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bioRxiv2017

Mosquito lipids regulate Plasmodium sporogony and infectivity to the mammalian host

Malaria is a fatal human parasitic disease transmitted by a mosquito vector. The evolution of within-host malaria virulence has been the focus of many empirical and theoretical studies. However, the vectors contribution to virulence evolution is not well understood. Here we explored how within-vector resource exploitation impacts evolutionary trajectories of within-host Plasmodium virulence. We developed a nested model of within-vector dynamics and malaria epidemiology, which predicted that non-competitive resource exploitation within-vector restricts within-host parasite virulence. To validate our model, we experimentally manipulated mosquito lipid trafficking and gauged within-vector parasite development, within-host infectivity and virulence. We found that mosquito-derived lipids determine within-host parasite virulence by shaping development and metabolic activity of transmissible sporozoites. Our findings uncover the role of within-vector environment in regulating within-host Plasmodium virulence and identify Plasmodium metabolic traits that may contribute to the evolution of malaria virulence.

microbiology↗

bioRxiv2017

Phylogeographic separation and formation of sexually discreet lineages in a global population of Yersinia pseudotuberculosis

Yersinia pseudotuberculosis is a Gram negative intestinal pathogen of humans and has been responsible for several nation-wide gastro-intestinal outbreaks. Large-scale population genomic studies have been performed on the other human pathogenic Yersinia, Y. pestis and Y. enterocolitica allowing a high-resolution understanding of the ecology, evolution and dissemination of these pathogens. However, to date no large-scale global population genomic analysis of Y. pseudotuberculosis has been performed. Here we present analyses of the genomes of 134 strains of Y. pseudotuberculosis isolated from around the world, from multiple ecosystems since 1960s. Our data display a phylogeographic split within the population, with an Asian ancestry and subsequent dispersal of successful clonal lineages into Europe and the rest of the world. These lineages can be differentiated by CRISPR cluster arrays, and we show that the lineages are limited with respect to inter-lineage genetic exchange. This restriction of genetic exchange maintains the discrete lineage structure in the population despite co-existence of lineages for thousands of years in multiple countries. Our data highlights how CRISPR can be informative of the evolutionary trajectory of bacterial lineages, and merits further study across bacteria.

microbiology↗

bioRxiv2017

Saccharomyces cerevisiae strain YH166: a novel wild yeast for the production of tropical fruit sensory attributes in fermented beverages

All ales are fermented by various strains of Saccharomyces cerevisiae. However, recent whole-genome sequencing has revealed that most commercially available ale yeasts are highly related and represent a small fraction of the genetic diversity found among S. cerevisiae isolates as a whole. This lack of diversity limits the phenotypic variations between these strains, which translates into a limited number of sensory compounds created during fermentation. Here, we characterized a collection of wild S. cerevisiae, S. kudriavzevii, and S. paradoxus strains for their ability to ferment wort into beer. Although many isolates performed well, S. cerevisiae strain YH166 was the most promising, displaying excellent fermentation kinetics and attenuation, as well as a tropical fruit sensory profile. Use of this strain in multiple styles of beer suggested that it is broadly applicable in the brewing industry. Thus, YH166 is a novel ale strain that can be used to lend fruity esters to beer and should pair well with citrusy hops in hop-forward ales.

microbiology↗

bioRxiv2017

Controlling microbial communities: a theoretical framework

Microbes comprise nearly half of all biomass on Earth. Almost every habitat on Earth is teeming with microbes, from hydrothermal vents to the human gastrointestinal tract. Those microbes form complex communities and play critical roles in maintaining the integrity of their environment or the well-being of their hosts. Controlling microbial communities can help us restore natural ecosystems and maintain healthy human microbiota. Yet, our ability to precisely manipulate microbial communities has been fundamentally impeded by the lack of a systematic framework to control them. Here we fill this gap by developing a control framework based on the new notion of structural accessibility. This framework allows identifying minimal sets of \"driver species\" through which we can achieve feasible control of the entire microbial community. We numerically validate our control framework on large microbial communities, and then we demonstrate its application for controlling the gut microbiota of gnotobiotic mice infected with Clostridium difficile and the core microbiota of the sea sponge Ircinia oros.

microbiology↗

bioRxiv2017

Distribution and inheritance of a gene cluster encoding a sulfated tyrosine peptide in Xanthomonas spp.

