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bioRxiv2017

Impaired competence in flagellar mutants of Bacillus subtilis is connected to the regulatory network governed by DegU

The competent state is a developmentally distinct phase, in which bacteria are able to take up and integrate exogenous DNA into their genome. Bacillus subtilis is one of the naturally competent bacterial species and the domesticated laboratory strain 168 is easily transformable. In this study, we report a reduced transformation frequency of B. subtilis mutants lacking functional and structural flagellar components. This includes hag, the gene encoding the flagellin protein forming the filament of the flagellum. We confirm that the observed decrease of the transformation frequency is due to reduced expression of competence genes, particularly of the main competence regulator comK. The impaired competence is due to an increase in the phosphorylated form of the response regulator DegU, which is involved in regulation of both flagellar motility and competence. Altogether, our study identified a close link between motility and natural competence in B. subtilis suggesting that hindrance in motility has great impact on differentiation of this bacterium not restricted only to the transition towards sessile growth stage.\n\nOriginality-Significance statementUnderstanding how versatile bacterial phenotypes influence each other is important for our basic understanding of microbial ecology. Our research highlights the novel intertwinement of bacterial differentiation and reveal how lack of single cell motility adjusts DNA exchange among bacterial strains.

microbiology↗

bioRxiv2017

BldC delays entry into development to produce a sustained period of vegetative growth in Streptomyces venezuelae

Streptomycetes are filamentous bacteria that differentiate by producing spore-bearing reproductive structures called aerial hyphae. The transition from vegetative to reproductive growth is controlled by the bld (bald) loci, and mutations in bld genes prevent the formation of aerial hyphae, either by blocking entry into development (typically mutations in activators) or by inducing precocious sporulation in the vegetative mycelium (typically mutations in repressors). One of the bld genes, bldC, encodes a 68-residue DNA-binding protein related to the DNA-binding domain of MerR-family transcription factors. Recent work has shown that BldC binds DNA by a novel mechanism, but there is less insight into its impact on Streptomyces development. Here we used ChIP-seq coupled with RNA-seq to define the BldC regulon in the model species Streptomyces venezuelae, showing that BldC can function both as a repressor and as an activator of transcription. Using electron microscopy and time-lapse imaging, we show that bldC mutants are bald because they initiate development prematurely, bypassing the formation of aerial hyphae. This is consistent with the premature expression of BldC target genes encoding proteins with key roles in development (e.g. whiD, whiI, sigF), chromosome condensation and segregation (e.g. smeA-sffA, hupS), and sporulation-specific cell division (e.g. dynAB), suggesting that BldC-mediated repression is critical to maintain a sustained period of vegetative growth prior to sporulation. We discuss the possible significance of BldC as an evolutionary link between MerR family transcription factors and DNA architectural proteins.\n\nIMPORTANCEUnderstanding the mechanisms that drive bacterial morphogenesis depends on the dissection of the regulatory networks that underpin the cell biological processes involved. Recently, Streptomyces venezuelae has emerged as an attractive new model system for the study of morphological differentiation in Streptomyces. This has led to significant progress in identifying the genes controlled by the transcription factors that regulate aerial mycelium formation (Bld regulators) and sporulation (Whi regulators). Taking advantage of S. venezuelae, we used ChIP-seq coupled with RNA-seq to identify the genes directly under the control of BldC. Because S. venezuelae sporulates in liquid culture, the complete spore-to-spore life cycle can be examined using time-lapse microscopy, and we applied this technique to the bldC mutant. These combined approaches reveal BldC to be a member of an emerging class of Bld regulators that function principally to repress key sporulation genes, thereby extending vegetative growth and blocking the onset of morphological differentiation.

microbiology↗

bioRxiv2017

A universal, genome-wide guide finder for CRISPR/Cas9 targeting in microbial genomes

BackgroundThe CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression or activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, no pan-bacterial, genome-wide tools exist for guide discovery. We have created Guide Finder: a customizable, user-friendly program that can design guides for any annotated bacterial genome.\n\nResultsGuide Finder designs guides from NGG PAM sites for any number of genes using an annotated genome and fasta file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, a feature unique to Guide Finder. Guide Finder has been tested on a variety of diverse bacterial genomes, on average finding guides for 95% of genes. Moreover, guides designed by the program are functionally useful--focusing on CRISPRi as a potential application--as demonstrated by essential gene knockdown in two staphylococcal species.\n\nConclusionsThrough the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies for a variety of bacterial species.

