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bioRxiv2016

Viral coinfection is shaped by bacterial ecology and virus-virus interactions across diverse microbial taxa and environments

Infection of more than one virus in a host, coinfection, is common across taxa and environments. Viral coinfection can enable genetic exchange, alter the dynamics of infections, and change the course of viral evolution. Yet, a systematic test of the factors explaining variation in viral coinfection across different taxa and environments awaits completion. Here I employ three microbial data sets of virus-host interactions covering cross-infectivity, culture coinfection, and single-cell coinfection (total: 6,564 microbial hosts, 13,103 viruses) to provide a broad, comprehensive picture of the ecological and biological factors shaping viral coinfection. I found evidence that ecology and virus-virus interactions are recurrent factors shaping coinfection patterns. Host ecology was a consistent and strong predictor of coinfection across all three datasets: cross-infectivity, culture coinfection, and single-cell coinfection. Host phylogeny or taxonomy was a less consistent predictor, being weak or absent in the cross-infectivity and single-cell coinfection models, yet it was the strongest predictor in the culture coinfection model. Virus-virus interactions strongly affected coinfection. In the largest test of superinfection exclusion to date, prophage sequences reduced culture coinfection by other prophages, with a weaker effect on extrachromosomal virus coinfection. At the single-cell level, prophage sequences eliminated coinfection. Virus-virus interactions also increased culture coinfection with ssDNA-dsDNA coinfections >2x more likely than ssDNA-only coinfections. The presence of CRISPR spacers was associated with a ~50% reduction in single-cell coinfection in a marine bacteria, despite the absence of exact spacer matches in any active infection. Collectively, these results suggest the environment bacteria inhabit and the interactions among surrounding viruses are two factors consistently shaping viral coinfection patterns. These findings highlight the role of virus-virus interactions in coinfection with implications for phage therapy, microbiome dynamics, and viral infection treatments.

Microbiology↗

bioRxiv2016

Sequential acquisition of virulence and fluoroquinolone resistance has shaped the evolution of Escherichia coli ST131

Escherichia coli ST131 is the most frequently isolated fluoroquinolone resistant (FQR) E. coli clone worldwide and a major cause of urinary tract and bloodstream infections. Although originally identified through its association with the CTX-M-15 extended-spectrum {beta}-lactamase resistance gene, global genomic epidemiology studies have failed to resolve the geographical and temporal origin of the ST131 ancestor. Here, we developed a framework for the reanalysis of publicly available genomes from different sources and used this dataset to reconstruct the evolutionary steps that led to the emergence of FQR ST131. Using Bayesian estimation, we show that point mutations in chromosomal genes that confer FQR coincide with the first clinical use of fluoroquinolone in 1986, and illustrate the impact of this pivotal event in the rapid population expansion of ST131 worldwide from an apparent origin in North America. Furthermore, we identify key virulence factor acquisition events that predate the development of FQR, suggesting that the gain of virulence-associated genes followed by the tandem development of antibiotic resistance primed the successful global dissemination of ST131.

Microbiology↗

bioRxiv2016

Identification and characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts reveals their constitutive expression in leukemic and pre-leukemic clones

Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells and produces a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about {bsim}5% develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The 5LTRs of both the BLV and HTLV-1 proviruses are transcriptionally silent in tumors, however they are not entirely quiescent, with the HLTV-1 antisense transcript HBZ and the BLV microRNAs constitutively expressed in tumors. Here, using RNA-seq, we demonstrate that in addition to microRNAs, the BLV provirus also constitutively expresses two antisense transcripts in all BLV infected samples examined. The first transcript (AS1) has alternate potential polyadenylation sites generating a short transcript of {bsim}600bp (aS1-S) and a less abundant longer transcript of {bsim}2200bp (AS1-L). Alternative splicing also creates a second transcript of {bsim}400bp (AS2) utilizing the first exon of AS1. Production of AS transcripts from the 3LTR was supported by reporter assays demonstrating that the BLV LTR has substantial and Tax-independent antisense promoter activity. BLV AS transcripts predominantly localize in the nucleus. Examination of protein coding potential showed AS2 to be non-coding, while the AS1-S/L transcripts coding potential is ambiguous, with a small potential open reading frame (ORF) of 264bp present. The AS1-L transcript overlaps the BLV microRNAs transcribed in the sense direction. Using high throughput sequencing of RNA-ligase-mediated (RLM) 5 RACE products, we show that the perfect complementary between the transcripts leads to RNA-induced silencing complex (RISC) mediated cleavage of AS1-L. Furthermore, experiments using BLV proviruses where the microRNAs were removed or inverted point to additional transcriptional interactions between the two viral RNA species. Knock down of AS1-S/L using locked nucleic acids (LNAs) showed no obvious effect on the cells phenotype. While a detailed elucidation of the BLV antisense transcripts function remains in the future, the constitutive expression in all samples examined, points to a vital role for the transcripts in the life cycle and oncogenic potential of BLV.

