Surface sensing for biofilm formation in Pseudomonas aeruginosa

YesAggregating and forming biofilms on biotic or abiotic surfaces are ubiquitous bacterial behaviors under various conditions. In clinical settings, persistent presence of biofilms increases the risks of healthcare-associated infections and imposes huge healthcare and economic burdens. Bacteria within biofilms are protected from external damage and attacks from the host immune system and can exchange genomic information including antibiotic-resistance genes. Dispersed bacterial cells from attached biofilms on medical devices or host tissues may also serve as the origin of further infections. Understanding how bacteria develop biofilms is pertinent to tackle biofilm-associated infections and transmission. Biofilms have been suggested as a continuum of growth modes for adapting to different environments, initiating from bacterial cells sensing their attachment to a surface and then switching cellular physiological status for mature biofilm development. It is crucial to understand bacterial gene regulatory networks and decision-making processes for biofilm formation upon initial surface attachment. Pseudomonas aeruginosa is one of the model microorganisms for studying bacterial population behaviors. Several hypotheses and studies have suggested that extracellular macromolecules and appendages play important roles in bacterial responses to the surface attachment. Here, I review recent studies on potential molecular mechanisms and signal transduction pathways for P. aeruginosa surface sensing.This work is supported by University of Bradfor

Chemoperception of Specific Amino Acids Controls Phytopathogenicity in Pseudomonas syringae pv. tomato

IMPORTANCE There is substantive evidence that chemotaxis is a key requisite for efficient pathogenesis in plant pathogens. However, information regarding particular bacterial chemoreceptors and the specific plant signal that they sense is scarce. Our work shows that the phytopathogenic bacterium Pseudomonas syringae pv. tomato mediates not only chemotaxis but also the control of pathogenicity through the perception of the plant abundant amino acids Asp and Glu. We describe the specificity of the perception of L- and D-Asp and L-Glu by the PsPto-PscA chemoreceptor and the involvement of this perception in the regulation of pathogenicity-related traits. Moreover, a saturating concentration of D-Asp reduces bacterial virulence, and we therefore propose that ligand-mediated interference of key chemoreceptors may be an alternative strategy to control virulence.Supplemental material for this article may be found at https://doi.org/10.1128/mBio .01868-19.We acknowledge M. Trini Gallegos for kindly provide plasmid pCdrA::gfpS and S. Nebreda for technical assistance.Chemotaxis has been associated with the pathogenicity of bacteria in plants and was found to facilitate bacterial entry through stomata and wounds. However, knowledge regarding the plant signals involved in this process is scarce. We have addressed this issue using Pseudomonas syringae pv. tomato, which is a foliar pathogen that causes bacterial speck in tomato. We show that the chemoreceptor P. syringae pv. tomato PscA (PsPto-PscA) recognizes specifically and with high affinity L-Asp, L-Glu, and D-Asp. The mutation of the chemoreceptor gene largely reduced chemotaxis to these ligands but also altered cyclic di-GMP (c-di-GMP) levels, biofilm formation, and motility, pointing to cross talk between different chemosensory pathways. Furthermore, the PsPto-PscA mutant strain showed reduced virulence in tomato. Asp and Glu are the most abundant amino acids in plants and in particular in tomato apoplasts, and we hypothesize that this receptor may have evolved to specifically recognize these compounds to facilitate bacterial entry into the plant. Infection assays with the wild-type strain showed that the presence of saturating concentrations of D-Asp also reduced bacterial virulence.This work was supported by grants AGL2015-63851-R and RTI2018-095222-B100 (to E.L.-S.) and BIO2016-76779-P (to T.K.) from the Ministerio de Economía y Competitividad, Spain. J.P.C.-V. was supported by the FPI program (BES-2016-076452, MINECOSpain)

Function, localization and regulation of the Pseudomonas aeruginosa diguanylate cyclase response regulator WspR

Thesis (Ph.D.)--University of Washington, 2013WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces c-di-GMP, which in turn stimulates biofilm formation. Previous work demonstrated that when phosphorylated in response to growth on surfaces, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. In this study, I sought to determine the physiological relevance of WspR and its subcellular clustering, as well as the mechanism of how WspR forms subcellular clusters. My results confirm that WspR contributes specifically to production of the Pel polysaccharide. Cluster formation appears to be an intrinsic property of WspR. Analysis of six single amino acid variants of WspR show that the formation of WspR-P subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR, making it more active in c-di-GMP production. Finally, I observed that c-di-GMP inhibition may play a role in the subcellular cluster formation of WspR. Oligomer formation visualized as subcellular clusters is a mechanism by which the activities of response regulator-diguanylate cyclases can be regulated. This work includes a supplementary movie depicting the movement of WspR subcellular clusters in a 5 s time frame

Stable Isotope Probing with 15N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density▿ †

Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with 15N, nearly all applications of this technique to date have used 13C. Successful application of SIP using 15N-DNA (15N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml−1 for 15N-labeled DNA, relative to 0.036 g ml−1 for 13C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml−1. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of 15N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate 15N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of 15N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use 15N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in 13C-DNA-SIP applications

Stable Isotope Probing with 15N2 Reveals Novel Noncultivated Diazotrophs in Soil▿

Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel 15N2-DNA stable isotope probing (5N2-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from 15N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the Rhizobiales while the other two represent deeply divergent lineages of noncultivated bacteria within the Betaproteobacteria and Actinobacteria, respectively. Analysis of nifH genes from 15N-labeled DNA also revealed three microbial groups, one of which was associated with Alphaproteobacteria while the others were associated with two noncultivated groups that are deeply divergent within nifH cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that 15N2-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by Actinobacteria outside of the order Actinomycetales

Diversity of Planctomycetes in Soil in Relation to Soil History and Environmental Heterogeneity

Members of the Planctomycetes, which were once thought to occur primarily in aquatic environments, have been discovered in soils on five continents, revealing that these Bacteria are a widespread and numerically abundant component of microbial communities in soil. We examined the diversity of Planctomycetes in soil samples obtained from experimental plots at an agricultural site in order to assess the extent of Planctomycetes diversity in soil, to determine whether management effects such as past land cover and compost addition affected the composition of the Planctomycetes community, and to determine whether the observations made could provide insight into the ecological distribution of these organisms. Analysis of Planctomycetes 16S rRNA gene sequences revealed a total of 312 ± 35 unique phylotypes in the soil at the site examined. The majority of these Planctomycetes sequences were unique, and the sequences had phylogenetic affiliations that included all major lineages in the Planctomycetaceae, as well as several novel groups of deeply divergent Planctomycetes. Both soil management history and compost amendment had significant effects on the Planctomycetes diversity, and variations in soil organic matter, Ca(2+) content, and pH were associated with variations in the Planctomycetes community composition. In addition, Planctomycetes richness increased in proportion to the area sampled and was correlated with the spatial heterogeneity of nitrate, which was associated with the soil management history at the orchard site examined. This report provides the first systematic assessment of the diversity of Planctomycetes in soil and also provides evidence that the diversity of this group increases with area as defined by the general power law description of the taxon-area relationship