Contribution of TAT System Translocated PhoX to Campylobacter jejuni Phosphate Metabolism and Resilience to Environmental Stresses

Campylobacter jejuni is a common gastrointestinal pathogen that colonizes food animals; it is transmitted via fecal contamination of food, and infections in immune-compromised people are more likely to result in serious long-term illness. Environmental phosphate is likely an important sensor of environmental fitness and the ability to obtain extracellular phosphate is central to the bacteria's core metabolic responses. PhoX is the sole alkaline phosphatase in C. jejuni, a substrate of the TAT transport system. Alkaline phosphatases mediate the hydrolytic removal of inorganic phosphate (Pi) from phospho-organic compounds and thereby contribute significantly to the polyphosphate kinase 1 (ppk1) mediated formation of poly P, a molecule that regulates bacterial response to stresses and virulence. Similarly, deletion of the tatC gene, a key component of the TAT system, results in diverse phenotypes in C. jejuni including reduced stress tolerance and in vivo colonization. Therefore, here we investigated the contribution of phoX in poly P synthesis and in TAT-system mediated responses. The phoX deletion mutant showed significant decrease (P<0.05) in poly P accumulation in stationary phase compared to the wild-type, suggesting that PhoX is a major contributor to the inorganic phosphate pool in the cell which is essential for poly P synthesis. The phoX deletion is sufficient for a nutrient stress defect similar to the defect previously described for the ΔtatC mutant. Additionally, the phoX deletion mutant has increased resistance to certain antimicrobials. The ΔphoX mutant was also moderately defective in invasion and intracellular survival within human intestinal epithelial cells as well as in chicken colonization. Further, the ΔphoX mutant produced increased biofilm that can be rescued with 1 mM inorganic phosphate. The qRT-PCR of the ΔphoX mutant revealed transcriptional changes that suggest potential mechanisms for the increased biofilm phenotype

Adaptive Evolution of the Lactose Utilization Network in Experimentally Evolved Populations of Escherichia coli

Adaptation to novel environments is often associated with changes in gene regulation. Nevertheless, few studies have been able both to identify the genetic basis of changes in regulation and to demonstrate why these changes are beneficial. To this end, we have focused on understanding both how and why the lactose utilization network has evolved in replicate populations of Escherichia coli. We found that lac operon regulation became strikingly variable, including changes in the mode of environmental response (bimodal, graded, and constitutive), sensitivity to inducer concentration, and maximum expression level. In addition, some classes of regulatory change were enriched in specific selective environments. Sequencing of evolved clones, combined with reconstruction of individual mutations in the ancestral background, identified mutations within the lac operon that recapitulate many of the evolved regulatory changes. These mutations conferred fitness benefits in environments containing lactose, indicating that the regulatory changes are adaptive. The same mutations conferred different fitness effects when present in an evolved clone, indicating that interactions between the lac operon and other evolved mutations also contribute to fitness. Similarly, changes in lac regulation not explained by lac operon mutations also point to important interactions with other evolved mutations. Together these results underline how dynamic regulatory interactions can be, in this case evolving through mutations both within and external to the canonical lactose utilization network

Population Analysis of the Fusarium graminearum Species Complex from Wheat in China Show a Shift to More Aggressive Isolates

A large number of Fusarium isolates was collected from blighted wheat spikes originating from 175 sampling sites, covering 15 provinces in China. Species and trichothecene chemotype determination by multilocus genotyping (MLGT) indicated that F. graminearum s. str. with the 15-acetyl deoxynivalenol (15ADON) chemotype and F. asiaticum with either the nivalenol (NIV) or the 3-acetyl deoxynivalenol (3ADON) chemotype were the dominant causal agents. Bayesian model-based clustering with allele data obtained with 12 variable number of tandem repeats (VNTR) markers, detected three genetic clusters that also show distinct chemotypes. High levels of population genetic differentiation and low levels of effective number of migrants were observed between these three clusters. Additional genotypic analyses revealed that F. graminearum s. str. and F. asiaticum are sympatric. In addition, composition analysis of these clusters indicated a biased gene flow from 3ADON to NIV producers in F. asiaticum. In phenotypic analyses, F. asiaticum that produce 3ADON revealed significant advantages over F. asiaticum that produce NIV in pathogenicity, growth rate, fecundity, conidial length, trichothecene accumulation and resistance to benzimidazole. These results suggest that natural selection drives the spread of a more vigorous, more toxigenic pathogen population which also shows higher levels of fungicide resistance