microbiology↗

bioRxiv2017

Stress granule-inducing eukaryotic translation initiation factor 4A inhibitors block influenza A virus replication

Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5 untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection results in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates the feasibility of targeting core host protein synthesis machinery to prevent viral replication.\n\nIMPORTANCEInfluenza A virus (IAV) relies on cellular protein synthesis to decode viral messenger RNAs. Pateamine A and silvestrol are natural products that inactivate an essential protein synthesis protein known as eIF4A. Here we show that IAV is sensitive to these eIF4A inhibitor drugs. Treatment of infected cells with pateamine A or silvestrol prevented synthesis of viral proteins, viral genome replication and release of infectious virions. The irreversible eIF4A inhibitor pateamine A sustained long-term blockade of viral replication, whereas viral protein synthesis quickly resumed after silvestrol was removed from infected cells. Prolonged incubation of either infected or uninfected cells with these drugs induced the programmed cell death cascade called apoptosis. Our findings suggest that core components of the host protein synthesis machinery are viable targets for antiviral drug discovery. The most promising drug candidates should selectively block protein synthesis in infected cells without perturbing bystander uninfected cells.

microbiology↗

bioRxiv2017

Abundance and diversity of the fecal resistome in slaughter pigs and broilers in nine European countries

EFFORT groupHaitske Graveland, Alieda van Essen, Bruno Gonzalez-Zorn, Gabriel Moyano, Pascal Sanders, Claire Chauvin, Julie David, Antonio Battisti, Andrea Caprioli, Jeroen Dewulf, Thomas Blaha, Katharina Wadepohl, Maximiliane Brandt, Dariusz Wasyl, Magdalena Skarzynska, Magdalena Zajac, Hristo Daskalov, Helmut W Saatkamp, Katharina D.C. Stark.\n\nAbstractAntimicrobial resistance (AMR) in bacteria and associated human morbidity and mortality is increasing. Use of antimicrobials in livestock selects for AMR that can subsequently be transferred to humans. This flow of AMR between reservoirs demands surveillance in livestock as well as in humans. As part of the EFFORT project (www.effort-against-amr.eu), we have quantified and characterized the acquired resistance gene pools (resistomes) of 181 pig and 178 poultry farms from nine European countries, generating more than 5,000 gigabases of DNA sequence, using shotgun metagenomics. We quantified acquired AMR using the ResFinder database and a database constructed for this study, consisting of AMR genes identified through screening environmental DNA. The pig and poultry resistomes were very different in abundance and composition. There was a significant country effect on the resistomes, more so in pigs than poultry. We found higher AMR loads in pigs, while poultry resistomes were more diverse. We detected several recently described, critical AMR genes, including mcr-1 and optrA, the abundance of which differed both between host species and countries. We found that the total acquired AMR level, was associated with the overall country-specific antimicrobial usage in livestock and that countries with comparable usage patterns had similar resistomes. Novel, functionally-determined AMR genes were, however, not associated with total drug use.

microbiology↗

bioRxiv2017

Traditional Norwegian Kveik Yeasts: Underexplored Domesticated Saccharomyces cerevisiae Yeasts

Human activity has resulted in the domestication of Saccharomyces cerevisiae yeasts specifically adapted to beer production. While there is evidence beer yeast domestication was accelerated by industrialization of beer, there also exists a home-brewing culture in western Norway which has passed down yeasts referred to as kveik for generations. This practice has resulted in ale yeasts which are typically highly flocculant, phenolic off flavour negative (POF-), and exhibit a high rate of fermentation, similar to previously characterized lineages of domesticated yeast. Additionally, kveik yeasts are highly temperature tolerant, likely due to the traditional practice of pitching yeast into warm (>30 {degrees}C) wort. Here, we characterize kveik yeasts from 9 different Norwegian sources via PCR fingerprinting, phenotypic screens, lab-scale fermentations, and flavour metabolite analysis using HS-SPME-GC-MS. Genetic fingerprinting via interdelta PCR suggests that kveik yeasts form a lineage distinct from other domesticated yeasts. Our analyses confirm that kveik yeasts display hallmarks of domestication such as loss of 4-vinylguaiacol production and high flocculation, and show superior thermotolerance, ethanol tolerance, fermentation rate, and unique flavour metabolite production profiles in comparison to other ale strains, suggesting a broad industrial potential for this group of yeasts.