Microbiology↗

bioRxiv2016

DNA from dust: comparative genomics of large DNA viruses in field surveillance samples

Mareks disease (MD) is a lymphoproliferative disease of chickens caused by airborne gallid herpesvirus type 2 (GaHV-2, aka MDV-1). Mature virions are formed in the feather follicle epithelium cells of infected chickens from which the virus is shed as fine particles of skin and feather debris, or poultry dust. Poultry dust is the major source of virus transmission between birds in agricultural settings. Despite both clinical and laboratory data that show increased virulence in field isolates of MDV-1 over the last 40 years, we do not yet understand the genetic basis of MDV-1 pathogenicity. Our present knowledge on genome-wide variation in the MDV-1 genome comes exclusively from laboratory-grown isolates. MDV-1 isolates tend to lose virulence with increasing passage number in vitro, raising concerns about their ability to accurately reflect virus in the field. The ability to rapidly and directly sequence field isolates of MDV-1 is critical to understanding the genetic basis of rising virulence in circulating wild strains. Here we present the first complete genomes of uncultured, field-isolated MDV-1. These five consensus genomes were derived directly from poultry dust or single chicken feather follicles without passage in cell culture. These sources represent the shed material that is transmitted to new hosts, vs. the virus produced by a point source in one animal. We developed a new procedure to extract and enrich viral DNA, while reducing host and environmental contamination. DNA was sequenced using Illumina MiSeq high-throughput approaches and processed through a recently described bioinformatics workflow for de novo assembly and curation of herpesvirus genomes. We comprehensively compared these genomes to one another and also to previously described MDV-1 genomes. The field-isolated genomes had remarkably high DNA identity when compared to one another, with few variant proteins between them. In an analysis of genetic distance, the five new field genomes grouped separately from all previously described genomes. Each consensus genome was also assessed to determine the level of polymorphisms within each sample, which revealed that MDV-1 exists in the wild as a polymorphic population. By tracking a new polymorphic locus in ICP4 over time, we found that MDV-1 genomes can evolve in short period of time. Together these approaches advance our ability to assess MDV-1 variation within and between hosts, over time, and during adaptation to changing conditions.

Microbiology↗

bioRxiv2016

Triclosan Exposure is Associated with Rapid Restructuring of the Microbiome in Adult Zebrafish

Growing evidence indicates that disrupting the microbial community that comprises the intestinal tract, known as the gut microbiome, can contribute to the development or severity of disease. As a result, it is important to discern the agents responsible for microbiome disruption. While animals are frequently exposed to a diverse array of environmental chemicals, little is known about their effects on gut microbiome stability and structure. Here, we demonstrate how zebrafish can be used to glean insight into the effects of environmental chemical exposure on the structure and ecological dynamics of the gut microbiome. Specifically, we exposed forty-five adult zebrafish to triclosan-laden food for four or seven days or a control diet, and analyzed their microbial communities using 16S rRNA amplicon sequencing. Triclosan exposure was associated with rapid shifts in microbiome structure and diversity. We find evidence that several operational taxonomic units (OTUs) associated with the family Enterobacteriaceae appear to be susceptible to triclosan exposure, while OTUs associated with the genus Pseudomonas appeared to be more resilient and resistant to exposure. We also found that triclosan exposure is associated with topological alterations to microbial interaction networks and results in an overall increase in the number of negative interactions per microbe in these networks. Together these data indicate that triclosan exposure results in altered composition and ecological dynamics of microbial communities in the gut. Our work demonstrates that because zebrafish afford rapid and inexpensive interrogation of a large number of individuals, it is a useful experimental system for the discovery of the gut microbiomes interaction with environmental chemicals.

