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bioRxiv2017

Modelling Phototaxis as a Collective Phenomenon inCyanobacterial Colonies

Cyanobacteria are a widely distributed, diverse group of photosynthetic bacteria that exhibit phototaxis, or motion in response to light. Cyanobacteria such as Synechocystis sp. secrete a mixture of complex polysaccharides that facilitate cell motion, while their type 4 pili allow them to physically attach to each other. Even though cells can respond individually to light, colonies of such bacteria are observed to move collectively towards the light source in dense finger-like projections. Agent-based models are especially useful in connecting individual cell behaviour with the emergent collective phenomena that arise out of their interactions. We present an agent-based model for cyanobacterial phototaxis that accounts for slime deposition as well as for direct physical links between bacteria, mediated through their type 4 pili. We reproduce the experimentally observed aggregation of cells at the colony boundary as a precursor to finger formation. Our model also describes the changes in colony morphology that occur when the location of the light source is abruptly changed. We find that the overall motion of cells toward light remains relatively unimpaired even if a fraction of them do not sense light, allowing heterogeneous populations to continue to mount a robust collective response to stimuli. Our work suggests that in addition to bio-chemical signalling via diffusible molecules in the context of bacterial quorum-sensing, short-ranged physical interactions may also contribute to collective effects in bacterial motility.

microbiology↗

bioRxiv2017

Evolution with a seed bank: the population genetic consequences of microbial dormancy

Dormancy is a bet-hedging strategy that allows organisms to persist through conditions that are sub-optimal for growth and reproduction by entering a reversible state of reduced metabolic activity. Dormancy allows a population to maintain a reservoir of genetic and phenotypic diversity (i.e., a seed bank) that can contribute to the long-term survival of a population. This strategy can be potentially adaptive and has long been of interest to ecologists and evolutionary biologists. However, comparatively little is known about how dormancy influences the fundamental evolutionary forces of genetic drift, mutation, selection, recombination, and gene flow. Here, we investigate how seed banks affect the processes underpinning evolution by reviewing existing theory, implementing novel simulations, and determining how and when dormancy can influence evolution as a population genetic process. We extend our analysis to examine how seed banks can alter macroevolutionary processes, including rates of speciation and extinction. Through the lens of population genetic theory, we can understand the extent that seed banks influence microbial evolutionary dynamics.

microbiology↗

bioRxiv2017

Bacterial community profiles and Vibrio parahaemolyticus abundance in individual oysters and their association with estuarine ecology

Oysters naturally harbor the human gastric pathogen Vibrio parahaemolyticus, but the nature of this association is unknown. Because microbial interactions could influence the accumulation of V. parahaemolyticus in oysters, we investigated the composition of the microbiome in water and oysters at two ecologically unique sites in the Great Bay Estuary, New Hampshire using 16s rRNA profiling. We then evaluated correlations between bacteria inhabiting the oyster with V. parahaemolyticus abundance quantified using a most probable number (MPN) analysis. Even though oysters filter-feed, their microbiomes were not a direct snapshot of the bacterial community in overlaying water, suggesting they selectively accumulate some bacterial phyla. The microbiome of individual oysters harvested more centrally in the bay were relatively more similar to each other and had fewer unique phylotypes, but overall more taxonomic and metabolic diversity, than the microbiomes from tributary-harvested oysters that were individually more variable with lower taxonomic and metabolic diversity. Oysters harvested from the same location varied in V. parahaemolyticus abundance, with the highest abundance oysters collected from one location. This study, which to our knowledge is the first of its kind to evaluate associations of V. parahaemolyticus abundance with members of individual oyster microbiomes, implies that sufficient sampling and depth of sequencing may reveal microbiome members that could impact V. parahaemolyticus abundance.