The rice XA21-mediated immune response is activated upon recognition of the RaxX peptide produced by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The 60 residue RaxX precursor is posttranslationally modified to form a sulfated tyrosine peptide that shares sequence and functional similarity with the plant sulfated tyrosine (PSY) peptide hormones. The five kb raxX-raxSTAB gene cluster of Xoo encodes RaxX, the RaxST tyrosylprotein sulfotransferase, and the RaxA and RaxB components of a predicted type one secretion system. The identified the complete raxX-raxSTAB gene cluster is present only in Xanthomonas spp., in five distinct lineages in addition to X. oryzae. The phylogenetic distribution of the raxX-raxSTAB gene cluster is consistent with the occurrence of multiple lateral transfer events during Xanthomonas speciation. RaxX variants representing each of the five lineages, and three Xoo RaxX variants, fail to activate the XA21-mediated immune response yet retain peptide hormone activity. These RaxX variants contain a restricted set of missense mutations, consistent with the hypothesis that selection acts to maintain peptide hormone-like function. These observations are also consistent with the hypothesis that the XA21 receptor evolved specifically to recognize Xoo RaxX.

microbiology↗

bioRxiv2017

Enterococcus faecalis and pathogenic streptococci inactivate daptomycin by releasing phospholipids

Daptomycin is a lipopeptide antibiotic with activity against Gram-positive bacteria. We have shown previously that Staphylococcus aureus can survive daptomycin exposure by releasing membrane phospholipids that inactivate the antibiotic. To determine whether other pathogens possess this defence mechanism, phospholipid release and daptomycin activity were measured after incubation of Staphylococcus epidermidis, Group A or B streptococci, Streptococcus gordonii or Enterococcus faecalis with the antibiotic. All bacteria released phospholipid in response to daptomycin, which resulted in at least partial inactivation of the antibiotic. However, E. faecalis showed the highest levels of lipid release and daptomycin inactivation. As shown previously for S. aureus, phospholipid release by E. faecalis was inhibited by the lipid biosynthesis inhibitor platensimycin. In conclusion, several pathogenic Gram-positive bacteria, including E. faecalis, inactivate daptomycin by releasing phospholipids, which may contribute to the failure of daptomycin to resolve infections caused by these pathogens.

microbiology↗

bioRxiv2017

Ecophysiology of freshwater Verrucomicrobia inferred from genomes recovered through time-series metagenomics

Microbes are critical in carbon and nutrient cycling in freshwater ecosystems. Members of the Verrucomicrobia are ubiquitous in such systems, yet their roles and ecophysiology are not well understood. In this study, we recovered 19 Verrucomicrobia draft genomes by sequencing 184 time-series metagenomes from a eutrophic lake and a humic bog that differ in carbon source and nutrient availabilities. These genomes span four of the seven previously defined Verrucomicrobia subdivisions, and greatly expand the known genomic diversity of freshwater Verrucomicrobia. Genome analysis revealed their potential role as (poly)saccharide-degraders in freshwater, uncovered interesting genomic features for this life style, and suggested their adaptation to nutrient availabilities in their environments. Between the two lakes, Verrucomicrobia populations differ significantly in glycoside hydrolase gene abundance and functional profiles, reflecting the autochthonous and terrestrially-derived allochthonous carbon sources of the two ecosystems respectively. Interestingly, a number of genomes recovered from the bog contained gene clusters that potentially encode a novel porin-multiheme cytochrome c complex and might be involved in extracellular electron transfer in the anoxic humic-rich environment. Notably, most epilimnion genomes have large numbers of so-called \"Planctomycete-specific\" cytochrome c-containing genes, which exhibited nearly opposite distribution patterns with glycoside hydrolase genes, probably associated with the different environmental oxygen availability and carbohydrate complexity between lakes/layers. Overall, the recovered genomes are a major step towards understanding the role, ecophysiology and distribution of Verrucomicrobia in freshwater.\n\nIMPORTANCEFreshwater Verrucomicrobia are cosmopolitan in lakes and rivers, yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia are restricted to one subdivision of this phylum. Here, we greatly expand the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia as potential (poly)saccharide-degraders, and suggested their adaptation to carbon source of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called \"Planctomycete-specific\" cytochrome c-containing genes, and found their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology and distribution of freshwater Verrucomicrobia, while highlighting their potential role in freshwater carbon cycling.