Microbiology↗

bioRxiv2016

Transhydrogenase and growth substrate influence lipid hydrogen isotope ratios in Desulfovibrio alaskensis G20

Microbial fatty acids preserve metabolic and environmental information in their hydrogen isotope ratios (2H/1H). This ratio is influenced by parameters that include the 2H/1H of water in the microbial growth environment, and biosynthetic fractionations between water and lipid. In some microbes, this biosynthetic fractionation has been shown to vary systematically with central energy metabolism, and controls on fatty acid 2H/1H may be linked to the intracellular production of NADPH. We examined the apparent fractionation between media water and the fatty acids produced by Desulfovibrio alaskensis G20. Growth was in batch culture with malate as an electron donor for sulfate respiration, and with pyruvate and fumarate as substrates for fermentation and for sulfate respiration. A larger fractionation was observed as a consequence of respiratory or fermentative growth on pyruvate than growth on fumarate or malate. This difference correlates with opposite apparent flows of electrons through the electron bifurcating/confurcating transhydrogenase NfnAB. When grown on malate or fumarate, mutant strains of D. alaskensis G20 containing transposon disruptions in a copy of nfnAB show different fractionations than the wild type strain. This phenotype is muted during fermentative growth on pyruvate, and it is absent when pyruvate is a substrate for sulfate reduction. All strains and conditions produced similar fatty acid profiles, and the 2H/1H of individual lipids changed in concert with the mass-weighted average. Unsaturated fatty acids were generally depleted in 2H relative to their saturated homologues, and anteiso-branched fatty acids were generally depleted in 2H relative to straight-chain fatty acids. Fractionation correlated with growth rate, a pattern that has also been observed in the fractionation of sulfur isotopes during dissimilatory sulfate reduction by sulfate reducing bacteria.

Microbiology↗

bioRxiv2016

16S rRNA amplicon sequencing for epidemiological surveys of bacteria in wildlife: the importance of cleaning post-sequencing data before estimating positivity, prevalence and co-infection

ImportanceSeveral recent public health crises have shown that the surveillance of zoonotic agents in wildlife is important to prevent pandemic risks. Rodents are intermediate hosts for numerous zoonotic bacteria. High-throughput sequencing (HTS) technologies are very useful for the detection and surveillance of zoonotic bacteria, but rigorous experimental processes are required for the use of these cheap and effective tools in such epidemiological contexts. In particular, HTS introduces biases into the raw dataset that might lead to incorrect interpretations. We describe here a procedure for cleaning data before estimating reliable biological parameters, such as bacterial positivity, prevalence and coinfection, by 16S rRNA amplicon sequencing on the MiSeq platform. This procedure, applied to 711 commensal rodents collected from 24 villages in Senegal, Africa, detected several emerging bacterial genera, some in high prevalence, while never before reported for West Africa. This study constitutes a step towards the use of HTS to improve our understanding of the risk of zoonotic disease transmission posed by wildlife, by providing a new strategy for the use of HTS platforms to monitor both bacterial diversity and infection dynamics in wildlife. In the future, this approach could be adapted for the monitoring of other microbes such as protists, fungi, and even viruses.\n\nSummaryHuman impact on natural habitats is increasing the complexity of human-wildlife interfaces and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential of recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of large numbers of samples, to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to eliminate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a trimming strategy for managing them and cleaning the dataset. We also analyzed 711 commensal rodents collected from 24 villages in Senegal, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis and 221 Mastomys erythroleucus. Seven major genera of pathogenic bacteria were detected: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus and Orientia. The last five of these genera have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90%, depending on the bacterial taxon, rodent species and site considered, and a mean of 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial pathogens without requiring assumptions about their presence in the samples. This approach is, thus, particularly suitable for continuous pathogen surveillance in the framework of disease monitoring programs

Microbiology↗

bioRxiv2016

Inducible colistin resistance via a disrupted plasmid-borne mcr-1 gene in a 2008 Vietnamese Shigella sonnei isolate

The mcr-1 gene, which confers resistance against the last-resort antimicrobial colistin, was recently discovered in Enterobacteriaceae circulating in China. Through genome sequencing we identified a plasmid-associated inactive form of mcr-1 in a 2008 Vietnamese isolate of Shigella sonnei. The plasmid was conjugated into E. coli and mcr-1 was activated upon exposure to colistin, suggesting the gene has been circulating in human-restricted pathogens for some time but carries a selective fitness cost.