microbiology↗

bioRxiv2017

A model for sheath formation coupled to motility in Leptothrix ochracea

Optical and atomic force microscopy (AFM) of naturally occurring Leptothrix ochracea was used to study the fine structure of sheaths and cells. Morphology of young sheaths suggests the scaffold chains have strong self-adhesion. Evidence from un-encapsulated cells indicates fresh scaffold production through cell walls. Simple diffusion arguments are used to explain the morphology of the sheath structure. We propose a novel cell motility model based on previously published video data, our AFM images of naked cells, and simple flow calculations. The model indicates that motility results from differential shear forces resulting from extrusion of sheath material that passively pushes a filament of connected cells forward as the surrounding sheath material hardens behind the cell train.

microbiology↗

bioRxiv2017

Disentangling metabolic functions of bacteria in the honey bee gut

It is presently unclear how much individual community members contribute to the overall metabolic output of a gut microbiota. To address this question, we used the honey bee, which harbors a relatively simple and remarkably conserved gut microbiota with striking parallels to the mammalian system and importance for bee health. Using untargeted metabolomics, we profiled metabolic changes in gnotobiotic bees that were colonized with the complete microbiota reconstituted from cultured strains. We then determined the contribution of individual community members in mono-colonized bees, and recapitulated our findings using in vitro cultures. Our results show that the honey bee gut microbiota utilizes a wide range of pollen-derived substrates including flavonoids and outer pollen wall components, suggesting a key role for degradation of recalcitrant secondary plant metabolites and pollen digestion. In turn, multiple species were responsible for the accumulation of organic acids and polyphenol degradation products, and a specific gut symbiont, Bifidobacterium asteroides, stimulated the production of host hormones known to impact bee development. While we found evidence for cross-feeding interactions, [~]80% of the identified metabolic changes were also observed in mono-colonized bees with Lactobacilli being responsible for the largest share of the metabolic output. These results suggest that bacteria in the honey bee gut colonize largely independent metabolic niches, which may be a general characteristic of gut microbiomes. Our study reveals diverse metabolic functions of gut bacteria that are likely to contribute to bee health, and provide fundamental insights into how metabolic functions are partitioned within gut communities.\n\nAuthor summaryHoney bees are important pollinators that harbor a simple gut microbiota with striking parallels to the mammalian system. This makes them relevant models to study gut microbiota functions and its impact on host health. We applied untargeted metabolomics to characterize metabolic changes induced by the gut microbiota, and to characterize contributions of the major community members. We find that the gut microbiota digests recalcitrant substrates derived from the bees pollen-diet. Most metabolic changes could be explained by the activity of individual community members suggesting substrate specificity and independent metabolic niches. Our study provides novel insights into the functional understanding of the bee gut microbiota and provides a framework for applying untargeted metabolomics to disentangle metabolic functions of gut bacteria.

microbiology↗

bioRxiv2017

Antidiabetic drugs “gliptin” affect biofilm formation by the cariogenic bacterium Streptococcus mutans through inhibition of the bacterial DPP IV-like enzyme

Streptococcus mutans, a dental caries causing odontopathogen, produces X-prolyl dipeptidyl peptidase (Sm-XPDAP, encoded by pepX), a serine protease known to have a nutritional role. Considering the potential of proteases as therapeutic targets in pathogens, this study was primarily aimed at investigating the role of Sm-XPDAP in contributing to virulence-related traits. Dipeptidyl peptidase (DPP IV), an XPDAP analogous enzyme found in mammalian tissues, is a well known therapeutic target in Type II diabetes. Based on the hypothesis that gliptins, commonly used as anti-human-DPP IV drugs, may affect bacterial growth upon inhibition of Sm-XPDAP, we have determined their ex vivo antimicrobial and anti-biofilm activity towards S. mutans. All three DPP IV drugs tested reduced biofilm formation as determined by crystal violet staining. To link the observed biofilm inhibition to the human-DPP IV analogue present in S. mutans UA159, a pepX isogenic mutant was generated. In addition to reduced biofilm formation, CLSM studies of the biofilm formed by the pepX isogenic mutant showed these were comparable to those formed in the presence of saxagliptin, suggesting a probable role of this enzyme in biofilm formation by S. mutans UA159. The effects of both pepX deletion and DPP IV drugs on the proteome were studied using LC-MS/MS. Overall, this study highlights the potential of Sm-XPDAP as a novel anti-biofilm target and suggests a template molecule to synthesize lead compounds effective against this enzyme.

