uses polyketides to regulate butanol production and differentiation

Polyketides are an important class of bioactive small molecules valued not only for their diverse therapeutic applications, but also for their role in controlling interesting biological phenotypes in their producing organisms. While numerous examples of polyketides derived from aerobic organisms exist, only a single family of polyketides has been identified from anaerobic organisms. Here we uncover a novel family of polyketides native to the anaerobic bacterium Clostridium acetobutylicum, an organism well-known for its historical use as an industrial producer of the organic solvents acetone, butanol, and ethanol. Through mutational analysis and chemical complementation assays, we demonstrate that these polyketides influence butanol production and act as chemical triggers of sporulation and granulose accumulation in this strain. This study represents a significant addition to the body of work demonstrating the existence and importance of polyketides in anaerobes, and showcases a novel strategy of manipulating the secondary metabolism of an organism to improve traits significant for industrial applications.

microbiology↗

bioRxiv2017

Functional Role Of Lanthanides In Enzymatic Activity And Transcriptional Regulation Of PQQ-Dependent Alcohol Dehydrogenases In Pseudomonas putida KT2440

The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs). Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems, based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs), which are often functionally redundant. Using purified enzymes from the soil-dwelling model organism Pseudomonas putida KT2440, the present study reports the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH) in a non-methylotrophic bacterium. PedH exhibits enzyme activity on a similar substrate range as its Ca2+-dependent counterpart PedE, including linear and aromatic primary and secondary alcohols as well as aldehydes, however, only in the presence of lanthanide ions including La3+, Ce3+, Pr3+, Sm3+ or Nd3+. Reporter assays revealed that PedH not only has a catalytic function, but is also involved in the transcriptional regulation of pedE and pedH, most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 - 10 nM of lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results we conclude that functional redundancy and inverse regulation of PedE and PedH represents an adaptive strategy of P. putida KT2440 to optimize growth with volatile alcohols in response to different lanthanide availability.\n\nIMPORTANCEDue to their low bioavailability, lanthanides have long been considered as biologically inert. In recent years however, the identification of lanthanides as a cofactor in methylotrophic bacteria has attracted tremendous interest among various biological fields. The present study reveals that one of the two PQQ-ADHs produced by the model organism P. putida KT2440 also utilizes lanthanides as a cofactor, thus expanding the scope of lanthanide employing bacteria beyond the methylotrophs. Similar to methyloptrophic bacteria, a complex regulatory network is involved in the lanthanide-responsive switch between the two PQQ-ADHs encoded by P. putida KT2440. We further show that functional production of at least one of the enzymes is crucial for efficient growth with several volatile alcohols. Overall, our study provides a novel understanding for the redundancy of PQQ-ADHs observed in many organisms and further highlights the importance of lanthanides for bacterial metabolism, particularly in soil environments.

microbiology↗

bioRxiv2017

The Resistome Of Important Human Pathogens

Genes capable of conferring resistance to clinically used antibiotics have been found in many different natural environments. However, a concise overview of the resistance genes found in common human bacterial pathogens is lacking, which complicates risk ranking of environmental reservoirs. Here, we present an analysis of potential antibiotic resistance genes in the 17 most common bacterial pathogens isolated from humans. We analyzed more than 20,000 bacterial genomes and defined a clinical resistome as the set of resistance genes found across these genomes. Using this database, we uncovered the co-occurrence frequencies of the resistance gene clusters within each species enabling identification of co-dissemination and co-selection patterns. The resistance genes identified in this study represent the subset of the environmental resistome that is clinically relevant and the dataset and approach provides a baseline for further investigations into the abundance of clinically relevant resistance genes across different environments. To facilitate an easy overview the data is presented at the species level at www.resistome.biosustain.dtu.dk.

microbiology↗

bioRxiv2017

In Vivo Host-Pathogen Interaction As Revealed By Global Proteomic Profiling Of Zebrafish Larvae

The outcome of a host-pathogen interaction is determined by the conditions of the host, the pathogen, and the environment. Although numerous proteomic studies of in vitro-grown microbial pathogens have been performed, in vivo proteomic approaches are still rare. In addition, increasing evidence supports that in vitro studies inadequately reflect in vivo conditions. Choosing the proper host is essential to detect the expression of proteins from the pathogen in vivo. Numerous studies have demonstrated the suitability of zebrafish (Danio rerio) embryos as a model to in vivo studies of Pseudomonas aeruginosa infection. In most zebrafish-pathogen studies, infection is achieved by microinjection of bacteria into the larvae. However, few reports using static immersion of bacterial pathogens have been published. In this study we infected 3 days post-fertilization (DPF) zebrafish larvae with P. aeruginosa PAO1 by immersion and injection and tracked the in vivo immune response by the zebrafish. Additionally, by using non-isotopic (Q-exactive) metaproteomics we simultaneously evaluated the proteomic response of the pathogen (P. aeruginosa PAO1) and the host (zebrafish). We found some zebrafish metabolic pathways, such as hypoxia response via HIF activation pathway, exclusively enriched in the larvae exposed by static immersion. In contrast, we found that inflammation mediated by chemokine and cytokine signaling pathways was exclusively enriched in the larvae exposed by injection, while the integrin signaling pathway and angiogenesis were solely enriched in the larvae exposed by immersion. We also found important virulence factors from P. aeruginosa that were enriched only after exposure by injection, such as the Type-III secretion system and flagella-associated proteins. On the other hand, P. aeruginosa proteins involved in processes like biofilm formation, cellular responses to antibiotic and starvation were enriched exclusively after an exposure by immersion.\n\nWe demonstrated the suitability of zebrafish embryos as a model for in vivo host-pathogen based proteomic studies in P. aeruginosa. Our global proteomic profiling identifies novel molecular signatures that give systematic insight into zebrafish-Pseudomonas interaction.