Microbiology↗

bioRxiv2016

Working with Zika and Usutu Viruses in Vitro

Usutu and Zika viruses are emerging arboviruses of significant medical and veterinary importance. These viruses have not been studied as well as other medically important arboviruses such as West Nile, dengue, or chikungunya viruses. As such, information regarding the behavior of Zika and Usutu viruses in the laboratory is dated. Usutu virus re-emerged in Austria in 2001 and has since spread throughout the European and Asian continents causing significant mortality among birds. Zika virus has recently appeared in the Americas and has exhibited unique characteristics of pathogenesis, including birth defects, and transmission. Information about the characteristics of Usutu and Zika viruses are needed to better understand the transmission, dispersal, and adaptation of these viruses in new environments. Since their initial characterization in the middle of last century, technologies and reagents have been developed that could enhance our abilities to study these pathogens. Currently, standard laboratory methods for these viruses are limited to 2-3 cell lines and many assays take several days to generate meaningful data. The goal of this study was to characterize these viruses in cell culture to provide some basic parameters to further their study. Cell lines from 17 species were permissive to both Zika and Usutu viruses. These viruses were able to replicate to significant titers in most of the cell lines tested. Moreover, cytopathic effects were observed in 8 of the cell lines tested. The data show that, unlike other flaviviruses, neither Zika nor Usutu viruses require an acidic environment to fuse with a host cell. This may provide a tool to help characterize events or components in the flavivirus fusion process. These data indicate that a variety of cell lines can be used to study Zika and Usutu viruses and may provide an updated foundation for the study of host-pathogen interactions, model development, and the development of therapeutics.\n\nAuthor SummaryUsutu and Zika viruses are arboviruses of identified in significant medical and veterinary outbreaks in recent years. Currently, standard laboratory methods for these viruses are limited to 2-3 cell lines and basic viral characterization has not been performed since the mid-20th century. Zika and Usutu viruses were characterized in cell culture. The data show that a variety of cell lines can be used to study the viruses. Neither Zika nor Usutu viruses require an acidic environment for host cell infection.

Microbiology↗

bioRxiv2016

Extrusions promote engulfment and Chlamydia survival within macrophages

All obligate intracellular pathogens must exit their host cells in order to propagate and survive as a species; the precise strategies they use have a direct impact on their ability to disseminate within a host, transmit to new hosts, and engage or avoid immune responses. The obligate intracellular bacterium Chlamydia trachomatis exits the host cell by two distinct exit strategies, lysis and extrusion. Despite being equally active pathways, lysis and extrusion differ greatly in their mechanisms. The defining characteristics of extrusions, and advantages gained by Chlamydia within this unique double-membrane structure are not well understood. Here, we present data that defines extrusions as being largely devoid of host organelles, comprised mostly of Chlamydia elementary bodies, and containing phosphatidylserine on the outer surface of the extrusion membrane. Towards defining a functional role for extrusions in Chlamydia pathogenesis, we demonstrate that extrusions confer significant infectious advantages for Chlamydia by serving as transient, intracellular-like niches for extracellular Chlamydia, as compared to Chlamydia that would otherwise exit by lysing the host cell. In addition to enhanced survival outside of the host cell, we report the key discovery that chlamydial extrusions can be engulfed by primary bone marrow-derived macrophages, after which they provide a protective microenvironment for Chlamydia. Extrusion-derived Chlamydia were able to stave off macrophage based killing beyond 8 h, and culminated in the release of infectious EB from the macrophage. Based on these findings, we propose a model in which a major outcome of Chlamydia exiting epithelial cells inside extrusions is to hijack macrophages as vehicles for dissemination within the host.

Microbiology↗

bioRxiv2016

South Asia as a reservoir for the global spread of ciprofloxacin resistant Shigella sonnei

BackgroundAntimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin resistant S. sonnei are being increasingly isolated in Asia, and sporadically reported on other continents.\n\nMethods and FindingsHypothesising that Asia is the hub for the recent international spread of ciprofloxacin resistant S. sonnei, we performed whole genome sequencing on a collection of contemporaneous ciprofloxacin resistant S. sonnei isolated in six countries from within and outside of Asia. We reconstructed the recent evolutionary history of these organisms and combined these data with their geographical location of isolation. Placing these sequences into a global phylogeny we found that all ciprofloxacin resistant S. sonnei formed a single clade within a Central Asian expansion of Lineage III. Further, our data show that resistance to ciprofloxacin within S. sonnei can be globally attributed to a single clonal emergence event, encompassing sequential gyrA-S83L, parC-S80I and gyrA-D87G mutations. Geographical data predict that South Asia is the likely primary source of these organisms, which are being regularly exported across Asia and intercontinentally into Australia, the USA and Europe.\n\nConclusionsThis study shows that a single clone, which is widespread in South Asia, is driving the current intercontinental surge of ciprofloxacin resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial resistant S. sonnei, and provides a tractable model for studying how antimicrobial resistant Gram-negative community acquired pathogens spread globally.