microbiology↗

bioRxiv2017

Gut microbiota in the burying beetle, Nicrophorus vespilloides, provide colonization resistance against larval bacterial pathogens

Carrion beetles, Nicrophorus vespilloides, are reared on decomposing vertebrate carrion where larvae are exposed to high-density populations of carcass-derived bacteria. We previously showed that larvae do not become colonized with these bacteria, but instead are colonized with the gut microbiome of their parents. These results suggested that bacteria in the beetle microbiome outcompete the carcass derived species for colonization of the larval gut. Here we test this hypothesis directly and quantify the fitness consequences of colonization of the Nicrophorus larval gut with different bacterial symbionts, including the insect pathogen Serratia marcescens. First, we show that beetles colonized by their endogenous microbiome produce significantly heavier broods than those colonized with carcass-bacteria. Next, we show that bacteria from the endogenous microbiome, including Providencia rettgeri and Morganella morganii, are better colonizers of the beetle gut and can outcompete non-endogenous species, including S. marcescens and Escherichia coli, during in vivo competition. Finally, we find that Providencia and Morganella provide beetles with colonization resistance against Serratia and thereby reduce Serratia-induced larval mortality during co-inoculation. Importantly, this effect is eliminated in larvae first colonized by Serratia, suggesting that while competition within the larval gut is strongly determined by priority effects, these effects are less important for Serratia-induced mortality. Our work supports the idea that bacterial gut symbionts provide direct benefits to Nicrophorus larvae by outcompeting potential bacterial pathogens. They further suggest that one benefit of parental care in Nicrophorus vespilloides is the social transmission of the microbiome from caring parents to their offspring.

microbiology↗

bioRxiv2017

Statistics and simulation of growth of single bacterial cells: illustrations with B. subtilis and E. coli

The inherent stochasticity of molecular reactions prevents us from predicting the exact state of single-cells in a population. However, when a population grows at steady-state, the probability to observe a cell with particular combinations of properties is fixed. Here we validate and exploit existing theory on the statistics of single-cell growth in order to predict the probability of phenotypic characteristics such as cell-cycle times, volumes, accuracy of division and cell-age distributions, using real-time imaging data for Escherichia coli and Bacillus subtilis. Our results show that single-cell growth-statistics can accurately be predicted from a few basic measurements. These equations relate different phenotypic characteristics, and can therefore be used in consistency tests of experimental single-cell growth data and prediction of single-cell statistics. We also exploit these statistical relations in the development of a fast stochastic-simulation algorithm of single-cell growth and protein expression. This algorithm greatly reduces computational burden, by recovering the statistics of growing cell-populations from the simulation of only one of its lineages. Our approach is validated by comparison of simulations and experimental data. This work illustrates a methodology for the prediction, analysis and tests of consistency of single-cell growth and protein expression data from a few basic statistical principles.

microbiology↗

bioRxiv2017

Identification of Rab18 as an Essential Host Factor for BKPyV Infection Using a Whole Genome RNA Interference Screen

BK polyomavirus (BKPyV) is a human pathogen first isolated in 1971. BKPyV infection is ubiquitous in the human population, with over 80% of adults worldwide being seropositive for BKPyV. BKPyV infection is usually asymptomatic; however, BKPyV reactivation in immunosuppressed transplant patients causes two diseases, polyomavirus-associated nephropathy and hemorrhagic cystitis. To establish a successful infection in its host cells, BKPyV must travel in retrograde transport vesicles to reach the nuclei. To make this happen, BKPyV requires the cooperation of host cell proteins. To further identify host factors associated with BKPyV entry and intracellular trafficking, we performed a whole-genome siRNA screen on BKPyV infection of primary human renal proximal tubular epithelial cells. The results revealed the importance of the Ras-related protein Rab18 and syntaxin 18 for BKPyV infection. Our subsequent experiments implicated additional factors that interact with this pathway, and suggest a more detailed model of the intracellular trafficking process, indicating that BKPyV reaches the ER lumen through a retrograde transport pathway between the late endosome and the ER.

