The Pseudomonas aeruginosa Transcriptome in Planktonic Cultures and Static Biofilms Using RNA Sequencing

In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5′-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms

Small-scale spatial variation in infection risk shapes the evolution of a Borrelia resistance gene in wild rodents

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record.Spatial variation in pathogen-mediated selection is predicted to influence the evolutionary trajectory of host populations and lead to spatial variation in their immunogenetic composition. However, to date few studies have been able to directly link small-scale spatial variation in infection risk to host immune gene evolution in natural, non-human populations. Here we use a natural rodent-Borrelia system to test for associations between landscape-level spatial variation in Borrelia infection risk along replicated elevational gradients in the Swiss Alps and Toll-like receptor 2 (TLR2) evolution, a candidate gene for Borrelia resistance, across bank vole (Myodes glareolus) populations. We found that Borrelia infection risk (i.e. the product of Borrelia prevalence in questing ticks and the average tick load of voles at a sampling site) was spatially variable and significantly negatively associated with elevation. Across sampling sites, Borrelia prevalence in bank voles was significantly positively associated with Borrelia infection risk along the elevational clines. We observed a significant association between naturally occurring TLR2 polymorphisms in hosts and their Borrelia infection status. The TLR2 variant associated with a reduced likelihood of Borrelia infection was most common in rodent populations at lower elevations that face a high Borrelia infection risk, and its frequency changed in accordance with the change in Borrelia infection risk along the elevational clines. These results suggest that small-scale spatial variation in parasite-mediated selection affects the immunogenetic composition of natural host populations, providing a striking example that the microbial environment shapes the evolution of the host’s immune system in the wild.The study was financially supported by the Swiss National Science Foundation (PP00P3_128386 and PP00P3_157455), the University of Zurich Research Priority Program ‘Evolution in Action: from Genomes to Ecosystems’, the Faculty of Science of the University of Zurich, the Baugarten Stiftung, the Stiftung für wissenschaftliche Forschung an der Universität Zürich (STWF 17_027) and the Georges und Antoine Claraz-Schenkung

RsmW, Pseudomonas aeruginosa small non-coding RsmA-binding RNA upregulated in biofilm versus planktonic growth conditions

BACKGROUND: Biofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state. RESULTS: A computational algorithm was devised to detect and categorize sRNAs into 5 types: intergenic, intragenic, 5′-UTR, 3′-UTR, and antisense. Here we report a novel RsmY/RsmZ-type sRNA, termed RsmW, in P. aeruginosa up-transcribed in biofilm versus planktonic growth. RNA-Seq, 5’-RACE and Mfold predictions suggest RsmW has a secondary structure with 3 of 7 GGA motifs located on outer stem loops. Northern blot revealed two RsmW binding bands of 400 and 120 bases, suggesting RsmW is derived from the 3’-UTR of the upstream hypothetical gene, PA4570. RsmW expression is elevated in late stationary versus logarithmic growth phase in PB minimal media, at higher temperatures (37°C versus 28°C), and in both gacA and rhlR transposon mutants versus wild-type. RsmW specifically binds to RsmA protein in vitro and restores biofilm production and reduces swarming in an rsmY/rsmZ double mutant. PA4570 weakly resembles an RsmA/RsmN homolog having 49% and 51% similarity, and 16% and 17% identity to RsmA and RsmN amino acid sequences, respectively. PA4570 was unable to restore biofilm and swarming phenotypes in ΔrsmA deficient strains. CONCLUSION: Collectively, our study reveals an interesting theme regarding another sRNA regulator of the Rsm system and further unravels the complexities regulating adaptive responses for Pseudomonas species

A Role for the RNA Chaperone Hfq in Controlling Adherent-Invasive Escherichia coli Colonization and Virulence