microbiology↗

bioRxiv2017

In vivo structures of the Helicobacter pylori cag type IV secretion system

The bacterial type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Using electron cryotomography, here we reveal the molecular architecture of the cancer-associated Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to previously studied T4SSs. When H. pylori encounters host cells, however, the bacterium elaborates rigid, membranous tubes perforated by lateral ports. Dense, pilus-like rod structures extending from the inner membrane were also observed. We propose that the membrane tubes assemble out of the T4SS and are the delivery system for cag T4SS cargo. These studies reveal the architecture of a dynamic molecular machine that evolved to function in the human gastric niche.

microbiology↗

bioRxiv2017

PHENOS: a high-throughput and flexible tool for microorganism growth phenotyping on solid media

BACKGROUNDMicrobial arrays, with a large number of different strains on a single plate printed with robotic precision, underpin an increasing number of genetic and genomic approaches. These include Synthetic Genetic Array analysis, high-throughput Quantitative Trait Loci (QTL) analysis and 2-hybrid techniques. Measuring the growth of individual colonies within these arrays is an essential part of many of these techniques but is useful for any work with arrays. Measurement is typically done using intermittent imagery fed into complex image analysis software, which is not especially accurate and is challenging to use effectively. We have developed a simple and fast alternative technique that uses a pinning robot and a commonplace microplate reader to continuously measure the thickness of colonies growing on solid agar, complemented by a technique for normalizing the amount of cells initially printed to each spot of the array in the first place. We have developed software to automate the process of combining multiple sets of readings, subtracting agar absorbance, and visualizing colony thickness changes in a number of informative ways.\n\nRESULTSThe \"PHENOS\" pipeline (PHENotyping On Solid media), optimized for Saccharomyces yeasts, produces highly reproducible growth curves and is particularly sensitive to low-level growth. We have empirically determined a formula to estimate colony cell count from an absorbance measurement, and shown this to be comparable with estimates from measurements in liquid. We have also validated the technique by reproducing the results of an earlier QTL study done with conventional liquid phenotyping, and found PHENOS to be considerably more sensitive.\n\nCONCLUSIONS\"PHENOS\" is a cost effective and reliable high-throughput technique for quantifying growth of yeast arrays, and is likely to be equally very useful for a range of other types of microbial arrays. A detailed guide to the pipeline and software is provided with the installation files at https://github.com/gact/phenos.

microbiology↗

bioRxiv2017

Probing the evolutionary robustness of anti-virulence treatments targeting iron uptake in Pseudomonas aeruginosa

Background and objectivesTreatments that inhibit the expression or functioning of bacterial virulence factors hold great promise to be both effective and exert weaker selection for resistance than conventional antibiotics. However, the evolutionary robustness argument, based on the idea that anti-virulence treatments disarm rather than kill pathogens, is controversial. Here we probe the evolutionary robustness of two repurposed drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine of the opportunistic human pathogen Pseudomonas aeruginosa.\n\nMethodologyWe subjected replicated cultures of bacteria to two concentrations of each drug for 20 consecutive days in human serum as an ex-vivo infection model. We screened evolved populations and clones for resistance phenotypes, including the restoration of growth and pyoverdine production, and the evolution of iron uptake by-passing mechanisms. We whole-genome sequenced evolved clones to identify the genetic basis of resistance.\n\nResultsWe found that mutants resistant against anti-virulence treatments readily arose, but their selective spreading varied between treatments. Flucytosine resistance quickly spread in all populations due to disruptive mutations in upp, a gene encoding an enzyme required for flucytosine activation. Conversely, resistance against gallium arose only sporadically, and was based on mutations in transcriptional regulators, upregulating pyocyanin production, a redox-active molecule promoting siderophore-independent iron acquisition. The spread of gallium resistance could be hampered because pyocyanin-mediated iron delivery benefits resistant and susceptible cells alike.\n\nConclusions and implicationsOur work highlights that anti-virulence treatments are not evolutionarily robust per se. Instead, evolutionary robustness is a relative measure, with specific treatments occupying different positions on a continuous scale.