microbiology↗

bioRxiv2017

Obstruction of pilus retraction stimulates bacterial surface sensing

Surface association provides numerous fitness advantages to bacteria. Thus, it is critical for bacteria to recognize surface contact and to consequently initiate physiological changes required for a surface-associated lifestyle (1). Ubiquitous microbial appendages called pili are involved in sensing surfaces and mediating downstream surface-associated behaviors (2-6). The mechanism by which pili mediate surface sensing remains unknown, largely due to the difficulty to visualize their dynamic nature and to directly modulate their activity without genetic modification. Here, we show that Caulobacter crescentus pili undergo dynamic cycles of extension and retraction that cease within seconds of surface contact, and this arrest of pilus activity coincides with surface-stimulated holdfast synthesis. By physically blocking pili, we show that imposing resistance to pilus retraction is sufficient to stimulate holdfast synthesis in the absence of surface contact. Thus, resistance to type IV pilus retraction upon surface attachment is used for surface sensing.\n\nOne Sentence SummaryBacteria use the tension imparted on retracting pilus fibers upon their binding to a surface for surface sensing.

microbiology↗

bioRxiv2017

Two phosphoglucomutase paralogs regulate triggered secretion of the Toxoplasma micronemes

Parafusin is a phosphoglucomutase (PGM) paralog that acts as a signaling scaffold protein in calcium mediated exocytosis across many eukaryotes. In Toxoplasma gondii the parafusin related protein 1 (PRP1) has been associated in indirect and heterologous studies with the regulated exocytosis of the micronemes, which are required for successful host cell invasion and egress. Here we directly assessed the role of PRP1 by deleting the gene from the parasite. We observed a specific defect in microneme secretion in response to high Ca2+ fluxes, but not to phosphatidic acid fluxes controlling microneme release. We observed no defect in constitutive microneme secretion which was sufficient to support completion of the lytic cycle. Furthermore, deletion of the other PGM in Toxoplasma, PGM2, as well as the double PRP1/PGM2 deletion resulted in a similar phenotype. This suggests a functional interaction between these two genes. Strikingly, tachyzoites without both paralogs are completely viable in vitro and during acute mice infections. This indicates that PGM activity is neither required for glycolysis. In conclusion, the PRP1-PGM2 pair is required for a burst in microneme secretion upon high Ca2+ fluxes, but this burst is not essential to complete the lytic cycle of the parasite.\n\nPlain Language SummaryCalcium mediated control of microneme secretion is essential for host cell invasion and egress of Toxoplasma gondii. Here it is shown that the two phosphoglucomutases in Toxoplasma both function in the translation of a spike in calcium into a burst in microneme secretion.

microbiology↗

bioRxiv2017

Nonpolio enteroviruses can also be recovered from non-reproducible cytopathology in L20B cell line.

Samples showing cytopathology (CPE) on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in L20B or RD are considered negative for both poliovirus and nonpolio enteroviruses (NPEVs). The phenomenon is termed non-reproducible CPE. Its occurrence is usually ascribed to the likely presence of reoviruses, adenoviruses and other non-enteroviruses. This study aimed to investigate the likelihood that NPEVs are also present in cases with non-reproducible CPE.\n\nTwenty-six (26) cell culture suspensions were analyzed in this study. The suspensions were collected from the WHO National Polio Laboratory, Department of Virology, College of Medicine, University of Ibadan. The suspensions emanated from 13 L20B cell culture tubes that showed cytopathology within 5 days of inoculation with fecal suspension from AFP cases. However, on passage into one each of RD and L20B cell lines, the CPE was not reproducible. All samples were subjected to RNA extraction, cDNA synthesis, the WHO recommended VP1 RT-seminestedPCR assay, species resolution PCR assay, sequencing and phylogenetic analysis.\n\nSix (6) samples were positive for the VP1 RT-seminested PCR assay. Only four of which were positive by the species resolution PCR assay. The four amplicons were sequenced, however, only three (3) were successfully identified as Coxsackievirus A20 (2 isolates) and Echovirus 29 (1 isolate).\n\nThe results of this study unambiguously showed the presence of NPEVs (particularly CVA20 and E29) in cell culture supernatants of samples with CPE on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in RD cell line. Therefore, like reoviruses, adenoviruses and other non-enteroviruses, NPEVs can also be recovered in cases with non-reproducible CPE.