microbiology↗

bioRxiv2017

Bacteria defend against phages through type IV pilus glycosylation

Bacterial surface structures such as type IV pili are common receptors for phage. Strains of the opportunistic pathogen Pseudomonas aeruginosa express one of five different major type IV pilin alleles, two of which are glycosylated with either lipopolysaccharide O-antigen units or polymers of D-arabinofuranose. Here we show that both these post-translational modifications protect P. aeruginosa from a variety of pilus-specific phages. We identified a phage capable of infecting strains expressing both non-glycosylated and glycosylated pilins, and through construction of a chimeric phage, traced this ability to its unique tail proteins. Alteration of pilin sequence, or masking of binding sites by glycosylation, both block phage infection. The energy invested by prokaryotes in glycosylating thousands of pilin subunits is thus explained by the protection against phage predation provided by these common decorations.\n\nSIGNIFICANCEPost-translational modification of bacterial and archaeal surface structures such as pili and flagella is widespread, but the function of these decorations is not clear. We propose that predation by bacteriophages that use these structures as receptors selects for strains that mask potential phage binding sites using glycosylation. Phages are of significant interest as alternative treatments for antibiotic-resistant pathogens, but the ways in which phage interact with host receptors are not well understood. We show that specific phage tail proteins allow for infection of strains with glycosylated pili, providing a foundation for the creation of designer phages that can circumvent first-line bacterial defenses.

microbiology↗

bioRxiv2017

Generation of Arctic-like Rabies Viruses Containing Chimeric Glycoproteins Enables Serological Potency Studies.

Rabies viruses have the highest case fatality rate of any known virus and are responsible for an estimated 60,000 deaths each year. This is despite the fact that there are highly efficacious vaccines and postexposure prophylaxis available. However, while it is assumed these biologics provide protection against all rabies virus isolates, there are certain subdivisions of RABV lineages, such as within the Arctic-like RABV (AL rabies virus lineage, where data is limited and thus the potency of existing biologics has not been thoroughly assessed. By fusing the Arctic-like rabies virus envelope glycoprotein ecto- and transmembrane domains with the vesicular stomatitis virus cytoplasmic domain, a high titre (7.7 x 105 - 6.1 x 106 RLU/ml) pseudotyped virus was generated that was subsequently used in a pseudotyped virus neutralisation assay. These results showed that Arctic-like rabies viruses are neutralised to human, canine and feline vaccines and human post-exposure prophylaxis and this was not influenced by the swapping of the cytoplasmic domains (CVS-11 vs CVS-11etmVSVc; r = 0.99, p < 0.0001). This study supports the concept that rabies virus vaccines and newly identified mAbs are able to neutralise rabies virus variants that cluster in a monophyletic clade, referred to as phylogroup I lyssaviruses.

microbiology↗

bioRxiv2017

Campylobacter jejuni transcriptome changes during loss of culturability in water

BackgroundThe natural environment serves as a potential reservoir for Campylobacter, the leading cause of bacterial gastroenteritis in humans. However, little is understood about the mechanisms underlying variations in survival characteristics between different strains of C. jejuni in natural environments, including water.\n\nResultsWe identified three Campylobacter jejuni strains that exhibited variability in their ability to retain culturability after suspension in water at two different temperatures (4{degrees}C and 25{degrees}C). Of the three, strains C. jejuni M1 exhibited the most rapid loss of culturability whilst retaining viability. Using RNAseq transcriptomics, we characterised C. jejuni M1 gene expression in response to suspension in water by analyzing bacterial suspensions recovered immediately after introduction into water (Time 0), and from two sampling time/temperature combinations where considerable loss of culturability was evident, namely (i) after 24 h at 25{degrees}C, and (ii) after 72 h at 4{degrees}C. Transcript data were compared with a culture-grown control. Some gene expression characteristics were shared amongst the three populations recovered from water, with more genes being up-regulated than down. Many of the up-regulated genes were identified in the Time 0 sample, whereas the majority of down-regulated genes occurred in the 25{degrees}C (24 h) sample.\n\nConclusionsVariations in expression were found amongst genes associated with oxygen tolerance, starvation and osmotic stress. However, we also found upregulation of flagellar assembly genes, accompanied by down-regulation of genes involved in chemotaxis. Our data also suggested a switch from secretion via the sec system to via the tat system, and that the quorum sensing gene luxS may be implicated in the survival of strain M1 in water. Variations in gene expression also occurred in accessory genome regions. Our data suggest that despite the loss of culturability, C. jejuni M1 remains viable and adapts via specific changes in gene expression.