microbiology↗

bioRxiv2017

Prediction of the intestinal resistome by a novel 3D-based method

The intestinal microbiota is considered to be a major reservoir of antibiotic resistance determinants (ARDs) that could potentially be transferred to bacterial pathogens. Yet, this question remains hypothetical because of the difficulty to identify ARDs from intestinal bacteria. Here, we developed and validated a new annotation method (called pairwise comparative modelling, PCM) based on homology modelling in order to characterize the Human resistome. We were able to predict 6,095 ARDs in a 3.9 million protein catalogue from the Human intestinal microbiota. We found that predicted ARDs (pdARDs) were distantly related to known ARDs (mean amino-acid identity 29.8%). Among 3,651 pdARDs that were identified in metagenomic species, 3,489 (95.6%) were assumed to be located on the bacterial chromosome. Furthermore, genes associated with mobility were found in the neighbourhood of only 7.9% (482/6,095) of pdARDs. According to the composition of their resistome, we were able to cluster subjects from the MetaHIT cohort (n=663) into 6 \"resistotypes\". Eventually, we found that the relative abundance of pdARDs was positively associated with gene richness, but not when subjects were exposed to antibiotics. Altogether, our results support that most ARDs in the intestinal microbiota should be considered as intrinsic genes of commensal microbiota with a low risk of transfer to bacterial pathogens.

microbiology↗

bioRxiv2017

A LaeA- and BrlA-dependent cellular network governs tissue-specific secondary metabolism in the human pathogen Aspergillus fumigatus

Biosynthesis of many ecologically important secondary metabolites (SMs) in filamentous fungi is controlled by several global transcriptional regulators, like the chromatin modifier LaeA, and tied to both development and vegetative growth. In Aspergillus molds, asexual development is regulated by the BrlA>AbaA>WetA transcriptional cascade. To elucidate BrlA pathway involvement in SM regulation, we examined the transcriptional and metabolic profiles of {Delta}brlA, {Delta}abaA, {Delta}wetA and wild-type strains of the human pathogen Aspergillus fumigatus. We find that BrlA, in addition to regulating production of developmental SMs, regulates vegetative SMs and the SrbA-regulated hypoxia stress response in a concordant fashion to LaeA. We further show that the transcriptional and metabolic equivalence of {Delta}brlA and {Delta}laeA is mediated by a LaeA requirement preventing heterochromatic marks in the brlA promoter. These results provide a framework for the cellular network regulating not only fungal SMs but diverse cellular processes linked to virulence of this pathogen.

microbiology↗

bioRxiv2017

Rice paddy Nitrospirae encode and express genes related to sulfate respiration: proposal of the new genus Candidatus Sulfobium

Nitrospirae spp. distantly related to thermophilic, sulfate-reducing Thermodesulfovibrio species are regularly observed in environmental surveys of anoxic marine and freshwater habitats. However, little is known about their genetic make-up and physiology. Here, we present the draft genome of Nitrospirae bacterium Nbg-4 as a representative of this clade and analyzed its in situ protein expression under sulfate-enriched and sulfate-depleted conditions in rice paddy soil. The genome of Nbg-4 was assembled from replicated metagenomes of rice paddy soil that was used to grow rice plants in the presence and absence of gypsum (CaSO4x2H2O). Nbg-4 encoded the full pathway of dissimilatory sulfate reduction and showed expression thereof in gypsum-amended anoxic bulk soil as revealed by parallel metaproteomics. In addition, Nbg-4 encoded the full pathway of dissimilatory nitrate reduction to ammonia, which was expressed in bulk soil without gypsum amendment. The relative abundance of Nbg-4-related metagenome reads was similar under both treatments indicating that it maintained stable populations while shifting its energy metabolism. Further genome reconstruction revealed the potential to utilize butyrate, formate, H2, or acetate as electron donor, with the Wood-Ljungdahl pathway being expressed under both conditions. Comparison to publicly available Nitrospirae genome bins confirmed that the pathway for dissimilatory sulfate reduction is also present in related Nitrospirae recovered from groundwater. Subsequent phylogenomics showed that such microorganisms form a novel genus within the phylum Nitrospirae, with Nbg-4 as a representative species. Based on the widespread occurrence of this novel genus, we propose for Nbg-4 the name Candidatus Sulfobium mesophilum, gen. nov., spec. nov.\n\nImportanceRice paddies are indispensable for food supply but are a major source of the greenhouse gas methane. If not counterbalanced by cryptic sulfur cycling, methane emission from rice paddy fields would be even higher. However, the microorganisms involved in this sulfur cycling are little understood. By using an environmental systems biology approach of Italian rice paddy soil, we could retrieve the population genome of a novel member of the phylum Nitrospirae. This microorganism encoded the full pathway of dissimilatory sulfate reduction and expressed it in situ under sulfate-enriched and anoxic conditions. Phylogenomics and comparison to environmental surveys showed that such microorganisms are actually widespread in freshwater and marine environments. At the same time, they represent a yet undiscovered genus within the little explored Nitrospirae. Our results will be important to design enrichment strategies and postgenomic studies to fully understand the contribution of these novel Nitrospirae to the global sulfur cycle.