microbiology↗

bioRxiv2017

Mechanisms of surface antigenic variation in the human pathogenic fungus Pneumocystis jirovecii

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii. This uncultivable fungus is an obligate pulmonary pathogen which causes pneumonia in immuno-compromised individuals, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen of a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily which included five families encoding adhesive GPI-anchored glycoproteins, and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination, and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favoured by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists in the continuous production of new subpopulations composed of cells which are antigenically different.

microbiology↗

bioRxiv2017

MCE domain proteins: conserved inner membrane lipid-binding proteins required for outer membrane homeostasis

Bacterial proteins with MCE domains were first described as being important for Mammalian Cell Entry. More recent evidence suggests they are components of lipid ABC transporters. In Escherichia coli, the single-domain protein MlaD is known to be part of an inner membrane transporter that is important for maintenance of outer membrane lipid asymmetry. Here we describe two multi MCE domain-containing proteins in Escherichia coli, PqiB and YebT, the latter of which is an orthologue of MAM-7 that was previously reported to be an outer membrane protein. We show that all three MCE domain-containing proteins localise to the inner membrane. Bioinformatic analyses revealed that MCE domains are widely distributed across bacterial phyla but multi MCE domain-containing proteins evolved in Proteobacteria from single-domain proteins. Mutants defective in mlaD, pqiAB and yebST were shown to have distinct but partially overlapping phenotypes, but the primary functions of PqiB and YebT differ from MlaD. Complementing our previous findings that all three proteins bind phospholipids, results presented here indicate that multi-domain proteins evolved in Proteobacteria for specific functions in maintaining cell envelope homeostasis.

microbiology↗

bioRxiv2017

Small subsets of highly connected taxa predict compositional change in microbial communities

For decades, ecological theory has predicted that the complexity of communities should be related to their stability. However, this prediction has rarely been tested empirically, because of both the difficulty of finding suitable systems where the question is tractable and the trouble of defining \"stability\" in real systems. Microbial communities provide the opportunity to investigate a related question: how does community connectivity relate to the rate of compositional turnover? We used a newly developed metric called community \"cohesion\" to test how microbial community connectivity relates to Bray-Curtis dissimilarity through time. In three long-term datasets, we found that stronger connectivity corresponded to lower rates of compositional turnover. Using two case studies of disturbed and reference communities, we found that the predictive power of community connectivity was diminished by external disturbance. Finally, we tested whether the highly connected taxa were disproportionately important in explaining compositional turnover. We found that subsets of highly connected \"keystone\" taxa, generally comprising 1-5% of community richness, explained community turnover better than using all taxa. Our results suggest that stronger biotic interactions within microbial community dynamics are stabilizing to community composition, and that highly connected taxa are good indicators of pending community shifts.

microbiology↗

bioRxiv2017

Mapping-based all-RNA-information sequencing analysis (ARIseq) pipeline simultaneously revealed taxonomic composition, gene expression, and their correlation in an acidic stream ecosystem