microbiology↗

bioRxiv2017

Phenotypic consequences of RNA polymerase dysregulation in Escherichia coli

Many bacterial adaptive responses to changes in growth conditions due to biotic and abiotic factors involve reprogramming of gene expression at the transcription level. The bacterial RNA polymerase (RNAP), which catalyzes transcription, can thus be considered as the major mediator of cellular adaptive strategies. But how do bacteria respond if a stress factor directly compromises the activity of the RNAP? We used a phage-derived small protein to specifically perturb bacterial RNAP activity in exponentially growing Escherichia coli. Using cytological profiling, tracking RNAP behavior at single-molecule level and transcriptome analysis, we reveal that adaptation to conditions that directly perturb bacterial RNAP performance can result in a biphasic growth behavior and thereby confer the adapted bacterial cells an enhanced ability to tolerate diverse antibacterial stresses. The results imply that while synthetic transcriptional rewiring may confer bacteria with the intended desirable properties, such approaches may also collaterally allow them to acquire undesirable traits.

microbiology↗

bioRxiv2017

Construction of soil defined media using quantitative exometabolomic analysis of soil metabolites

Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil isolates. Environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil metabolites. To broadly characterize soil metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids, osmolytes, nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Water soluble organic carbon was fractionated by molecular weight and measured to determine the fraction of carbon accounted for by the quantified metabolites. This revealed that, community structures, these soil metabolites have an uneven quantitative distribution, with a single metabolite, trehalose accounting for 9.9 percent of much like soil microbial the (< 1 kDa) water extractable organic carbon. This quantitative information was used to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate SDM for supporting the growth of bacteria found at this field site, we examined the growth of 30 phylogenetically diverse soil isolates obtained using standard R2A medium. The simpler SDM1 supported the growth of up to 13 isolates while the more complex SDM2 supported up to 25 isolates. One isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series exometabolomics analysis to investigate SDM1 substrate preferences. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a tractable environmentally relevant media. We anticipate that this approach can be expanded to other environments to enhance isolation and characterization of diverse microbial communities.\n\nHighlights O_LILC/MS and GC/MS analyses of soil extracts revealed a diversity of 96 metabolites.\nC_LIO_LISoil defined media were constructed based on water extractable soil metabolomics data.\nC_LIO_LIThe defined media supported the growth of 25 out of 30 bacterial isolates.\nC_LIO_LIExometabolomics demonstrated preferential consumption of amino acids for one isolate.\nC_LIO_LIThese media can be used to understand environmentally relevant microbial substrate preferences.\nC_LI\n\nAbbreviationsDOM dissolved organic matter; SOM soil organic matter; WEOC water extractable organic carbon; LMWOS low molecular weight organic substances; SDM(1/2) soil defined media (1 and 2); R2A Reasoners 2A agar medium; ORFRC Oak Ridge Field Research Center; LC/MS liquid chromatography mass spectrometry; GC/MS gas chromatography/ mass spectrometry; TOC total organic carbon; HILIC hydrophilic interaction liquid chromatography

microbiology↗

bioRxiv2017

EsaB is a core component of the Staphylococcus aureus Type VII secretion system

Type VII secretion systems (T7SS) are found in many bacteria and secrete proteins involved in virulence and bacterial competition. In Staphylococcus aureus the small ubiquitin-like EsaB protein has been previously implicated as having a regulatory role in the production of the EsxC substrate. Here we show that in the S. aureus RN6390 strain, EsaB does not genetically regulate production of any T7 substrates or components, but is indispensable for secretion activity. Consistent with EsaB being a core component of the T7SS, loss of either EsaB or EssC are associated with upregulation of a common set of iron acquisition genes. However, a further subset of genes were dysregulated only in the absence of EsaB. In addition, fractionation revealed that although an EsaB fusion to yellow fluorescent protein partially localised to the membrane, it was still membrane-localised when the T7SS was absent. Taken together our findings suggest that EsaB has T7SS-dependent and T7SS-independent roles in S. aureus.

microbiology↗

bioRxiv2017

Increased ParB level affects expression of stress response, adaptation and virulence operons and potentiates repression of promoters adjacent to the high affinity binding sites parS3 and parS4 in Pseudomonas aeruginosa

Similarly to its homologs in other bacteria, Pseudomonas aeruginosa partitioning protein ParB facilitates segregation of newly replicated chromosomes. Lack of ParB is not lethal but results in increased frequency of anucleate cells production, longer division time, cell elongation, altered colony morphology and defective swarming and swimming motility. Unlike in other bacteria, inactivation of parB leads to major changes of the transcriptome, suggesting that, directly or indirectly, ParB plays a role in regulation of gene expression in this organism.\n\nParB overproduction affects growth rate, cell division and motility in a similar way as ParB deficiency. To identify primary ParB targets, here we analysed the impact of a slight increase in ParB level on P. aeruginosa transcriptome. ParB excess, which does not cause changes in growth rate and chromosome segregation, significantly alters the expression of 176 loci. Most notably, the mRNA level of genes adjacent to high affinity ParB binding sites parS1-4 close to oriC is reduced. Conversely, in cells lacking either parB or functional parS sequences the orfs adjacent to parS3 and parS4 are upregulated, indicating that direct ParB-parS3/parS4 interactions repress the transcription in this region. In addition, increased ParB level brings about repression or activation of numerous genes including several transcriptional regulators involved in SOS response, virulence and adaptation. Overall, our data support the role of partitioning protein ParB as a transcriptional regulator in Pseudomonas aeruginosa.