Combinatorial discovery of polymers resistant to bacterial attachment

Bacterial attachment and subsequent biofilm formation are key challenges to the long term performance of many medical devices. Here, a high throughput approach coupled with the analysis of surface structure-property relationships using a chemometics approach has been developed to simultaneously investigate the interaction of bacteria with hundreds of polymeric materials on a microarray format. Using this system, a new group of materials comprising ester and hydrophobic moieties are identified that dramatically reduce the attachment of clinically relevant, pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus aureus and uropathogenic Escherichia coli). Hit materials coated on silicone catheters resulted in up to a 30 fold reduction in coverage compared to a commercial silver embedded catheter, which has been proven to half the incidence of clinically acquired infection. These polymers represent a new class of materials resistant to bacterial attachment that could not have been predicted from the current understanding of bacteria-surface interactions

A Filamentous Hemagglutinin-Like Protein of Xanthomonas axonopodis pv. citri, the Phytopathogen Responsible for Citrus Canker, Is Involved in Bacterial Virulence

Xanthomonas axonopodis pv. citri, the phytopathogen responsible for citrus canker has a number of protein secretion systems and among them, at least one type V protein secretion system belonging to the two-partner secretion pathway. This system is mainly associated to the translocation of large proteins such as adhesins to the outer membrane of several pathogens. Xanthomonas axonopodis pv. citri possess a filamentous hemagglutinin-like protein in close vicinity to its putative transporter protein, XacFhaB and XacFhaC, respectively. Expression analysis indicated that XacFhaB was induced in planta during plant-pathogen interaction. By mutation analysis of XacFhaB and XacFhaC genes we determined that XacFhaB is involved in virulence both in epiphytic and wound inoculations, displaying more dispersed and fewer canker lesions. Unexpectedly, the XacFhaC mutant in the transporter protein produced an intermediate virulence phenotype resembling wild type infection, suggesting that XacFhaB could be secreted by another partner different from XacFhaC. Moreover, XacFhaB mutants showed a general lack of adhesion and were affected in leaf surface attachment and biofilm formation. In agreement with the in planta phenotype, adhesin lacking cells moved faster in swarming plates. Since no hyperflagellation phenotype was observed in this bacteria, the faster movement may be attributed to the lack of cell-to-cell aggregation. Moreover, XacFhaB mutants secreted more exopolysaccharide that in turn may facilitate its motility. Our results suggest that this hemagglutinin-like protein is required for tissue colonization being mainly involved in surface attachment and biofilm formation, and that plant tissue attachment and cell-to-cell aggregation are dependent on the coordinated action of adhesin molecules and exopolysaccharides

A eukaryotic-type signalling system of Pseudomonas aeruginosa contributes to oxidative stress resistance, intracellular survival and virulence

<p>Abstract</p> <p>Background</p> <p>The genome of <it>Pseudomonas aeruginosa </it>contains at least three genes encoding eukaryotic-type Ser/Thr protein kinases, one of which, <it>ppkA</it>, has been implicated in <it>P. aeruginosa </it>virulence. Together with the adjacent <it>pppA </it>phosphatase gene, they belong to the type VI secretion system (H1-T6SS) locus, which is important for bacterial pathogenesis. To determine the biological function of this protein pair, we prepared a <it>pppA-ppkA </it>double mutant and characterised its phenotype and transcriptomic profiles.</p> <p>Results</p> <p>Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pyoverdine. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the plant model of infection. Whole-genome transcriptome analysis revealed that <it>pppA-ppkA </it>deletion affects the expression of oxidative stress-responsive genes, stationary phase σ-factor RpoS-regulated genes, and quorum-sensing regulons. The transcriptome of the <it>pppA-ppkA </it>mutant was also analysed under conditions of oxidative stress and showed an impaired response to the stress, manifested by a weaker induction of stress adaptation genes as well as the genes of the SOS regulon. In addition, expression of either RpoS-regulated genes or quorum-sensing-dependent genes was also affected. Complementation analysis confirmed that the transcription levels of the differentially expressed genes were specifically restored when the <it>pppA </it>and <it>ppkA </it>genes were expressed ectopically.</p> <p>Conclusions</p> <p>Our results suggest that in addition to its crucial role in controlling the activity of <it>P. aeruginosa </it>H1-T6SS at the post-translational level, the PppA-PpkA pair also affects the transcription of stress-responsive genes. Based on these data, it is likely that the reduced virulence of the mutant strain results from an impaired ability to survive in the host due to the limited response to stress conditions.</p