We developed a new pipeline for simultaneous analyses of both rRNA profile as a taxonomic marker and mRNA profile as a functional marker, to understand microbial ecosystems in natural environments. Our pipeline, named All-RNA-Information sequencing (ARIseq), comprises a high-throughput sequencing of reverse transcribed total RNA and several widely used computational tools, and generates quantitatively reliable information on both community structures and gene expression patterns, which were verified by quantitative PCR analyses in this study. Particularly, correlation network analysis in the pipeline can reveal microbial taxa and expressed genes that share patterns of dynamics among different time and/or geographical points. The pipeline is primarily mapping-based, using a public database for small subunit rRNA genes and obtained contigs as the reference database for protein-coding genes. We applied this pipeline to biofilm samples, as examples, collected from an acidic spring water stream in the Chyatsubomi-goke Park in Gunma prefecture, Japan. Our analyses revealed the predominance of iron and sulfur-oxidizing bacteria and Pinnularia diatoms, and also indicated that the distributions of the iron-sulfur-oxidizing bacterial consortium and the Pinnularia diatoms largely overlapped but showed distinct patterns. In addition, our analyses showed that the iron-oxidizing bacterial genus Acidithiobacillus and co-occurring Acidiphilium shared similar distribution pattern whereas another iron-oxidizing genus Leptospirillum exhibited a distinct pattern. Our pipeline enables researchers to more easily capture the outline of microbial ecosystems based on the taxonomic composition, protein-coding gene expression, and their correlations.

microbiology↗

bioRxiv2017

SParticle, an algorithm for the analysis of filamentous microorganisms in submerged cultures

Streptomycetes are filamentous bacteria that produce a plethora of bioactive natural products and industrial enzymes. Their mycelial lifestyle typically results in high heterogeneity in bioreactors, with morphologies ranging from fragments and open mycelial mats to dense pellets. There is a strong correlation between morphology and production in submerged cultures, with small and open mycelia favoring enzyme production, while most antibiotics are produced mainly in pellets. Here we describe SParticle, a Streptomyces Particle analysis method that combines whole slide imaging with automated image analysis to characterize the morphology of submerged grown Streptomyces cultures. SParticle allows the analysis of over a thousand particles per hour, offering a high throughput method for the imaging and statistical analysis of mycelial morphologies. The software is available as a plugin for the open source software ImageJ and allows users to create custom filters for other microbes. Therefore, SParticle is a widely applicable tool for the analysis of filamentous microorganisms in submerged cultures.

microbiology↗

bioRxiv2017

Reactive Oxygen Species-Dependent Essential Genes in Salmonella Typhimurium

BackgroundThe molecular mechanisms underlying bacterial cell death due to stresses or bactericidal antibiotics are complex and remain puzzling. Previously, it was shown that iron is required for effective killing of bacterial cells by numerous bactericidal antibiotics. Here, our high-resolution Tn-seq analysis demonstrated that transposon mutants of S. Typhimurium with insertions in essential genes escaped immediate killing or growth inhibition under iron-restricted conditions for approximately one-third of all essential genes.\n\nResultsWe grouped essential genes into two categories, iron-dependent and iron-independent essential genes. The iron-dependency of the iron-dependent essential genes was further validated by the fact that the relative abundance of these essential gene mutants increased further with more severe iron restrictions. Our unexpected observation can be explained well by the recently proposed common killing mechanisms of bactericidal antibiotics via production of reactive oxygen species (ROS). In this model iron restriction would inhibit production of ROS, leading to reduced killing activity following blocking of an essential function. Interestingly, the targets of most antibiotics currently in use clinically, whether bacteriostatic or bactericidal, are iron-dependent essential genes.\n\nConclusionsOur result suggests that targeting iron-independent essential genes may be a better strategy for future antibiotic development, because blocking these genes would lead to immediate cell death regardless of iron concentration. On the contrary, blocking iron-dependent pathways under iron limited in vivo environment could lead to reduced killing action, which might increase drug-resistance by mutagenic action of sublethal concentrations of ROS. This work expands our knowledge on the role of iron to a broader range of essential pathways, and provides novel insights for development of more effective antibiotics.

microbiology↗