microbiology↗

bioRxiv2017

Metagenomics reveals niche partitioning within the phototrophic zone of a microbial mat

Hypersaline photosynthetic microbial mats are stratified microbial communities known for their taxonomic and metabolic diversity and strong light-driven day-night environmental gradients. In this study of the upper photosynthetic zone of hypersaline microbial mats of Elkhorn Slough, California (USA), we show how reference-based and reference-free methods can be used to meaningfully assess microbial ecology and genetic partitioning in these complex microbial systems. Mapping of metagenome reads to the dominant Cyanobacteria observed in the system, Coleofasciculus (Microcoleus) chthonoplastes, was used to examine strain variants within these metagenomes. Highly conserved gene subsystems indicate a core genome for the species, and a number of variant genes and subsystems suggest strain level differentiation, especially for carbohydrate utilization. Metagenome sequence coverage binning was used to assess ecosystem partitioning of remaining microbes. Functional gene annotation of these bins (primarily of Proteobacteria, Bacteroidetes, and Cyanobacteria) recapitulated the known biogeochemical functions in microbial mats using a genetic basis, and also revealed evidence of novel functional diversity within the Gemmatimonadetes and Gammaproteobacteria. Combined, these two approaches show how genetic partitioning can inform biogeochemical partitioning of the metabolic diversity within microbial ecosystems.

microbiology↗

bioRxiv2017

Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico

Zika virus (ZIKV) is a mosquito-borne virus and Aedes agypti has been mentioned as the main vector of the disease. Other mosquito species in the Aedes and Culex genera have been suggested to have the potential for being competent vectors, based on experimental exposition of mosquitoes to an infectious blood meal containing ZIKV. Here, we report the isolation in cell culture of ZIKV from different body parts of wild-caught female mosquitoes (Ae. aegypti, Ae. vexans, Culex quinquefasciatus, Cx. coronator, and Cx. tarsalis) and whole male mosquitoes (Ae. aegypti and Cx. quinquefasciatus) in Mexico. Importantly, the virus was isolated from the salivary glands of all of these mosquitoes, strongly suggesting that these species are potential vectors for ZIKV.

microbiology↗

bioRxiv2017

A single point mutation in a TssB/VipA homolog disrupts sheath formation in the type VI secretion system of Proteus mirabilis

The type VI secretion (T6S) system is a molecular device for the delivery of proteins from one cell into another. T6S function depends on the contractile sheath comprised of TssB/VipA and TssC/VipB proteins. We previously reported on a mutant variant of TssB that disrupts T6S-dependent export of the self-identity protein, IdsD, in the bacterium Proteus mirabilis. Here we determined the mechanism underlying that initial observation. We show that T6S-dependent export of multiple self-recognition proteins is abrogated in this mutant strain. We have mapped the mutation, which is a single amino acid change, to a region predicted to be involved in the formation of the TssB-TssC sheath. We have demonstrated that this mutation does indeed inhibit sheath formation, thereby explaining the global disruption of T6S activity. We propose that this mutation could be utilized as an important tool for studying functions and behaviors associated with T6S systems.

microbiology↗

bioRxiv2017

VipA N-terminal linker and VipB-VipB interaction modulate the contraction of Type VI secretion system sheath

Secretion systems are essential for bacteria to survive and manipulate their environment. The bacterial Type VI Secretion System (T6SS) generates the force needed for protein translocation by the contraction of a long polymer called sheath, which is composed of interconnected VipA/VipB subunits forming a six-start helix. The mechanism of T6SS sheath contraction and the structure of its extended state are unknown. Here we show that elongating the N-terminal VipA linker or eliminating charge of a specific VipB residue abolished sheath contraction and delivery of effectors into target cells. The assembly of the non-contractile sheaths was dependent on the baseplate component TssE and mass-spectrometry analysis identified Hcp, VgrG and other components of the T6SS baseplate specifically associated with stable non-contractile sheaths. The ability to lock T6SS in the pre-firing state opens new possibilities for understanding its mode of action.

microbiology↗