Adherent-invasive Escherichia coli (AIEC) has been linked with the onset and perpetuation of inflammatory bowel diseases. The AIEC strain LF82 was originally isolated from an ileal biopsy from a patient with Crohn's disease. The pathogenesis of LF82 results from its abnormal adherence to and subsequent invasion of the intestinal epithelium coupled with its ability to survive phagocytosis by macrophages once it has crossed the intestinal barrier. To gain further insight into AIEC pathogenesis we employed the nematode Caenorhabditis elegans as an in vivo infection model. We demonstrate that AIEC strain LF82 forms a persistent infection in C. elegans, thereby reducing the host lifespan significantly. This host killing phenotype was associated with massive bacterial colonization of the nematode intestine and damage to the intestinal epithelial surface. C. elegans killing was independent of known LF82 virulence determinants but was abolished by deletion of the LF82 hfq gene, which encodes an RNA chaperone involved in mediating posttranscriptional gene regulation by small non-coding RNAs. This finding reveals that important aspects of LF82 pathogenesis are controlled at the posttranscriptional level by riboregulation. The role of Hfq in LF82 virulence was independent of its function in regulating RpoS and RpoE activity. Further, LF82Δhfq mutants were non-motile, impaired in cell invasion and highly sensitive to various chemical stress conditions, reinforcing the multifaceted function of Hfq in mediating bacterial adaptation. This study highlights the usefulness of simple non-mammalian infection systems for the identification and analysis of bacterial virulence factors

In-Vivo Expression Profiling of Pseudomonas aeruginosa Infections Reveals Niche-Specific and Strain-Independent Transcriptional Programs

Pseudomonas aeruginosa is a threatening, opportunistic pathogen causing disease in immunocompromised individuals. The hallmark of P. aeruginosa virulence is its multi-factorial and combinatorial nature. It renders such bacteria infectious for many organisms and it is often resistant to antibiotics. To gain insights into the physiology of P. aeruginosa during infection, we assessed the transcriptional programs of three different P. aeruginosa strains directly after isolation from burn wounds of humans. We compared the programs to those of the same strains using two infection models: a plant model, which consisted of the infection of the midrib of lettuce leaves, and a murine tumor model, which was obtained by infection of mice with an induced tumor in the abdomen. All control conditions of P. aeruginosa cells growing in suspension and as a biofilm were added to the analysis. We found that these different P. aeruginosa strains express a pool of distinct genetic traits that are activated under particular infection conditions regardless of their genetic variability. The knowledge herein generated will advance our understanding of P. aeruginosa virulence and provide valuable cues for the definition of prospective targets to develop novel intervention strategies

Identification and functional characterization of small non-coding RNAs in Xanthomonas oryzae pathovar oryzae

<p>Abstract</p> <p>Background</p> <p>Small non-coding RNAs (sRNAs) are regarded as important regulators in prokaryotes and play essential roles in diverse cellular processes. <it>Xanthomonas oryzae </it>pathovar <it>oryzae </it>(<it>Xoo</it>) is an important plant pathogenic bacterium which causes serious bacterial blight of rice. However, little is known about the number, genomic distribution and biological functions of sRNAs in <it>Xoo</it>.</p> <p>Results</p> <p>Here, we performed a systematic screen to identify sRNAs in the <it>Xoo </it>strain PXO99. A total of 850 putative non-coding RNA sequences originated from intergenic and gene antisense regions were identified by cloning, of which 63 were also identified as sRNA candidates by computational prediction, thus were considered as <it>Xoo </it>sRNA candidates. Northern blot hybridization confirmed the size and expression of 6 sRNA candidates and other 2 cloned small RNA sequences, which were then added to the sRNA candidate list. We further examined the expression profiles of the eight sRNAs in an <it>hfq </it>deletion mutant and found that two of them showed drastically decreased expression levels, and another exhibited an Hfq-dependent transcript processing pattern. Deletion mutants were obtained for seven of the Northern confirmed sRNAs, but none of them exhibited obvious phenotypes. Comparison of the proteomic differences between three of the ΔsRNA mutants and the wild-type strain by two-dimensional gel electrophoresis (2-DE) analysis showed that these sRNAs are involved in multiple physiological and biochemical processes.</p> <p>Conclusions</p> <p>We experimentally verified eight sRNAs in a genome-wide screen and uncovered three Hfq-dependent sRNAs in <it>Xoo</it>. Proteomics analysis revealed <it>Xoo </it>sRNAs may take part in various metabolic processes. Taken together, this work represents the first comprehensive screen and functional analysis of sRNAs in rice pathogenic bacteria and facilitates future studies on sRNA-mediated regulatory networks in this important phytopathogen.</p

Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation

Using sample-matched transcriptomics and proteomics measurements it is now possible to begin to understand the impact of post-transcriptional regulatory programs in Enterobacteria. In bacteria post-transcriptional regulation is mediated by relatively few identified RNA-binding protein factors including CsrA, Hfq and SmpB. A mutation in any one of these three genes, csrA, hfq, and smpB, in Salmonella is attenuated for mouse virulence and unable to survive in macrophages. CsrA has a clearly defined specificity based on binding to a specific mRNA sequence to inhibit translation. However, the proteins regulated by Hfq and SmpB are not as clearly defined. Previous work identified proteins regulated by hfq using purification of the RNA-protein complex with direct sequencing of the bound RNAs and found binding to a surprisingly large number of transcripts. In this report we have used global proteomics to directly identify proteins regulated by Hfq or SmpB by comparing protein abundance in the parent and isogenic hfq or smpB mutant. From these same samples we also prepared RNA for microarray analysis to determine if alteration of protein expression was mediated post-transcriptionally. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all possible Salmonella proteins, respectively, with limited correlation between transcription and protein expression. These proteins represent a broad spectrum of Salmonella proteins required for many biological processes including host cell invasion, motility, central metabolism, LPS biosynthesis, two-component regulatory systems, and fatty acid metabolism. Our results represent one of the first global analyses of post-transcriptional regulons in any organism and suggest that regulation at the translational level is widespread and plays an important role in virulence regulation and environmental adaptation for Salmonella

The Gac-Rsm and SadB Signal Transduction Pathways Converge on AlgU to Downregulate Motility in Pseudomonas fluorescens

Flagella mediated motility in Pseudomonas fluorescens F113 is tightly regulated. We have previously shown that motility is repressed by the GacA/GacS system and by SadB through downregulation of the fleQ gene, encoding the master regulator of the synthesis of flagellar components, including the flagellin FliC. Here we show that both regulatory pathways converge in the regulation of transcription and possibly translation of the algU gene, which encodes a sigma factor. AlgU is required for multiple functions, including the expression of the amrZ gene which encodes a transcriptional repressor of fleQ. Gac regulation of algU occurs during exponential growth and is exerted through the RNA binding proteins RsmA and RsmE but not RsmI. RNA immunoprecipitation assays have shown that the RsmA protein binds to a polycistronic mRNA encoding algU, mucA, mucB and mucD, resulting in lower levels of algU. We propose a model for repression of the synthesis of the flagellar apparatus linking extracellular and intracellular signalling with the levels of AlgU and a new physiological role for the Gac system in the downregulation of flagella biosynthesis during exponential growth

Production of mannosylglycerate in Saccharomyces cerevisiae by metabolic engineering and bioprocess optimization

Mannosylglycerate (MG) is one of the most widespread compatible solutes among marine microorganisms adapted to hot environments. This ionic solute holds excellent ability to protect proteins against thermal denaturation, hence a large number of biotechnological and clinical applications have been put forward. However, the current prohibitive production costs impose severe constraints towards large-scale applications. All known microbial producers synthesize MG from GDP-mannose and 3-phosphoglycerate via a two-step pathway in which mannosyl-3-phosphoglycerate is the intermediate metabolite. In an early work, this pathway was expressed in Saccharomyces cerevisiae with the goal to confirm gene function (Empadinhas et al. in J Bacteriol 186:4075--4084, 2004), but the level of MG accumulation was low. Therefore, in view of the potential biotechnological value of this compound, we decided to invest further effort to convert S. cerevisiae into an efficient cell factory for MG production.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and also by project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI). Cristiana Faria was supported by a Ph.D. Grant from FCT (Ref. SFRH/ BD/79552/2011).info:eu-repo/semantics/publishedVersio