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Role of σ54 in the regulation of genes involved in biogenesis of type I and type IV pili in Xylella fastidiosa

ABSTRACT: The phytopathogen Xylella fastidiosa produces two classes of pili, long type IV pili and short type I pili, which are involved in motility and adhesion. In this work, we have investigated the role of σ54 factor and its involvement in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from citrus strain J1a12, and analyses of global gene expression profile by microarrays comparing the wild type and rpoN mutant strains showed that few genes exhibited differential expression. At least one gene, pilA1 (XF2542), which encodes the structural pilin protein of type IV fimbriae, showed decreased expression in the rpoN mutant whereas an operon encoding proteins of type I fimbriae were twofold more expressed in the mutant. Quantitative real time RT-PCR (qRT-PCR) analysis confirmed that pilA1 transcript was significantly reduced in the rpoN mutant. The transcriptional start site of pilA1 was determined by primer extension, and a canonical σ54-dependent promoter was found upstream of the start site. Genome sequence analysis of X. fastidiosa revealed five paralogues of pilA, but microarray and qRT-PCR data demonstrated that only the pilA1 transcript was significantly affected in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that σ54 regulates biofilm formation, probably via differential regulation of genes involved in type IV and type I fimbrial biogenesis. Keywords: rpoN mutant strain, pili

ORGANISM(S): Xylella fastidiosa

PROVIDER: GSE8097 | GEO | 2007/10/22

SECONDARY ACCESSION(S): PRJNA100951

REPOSITORIES: GEO

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Role of Ï54 in the regulation of genes involved in biogenesis of type I and type IV pili in Xylella fastidiosa

Project description:The phytopathogen Xylella fastidiosa produces two classes of pili, long type IV pili and short type I pili, which are involved in motility and adhesion. In this work, we have investigated the role of Ï54 factor and its involvement in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from citrus strain J1a12, and analyses of global gene expression profile by microarrays comparing the wild type and rpoN mutant strains showed that few genes exhibited differential expression. At least one gene, pilA1 (XF2542), which encodes the structural pilin protein of type IV fimbriae, showed decreased expression in the rpoN mutant whereas an operon encoding proteins of type I fimbriae were twofold more expressed in the mutant. Quantitative real time RT-PCR (qRT-PCR) analysis confirmed that pilA1 transcript was significantly reduced in the rpoN mutant. The transcriptional start site of pilA1 was determined by primer extension, and a canonical Ï54-dependent promoter was found upstream of the start site. Genome sequence analysis of X. fastidiosa revealed five paralogues of pilA, but microarray and qRT-PCR data demonstrated that only the pilA1 transcript was significantly affected in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that Ï54 regulates biofilm formation, probably via differential regulation of genes involved in type IV and type I fimbrial biogenesis. Direct comparison between wild type strain J1a12 and mutant strain rpoN-.

2010-06-25 | E-GEOD-8097 | biostudies-arrayexpress

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon

Project description:Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows exclusively in the xylem of several plants, causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time-course experiment (2, 8 and 12 hours) revealed many differentially expressed genes under nitrogen starvation, such as genes related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding a glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter and contains a predicted NtrC binding site. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon.

2010-12-21 | GSE21647 | GEO

Salmonella Typhimurium stbC mutant

Project description:Salmonella enterica serotype Typhimurium produces a variety of fimbrial appendages, among which the type 1 fimbriae is the most common type. In vitro static broth culture favors S. Typhimurium to produce type 1 fimbriae, while solid agar inhibits its expression. A transposon inserted in the stbC gene, which would encode an usher protein for Stb fimbriae of a non-flagellar S. Typhimurium LB5010 strain, conferred it to agglutinate yeast cells on both cultures, and was mannose-sensitive. Reverse transcription polymerase chain reaction (RT-PCR) revealed that the expression of the fimbrial major subunit gene fimA, and fimZ, a positive regulator gene of fimA, were both increased in the stbC mutant strain when grown on LB agar; fimW, a repressor gene of fimA, exhibited lower expression. Flagella were observed in the stbC mutant and this phenotype was correlated with the motile phenotype detected by MSRV agar medium and reaction with flagella antiserum. Microarray data and RT-PCR also indicated that the expression of three genes, motA, motB, and cheM, was enhanced in the stbC mutant. The S. Typhimurium stbC mutant was resistant to a variety of antibiotics, consistent with the finding that expression of yhcQ and ramA, two genes involved in multidrug resistance, was enhanced. A complementation test revealed that transforming a recombinant plasmid possessing the coding sequence of the stbC gene restored the mannose-sensitive agglutination phenotype to the stbC mutant much as that in the parental S. Typhimurium LB5010 strain, indicating the possibility of an interplay of different fimbrial systems in coordinating their expression. Key Words: Salmonella enterica serotype Typhimurium, fimbriae, type 1 fimbriae, stbC, transposon, multidrug resistant, flagella

2011-12-25 | GSE34685 | GEO

Salmonella Typhimurium stbC mutant

2011-12-25 | E-GEOD-34685 | biostudies-arrayexpress

Paraburkholderia phymatum σ54 controls utilization of dicarboxylates, motility and interbacterial competition