Microbiology↗

bioRxiv2016

Productive infection of field strains of avian coronavirus infectious bronchitis virus in chicken peripheral blood-derived monocyte

The avian coronavirus infectious bronchitis virus (IBV) typically infects the respiratory tract of chickens, but can also spread to other organs. However, the mechanisms of virus dissemination are presently unclear. We show that peripheral blood-derived monocytes/macrophages from chickens (chPBMC) are productively infected by clinical strains of IBV, accompanied by induction of apoptosis. Our data suggest that chPBMCs play a role in the dissemination of IBV, and may be important for viral pathogenesis.

Microbiology↗

bioRxiv2016

Twenty five new viruses associated with the Drosophilidae (Diptera)

Drosophila melanogaster is an important laboratory model for studies of antiviral immunity in invertebrates, and Drosophila species provide a valuable system to study virus host range and host switching. Here we use metagenomic RNA sequencing of ca. 1600 adult flies to discover 25 new RNA viruses associated with six different drosophilid hosts in the wild. We also provide a comprehensive listing of viruses previously reported from the Drosophilidae. The new viruses include Iflaviruses, Rhabdoviruses, Nodaviruses, and Reoviruses, and members of unclassified lineages distantly related to Negeviruses, Sobemoviruses and Poleroviruses, Flaviviridae, and Tombusviridae. Among these are close relatives of Drosophila X virus and Flock House virus, which we find in association with wild Drosophila immigrans. These two viruses are widely used in experimental studies but have not previously been reported to naturally infect Drosophila. Although we detect no new DNA viruses, in D. immigrans and D. obscura we identify sequences very closely related to Armadillidium vulgare Iridescent virus (Invertebrate Iridescent virus 31), bringing the total number of DNA viruses found in the Drosophilidae to three.

Microbiology↗

bioRxiv2016

The viral polymerase inhibitor 7-deaza-2’-C-methyladenosine is a potent inhibitor of in vitro Zika virus replication and delays disease progression in a robust mouse infection model

Zika virus (ZIKV) is an emerging flavivirus typically causing a dengue-like febrile illness, but neurological complications, such as microcephaly in newborns, have potentially been linked to this viral infection. We established a panel of in vitro assays to allow the identification of ZIKV inhibitors and demonstrate that the viral polymerase inhibitor 7-deaza-2-C-methyladenosine (7DMA) efficiently inhibits replication. Infection of AG129 (IFN-/{beta} and IFN-{gamma} receptor knockout) mice with ZIKV resulted in acute neutrophilic encephalitis with viral antigens accumulating in neurons of the brain and spinal cord. Additionally, high levels of viral RNA were detected in the spleen, liver and kidney, and levels of IFN-{gamma} and IL-18 were systematically increased in serum of ZIKV-infected mice. Interestingly, the virus was also detected in testicles of infected mice. In line with its in vitro anti-ZIKV activity, 7DMA reduced viremia and delayed virus-induced morbidity and mortality in infected mice, which also validates this small animal model to assess the in vivo efficacy of novel ZIKV inhibitors. Since AG129 mice can generate an antibody response, and have been used in dengue vaccine studies, the model can also be used to assess the efficacy of ZIKV vaccines.\n\nArticle Summary LineA robust cell-based antiviral assay was developed that allows to screen for and validate novel inhibitors of Zika virus (ZIKV) replication. The viral polymerase inhibitor 7-deaza-2-C-methyladenosine (7DMA) was identified as a potent ZIKV inhibitor. A mouse model for ZIKV infections, which was validated for antiviral studies, demonstrated that 7DMA markedly delays virus-induced disease in this model.