microbiology↗

bioRxiv2017

Novel genes required for the fitness of Streptococcus pyogenes in human saliva

Streptococcus pyogenes (group A Streptococcus, or GAS) causes 600 million cases of pharyngitis each year. Despite this considerable disease burden, the molecular mechanisms used by GAS to infect, cause clinical pharyngitis, and persist in the human oropharynx are poorly understood. Saliva is ubiquitous in the human oropharynx and is the first material GAS encounters in the upper respiratory tract. Thus, a fuller understanding of how GAS survives and proliferates in saliva may provide valuable insights into the molecular mechanisms at work in the human oropharynx. We generated a highly saturated transposon insertion mutant library in serotype M1 strain MGAS2221, a strain genetically representative of a pandemic clone that arose in the 1980s and spread globally. The transposon mutant library was exposed to human saliva to screen for GAS genes required for wild-type fitness in this clinically relevant fluid. Using transposon-directed insertion site sequencing (TraDIS), we identified 92 genes required for GAS fitness in saliva. The more prevalent categories represented are genes involved in carbohydrate transport/metabolism, amino acid transport/metabolism, and inorganic ion transport/metabolism. Using six isogenic mutant strains, we confirmed that each of the mutants are significantly impaired for growth or persistence in human saliva ex vivo. Mutants with an inactivated spy0644 (sptA) or spy0646 (sptC) gene have especially severe persistence defects. This study is the first use of TraDIS to study bacterial fitness in human saliva. The new information we obtained is valuable for future translational maneuvers designed to prevent or treat human GAS infections.\n\nIMPORTANCEThe human bacterial pathogen Streptococcus pyogenes (group A streptococcus, GAS) causes more than 600 million cases of pharyngitis annually worldwide, 15 million of which occur in the United States. The human oropharynx is the primary anatomic site for GAS colonization and infection, and saliva is the first material encountered. Using a genome-wide transposon mutant screen, we identified 92 GAS genes required for wild-type fitness in human saliva. Many of the identified genes are involved in carbohydrate transport/metabolism, amino acid transport/metabolism, and inorganic ion transport/metabolism. The new information is potentially valuable for developing novel GAS therapeutics and vaccine research.

microbiology↗

bioRxiv2017

Hunters or gardeners?Linking community structure and function of trap-associated microbes to the nutrient acquisition strategy of a carnivorous plant

All higher eukaryotes live in a relationship with diverse microorganisms which colonize their bodily surfaces; plants are no exception. However, we still lack a satisfactory understanding of how these loosely associated microbiomes with immense diversity and functional potential interact with their hosts or how these interactions shape processes within populations and ecosystems. There is considerable similarity between microbial communities colonizing plant surfaces such as roots, and those of the animal gut. This often overlooked parallel allows us to look at microbial as well as host ecophysiology from a fresh perspective. The traps of carnivorous plants are sophisticated digestive organs and interface environments between the supply and the demand for nutrients. We selected the miniature ecosystem in the traps of aquatic carnivorous Utricularia plants as our model system. By assessing the trap-associated microbial community structure, diversity, function, as well as the nutrient recycling potential of bacterivory, we gained insight into the nutrient acquisition strategies of the Utricularia hosts. We conclude that trap ecophysiological function is in many aspects highly analogous to that of the herbivore gut and centers around complex microbial consortia, which act synergistically to covert complex organic matter, often of algal origin, into a source of nutrients for the plants.