Voronoi Tessellation Captures Very Early Clustering of Single Primary Cells as Induced by Interactions in Nascent Biofilms

Biofilms dominate microbial life in numerous aquatic ecosystems, and in engineered and medical systems, as well. The formation of biofilms is initiated by single primary cells colonizing surfaces from the bulk liquid. The next steps from primary cells towards the first cell clusters as the initial step of biofilm formation remain relatively poorly studied. Clonal growth and random migration of primary cells are traditionally considered as the dominant processes leading to organized microcolonies in laboratory grown monocultures. Using Voronoi tessellation, we show that the spatial distribution of primary cells colonizing initially sterile surfaces from natural streamwater community deviates from uniform randomness already during the very early colonisation. The deviation from uniform randomness increased with colonisation — despite the absence of cell reproduction — and was even more pronounced when the flow of water above biofilms was multidirectional and shear stress elevated. We propose a simple mechanistic model that captures interactions, such as cell-to-cell signalling or chemical surface conditioning, to simulate the observed distribution patterns. Model predictions match empirical observations reasonably well, highlighting the role of biotic interactions even already during very early biofilm formation despite few and distant cells. The transition from single primary cells to clustering accelerated by biotic interactions rather than by reproduction may be particularly advantageous in harsh environments — the rule rather than the exception outside the laboratory

Shaping the growth behaviour of biofilms initiated from bacterial aggregates

Bacterial biofilms are usually assumed to originate from individual cells deposited on a surface. However, many biofilm-forming bacteria tend to aggregate in the planktonic phase so that it is possible that many natural and infectious biofilms originate wholly or partially from pre-formed cell aggregates. Here, we use agent-based computer simulations to investigate the role of pre-formed aggregates in biofilm development. Focusing on the initial shape the aggregate forms on the surface, we find that the degree of spreading of an aggregate on a surface can play an important role in determining its eventual fate during biofilm development. Specifically, initially spread aggregates perform better when competition with surrounding unaggregated bacterial cells is low, while initially rounded aggregates perform better when competition with surrounding unaggregated cells is high. These contrasting outcomes are governed by a trade-off between aggregate surface area and height. Our results provide new insight into biofilm formation and development, and reveal new factors that may be at play in the social evolution of biofilm communities

Phenotypic Heterogeneity and the Evolution of Bacterial Life Cycles

Most bacteria live in colonies, where they often express different cell types. The ecological significance of these cell types and their evolutionary origin are often unknown. Here, we study the evolution of cell differentiation in the context of surface colonization. We particularly focus on the evolution of a ‘sticky’ cell type that is required for surface attachment, but is costly to express. The sticky cells not only facilitate their own attachment, but also that of non-sticky cells. Using individual-based simulations, we show that surface colonization rapidly evolves and in most cases leads to phenotypic heterogeneity, in which sticky and non-sticky cells occur side by side on the surface. In the presence of regulation, cell differentiation leads to a remarkable set of bacterial life cycles, in which cells alternate between living in the liquid and living on the surface. The dominant life stage is formed by the surface-attached colony that shows many complex features: colonies reproduce via fission and by producing migratory propagules; cells inside the colony divide labour; and colonies can produce filaments to facilitate expansion. Overall, our model illustrates how the evolution of an adhesive cell type goes hand in hand with the evolution of complex bacterial life cycles