Microbiology↗

bioRxiv2016

A susceptible mouse model for Zika virus infection

Zika virus (ZIKV) is a mosquito-borne pathogen which has recently spread beyond Africa and into Pacific and South American regions. Despite first being detected in 1947, very little information is known about the virus and its spread has been associated with increases in Guillain-Barre syndrome and microcephaly. There are currently no known vaccines or antivirals against ZIKV infection. Progress in assessing interventions will require the development of animal models to test efficacies; however, there are only limited reports on in vivo studies. The only susceptible murine models have involved intracerebral inoculations or juvenile animals, which do not replicate natural infection. Our report has studied the effect of ZIKV infection in type-I interferon receptor deficient (A129) mice and the parent strain (129Sv/Ev) after subcutaneous challenge in the lower leg to mimic a mosquito bite. A129 mice developed severe symptoms with viral RNA being detected widespread in the blood, brain, spleen, liver and ovaries. Histological changes were also striking in these animals. 129Sv/Ev mice developed no clinical symptoms or histological changes, despite viral RNA being detectable in the blood, spleen and ovaries, albeit at lower levels to those seen in A129 mice. Our results identify A129 mice as being highly susceptible to ZIKV and thus a suitable small animal model for the testing of vaccines and antivirals which are urgently required.\n\nAuthor summarySince first being recognised in 1947, Zika virus (ZIKV) has mainly been a mild illness with symptoms including a limited fever and rash. In 2007 the virus spread from Africa into French Polynesia and then onwards across Pacific regions and into South America. In these new regions, ZIKV has been associated with more severe clinical conditions including Gullain-Barre syndrome and microcephaly. There are no currently approved antivirals or vaccines available with proven activity against ZIKV, and the World Health Organisation declared the spread of ZIKV as a Public Health Emergency of International Concern. Here, we have used a mouse strain with a deficiency in the type-I interferon receptor (A129) and shown that they are susceptible to ZIKV infection after inoculation that closely resembles the natural route via mosquito bite. Although A129 mice are deficient in the innate interferon response, they retain their adaptive immunity and thus have successfully been used as suitable models for the testing of vaccinations and antivirals. Our study provides details on a suitable model for the testing of future interventions against ZIKV.

Microbiology↗

bioRxiv2016

Functional metagenomics using Pseudomonas putida expands the known diversity of polyhydroxyalkanoate synthases and enables the production of novel polyhydroxyalkanoate copolymers

Bacterially produced biodegradable polyhydroxyalkanoates with versatile properties can be achieved using different PHA synthase enzymes. This work aims to expand the diversity of known PHA synthases via functional metagenomics, and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either Class I, Class II or unclassified PHA synthases, and many did not have close sequence matches to known PHA synthases. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of SCL-and MCL-PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics, and their use for the production of a variety of PHA polymer and copolymer mixtures.

Microbiology↗

bioRxiv2016

Adaptive significance of quorum sensing-dependent regulation of public goods by integration of growth rate: a trade-off between survival and efficiency

Quorum sensing (QS) is a mechanism of communication used by bacteria to monitor cell density and coordinate cooperative behaviors. An emerging framework is that the adaptive significance of QS in regulation of production of costly extracellular metabolites (public goods) is to maintain the homeostasis of cooperation. We investigated the functionality of QS-dependent regulation of rhamnolipids, extracellular surface-active glycolipids promoting the social swarming motility behavior, in Burkholderia glumae and found that QS is superfluous under rich nutritional conditions. In contrast, decreasing nutrient concentrations to reduce the growth rate amplifies rhamnolipid biosynthesis gene expression, revealing a system where QS-dependent regulation is triggered by the growth rate of the population rather than by its cell density. Our results provide evidence that the adaptive significance of QS in regulation of public goods is to maintain an optimized demand-driven supply of target cooperative behavior, wherein efficiency can be traded off against survival.

Microbiology↗

bioRxiv2016

Application of database-independent approach to assess the quality of OTU picking methods

Assigning 16S rRNA gene sequences to operational taxonomic units (OTUs) allows microbial ecologists to overcome the inconsistencies and biases within bacterial taxonomy and provides a strategy for clustering similar sequences that do not have representatives in a reference database. I have applied the Matthews correlation coefficient to assess the ability of 15 reference-independent and -dependent clustering algorithms to assign sequences to OTUs. This metric quantifies the ability of an algorithm to reflect the relationships between sequences without the use of a reference and can be applied to any dataset or method. The most consistently robust method was the average neighbor algorithm; however, for some datasets other algorithms matched its performance.

Microbiology↗