microbiology↗

bioRxiv2017

Initiation, elongation and realignment during influenza virus mRNA synthesis

The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the RdRp snatches capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to the ultimate or penultimate nucleotide of the segments for the initiation of viral mRNA synthesis. It has been proposed that this initiation process is not processive and that the RdRp uses a prime-realign mechanism during transcription. Here we provide in vitro evidence for the existence of this transcriptional prime-realign mechanism, but show that it only functions efficiently for primers that are short or can not stably base pair with the template. In addition, we demonstrate that transcriptional elongation is dependent on the priming loop of the PB1 subunit of the RdRp. We propose that the prime-realign mechanism may be used to rescue abortive transcription initiation events or cope with sequence variation among primers. Overall, these observations advance our mechanistic understanding of how the influenza A virus initiates transcription correctly and efficiently.\n\nImportanceThe influenza A virus causes severe disease in humans and is considered a major global health threat. The virus replicates and transcribes its genome using an enzyme called the RNA polymerase. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the viral RNA polymerase must transcribe the viral genome efficiently. In this report, we characterise a structure inside the polymerase that contributes to the efficiency of viral mRNA synthesis.

microbiology↗

bioRxiv2017

Peatland Acidobacteria with a dissimilatory sulfur metabolism

Sulfur-cycling microorganisms impact organic matter decomposition in wetlands and consequently greenhouse gas emissions from these globally relevant environments. However,their identities and physiological properties are largely unknown. By applying a functional metagenomics approach to an acidic peatland, we recovered draft genomes of seven novel Acidobacteria species with the potential for dissimilatory sulfite (dsrAB, dsrC, dsrD, dsrN, dsrT, dsrMKJOP) or sulfate respiration (sat, aprBA, qmoABC plus dsr genes). Surprisingly, the genomes also encoded dsrL, a unique gene of the sulfur oxidation pathway. Metatranscriptome analysis demonstrated expression of acidobacterial sulfur-metabolism genes in native peat soil and their upregulation in diverse anoxic microcosms. This indicated an active sulfate respiration pathway, which, however, could also operate in reverse for sulfur oxidation as recently shown for other microorganisms. Acidobacteria that only harbored genes for sulfite reduction additionally encoded enzymes that liberate sulfite from organosulfonates, which suggested organic sulfur compounds as complementary energy sources. Further metabolic potentials included polysaccharide hydrolysis and sugar utilization, aerobic respiration, several fermentative capabilities, and hydrogen oxidation. Our findings extend both, the known physiological and genetic properties of Acidobacteria and the known taxonomic diversity of microorganisms with a DsrAB-based sulfur metabolism, and highlight new fundamental niches for facultative anaerobic Acidobacteria in wetlands based on exploitation of inorganic and organic sulfur molecules for energy conservation.

microbiology↗

bioRxiv2017

MreB Filaments Create Rod Shape By Aligning Along Principal Membrane Curvature.

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion and increased glycan crosslinking. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.

microbiology↗

bioRxiv2017

Temporal differentiation of bovine airway epithelial cells grown at an air-liquid interface

The respiratory epithelium is exposed to assault by toxins and pathogens through the process of inhalation, which has numerous implications on both human and animal health. As such, there is a need to develop and characterise an in vitro model of the airway epithelium to study respiratory pathologies during infection or toxicology experiments. This has been achieved by growing airway epithelial cells at an air-liquid interface (ALI). Characterisation of ALI models are not well-defined for airway epithelial cells derived from non-human species. In this study we have fully characterised a bovine airway epithelial cell models (AECM) grown at an ALI in relation to ex vivo tissue. The morphology of the model was monitored at three day intervals, to identify the time-period at which the culture was optimally differentiated. The model was shown to be fully-differentiated by day 21 post-ALI. The culture formed a stereotypical pseudostratified, columnar epithelium containing the major cell types of the bronchial epithelium (ciliated-, goblet- and basal cells). Once fully differentiated the bovine AECM displayed both barrier function, through the formation of tight-junctions, and active mucociliary clearance, important properties of the mucosal barrier. The bovine bronchial epithelial cells remained stable for three weeks, with no evidence of deterioration or dedifferentiation. The window in which the model displayed full differentiation was determined to be between day 21-42 post-ALI. Through comparison with ex vivo tissue derived from donor animals, our bovine AECM was shown to be highly representative of the in vivo bovine bronchial epithelium and can be utilised in the study of respiratory pathologies.

microbiology↗