microbiology↗

bioRxiv2017

Development Of RNAseq Methodologies To Profile The In Vivo Transcriptome Of Bordetella pertussis During Murine Lung Infection

Bordetella pertussis is an obligate human respiratory pathogen that causes the disease whooping cough. A whole cell vaccine (DTP) was developed in the 1940s and was subsequently replaced in the 1990s with a protein-based subunit acellular vaccine (DTaP; tdap). Today, we are observing a resurgence of whooping cough due to evolution of the pathogen and waning vaccine immunity. The use of vaccines decreased the need for basic research on this pathogen. As a result, numerous questions on the basic pathogenesis of B. pertussis remain to be answered. Microarrays and more recently, RNA sequencing (RNAseq), have allowed the field to describe the in vitro gene expression profiles of the pathogen growing in both virulent and avirulent phases; however, no published studies have described an in vivo transcriptome of the pathogen. To address this need, we have designed and evaluated workflows to characterize the in vivo transcriptome of B. pertussis during infection of the murine lung. During our initial studies, we observed that only 0.014% of the ~100 million 2x50bp illumina reads corresponded to the pathogen, which is insufficient for analysis. Therefore, we developed a simple protocol to filter the bacteria out of the tissue homogenates and separate bacterial cells from the host tissue. RNA is then prepared, quantified, and the B. pertussis to host RNA ratio is determined. Here, we present the protocol and discuss the uses and next directions for which this RNAseq workflow can be applied. With this strategy we plan to fully characterize the B. pertussis transcriptome when the pathogen is infecting the murine lung in order to identify expressed genes that encode potential new vaccine antigens that will facilitate the development of the next generation of pertussis vaccines.

microbiology↗

bioRxiv2017

Undocumented Potential For Primary Productivity In A Globally-Distributed Bacterial Photoautotroph

Aerobic anoxygenic phototrophs (AAnPs) are common in the global oceans and are associated with photoheterotrophic activity. To date, AAnPs have not been identified in the surface ocean that possess the potential for carbon fixation. Using the Tara Oceans metagenomic dataset, we have reconstructed draft genomes of four bacteria that possess the genomic potential for anoxygenic phototrophy, carbon fixation via the Calvin-Benson-Bassham cycle, and the oxidation of sulfite and thiosulfate. Forming a monophyletic clade within the Alphaproteobacteria and lacking cultured representatives, the organisms compose minor constituents of local microbial communities (0.1-1.0%), but are globally distributed, present in multiple samples from the North Pacific, Mediterranean Sea, the East Africa Coastal Province, and the South Atlantic. These organisms represent a shift in our understanding of microbially-mediated photoautotrophy in the global oceans and provide a previously undiscovered route of primary productivity.\n\nSignificance StatementIn examining the genomic content of organisms collected during the Tara Oceans expedition, we have identified a novel clade within the Alphaproteobacteria that has the potential for photoautotrophy. Based on genome observations, these organisms have the potential to couple inorganic sulfur compounds as electron donors to fix carbon into biomass. They are globally distributed, present in samples from the North Pacific, Mediterranean Sea, East Africa Coastal Current, and the South Atlantic. This discovery may require re-examination of the microbial communities in the global ocean to understand and constrain the impacts of this group of organisms on the global carbon cycle.

microbiology↗

bioRxiv2017

A Phylogenetic Method To Perform Genome-Wide Association Studies In Microbes That Accounts For Population Structure And Recombination

Genome-Wide Association Studies (GWAS) in microbial organisms have the potential to vastly improve the way we understand, manage, and treat infectious diseases. Yet, GWAS methods established thus far remain insufficiently able to capitalise on the growing wealth of bacterial and viral genetic sequence data. Facing clonal population structure and homologous recombination, existing GWAS methods struggle to achieve both the precision necessary to reject spurious findings and the power required to detect associations in microbes. In this paper, we introduce a novel phylogenetic approach that has been tailor-made for microbial GWAS, which is applicable to organisms ranging from purely clonal to frequently recombining, and to both binary and continuous phenotypes. Our approach is robust to the confounding effects of both population structure and recombination, while maintaining high statistical power to detect associations. Thorough testing via application to simulated data provides strong support for the power and specificity of our approach and demonstrates the advantages offered over alternative cluster-based and dimension-reduction methods. Two applications to Neisseria meningitidis illustrate the versatility and potential of our method, confirming previously-identified penicillin resistance loci and resulting in the identification of both well-characterised and novel drivers of invasive disease. Our method is implemented as an open-source R package called treeWAS which is freely available at https://github.com/caitiecollins/treeWAS.

microbiology↗