Project description:Paraburkholderia phymatum is a beta-proteobacterium, which lives in the soil and is able to enter nitrogen-fixing symbiosis with different legumes. The biological nitrogen fixation (BNF) process is of great ecological and agronomic importance. We previously showed that the expression of the key P. phymatum BNF enzyme – the nitrogenase –is regulated by the sigma factor σ54 (or RpoN) inside root nodules. This study focused on identifying the σ54 regulon of P. phymatum grown in nitrogen limited conditions using RNA-Sequencing. Among the genes significantly down-regulated in absence of σ54 we found those coding for a C4-dicarboxylate transport system (Bphy_0225-27), a flagellar biosynthesis cluster (Bphy_2926-64) and one of the two type 6 secretion system (T6SS-b) present in P. phymatum genome (Bphy_5978-97). Indeed, the σ54 mutant was unable to grow on C4 dicarboxylates (fumarate, malate and succinate) as the sole carbon source and was less motile compared to the wild-type strain. Both defects were complemented by adding rpoN in trans. Additionally, using reporter fusions we confirmed that T6SS-b expression is regulated by σ54. Finally, a σ54 mutant was less competitive than its parental strain against P. diazotrophica, suggesting a role of σ54 in controlling interbacterial competition.

2020-08-12 | GSE156048 | GEO

Salmonella Typhimurium fimZ mutant

Project description:Salmonella enterica serovar Typhimurium produces type 1 fimbriae on the outer membrane and such hair-like appendages is proposed to play a significant role in the attachment of the bacteria to host cells and tissues. S. Typhimurium cultured in static broth favors expression of type 1 fimbriae. Conversely, solid agar medium represses type 1 fimbrial expression. Production of type 1 fimbriae is cooperatively regulated by fimZ, fimY, stm0551, fimW, and fimU within the fim gene cluster. FimZ belongs to the response regulator of the two-component regulatory system in bacteria and functions as a DNA binding protein. FimZ can bind to the promoter of the fimbrial major subunit gene fimA to activate its expression and produce type 1 fimbriae. A fimZ mutant in LB5010 strain constructed by allelic exchange was found to be non-fimbriate. Total RNA was extracted from the parental LB5010 and the fimZ mutant strain cultured in static broth and analyzed by hybridization to a S. Typhimurium LT2 DNA microarray. Transcriptomic analysis indicated that the stress response related gene cpxP, soxS and type 1 fimbriae related genes were down-regulated, whereas plasmid-encoded fimbriae, flagella associated genes and virulence genes like virK and mgtC were up-regulated in the fimZ mutant strain. It is possible that FimZ protein may play a role to interact with other genes besides fim genes. Mechanisms of this crosstalk warrant further elucidation.

2017-01-20 | GSE93882 | GEO

Salmonella Typhimurium ATCC14028 fimZ mutant

Project description:Fimbriae are hair-like structures present on the outer membrane of many Enterobacteriaceae and such appendages are proposed to play a significant role in the attachment of the bacteria to host cells and tissues. Salmonella enterica serovar Typhimurium has the potential to produce 13 different fimbrial types, among which type 1 fimbriae is the most common found. Expression of type 1 fimbriae is cooperatively controlled by fimZ, fimY, stm0551, fimW, and fimU within the fim gene cluster. FimZ belongs to the response regulator of the two-component regulatory system in bacteria and functions as a DNA binding protein. FimZ is a positive regulator for type 1 fimbrial expression in S. Typhimurium. A fimZ mutant in ATCC 14028 strain constructed by allelic exchange did not produce type 1 fimbriae. Total RNA was extracted from the parental ATCC 14028 and its fimZ mutant cultured in static broth and analyzed by hybridization to a S. Typhimurium LT2 DNA microarray. Transcriptomic analysis indicated that the type 1 fimbriae related genes, multidrug efflux protein gene acrF were down-regulated, whereas flagella associated genes, chemotaxis and virulence genes such as invF and invH genes were up-regulated in the fimZ mutant strain. It is possible that FimZ protein may play a role to interact with other genes besides regulating type 1 fimbriae.

2017-08-12 | GSE102575 | GEO

Metabolomics and transcriptomics identify multiple downstream targets of Paraburkholderia phymatum σ54 during symbiosis with Phaseolus vulgaris

Project description:RpoN (σ54) is the key sigma factor for the regulation of transcription of nitrogen fixation genes in diazotrophic bacteria, which include alpha- and beta-rhizobia. Our previous studies showed that a rpoN mutant of the beta-rhizobial strain Paraburkholderia phymatum formed root nodules on Phaseolus vulgaris that were unable to reduce atmospheric nitrogen into ammonia. In an effort to further characterize the RpoN regulon of P. phymatum, transcriptomics was combined with a powerful metabolomics approach. The metabolome of P. vulgaris root nodules infected by the P. phymatum rpoN Fix- mutant revealed statistically significant metabolic changes compared to wild-type Fix+ nodules, including reduced amounts of chorismate and elevated levels of flavonoids. A transcriptome analysis on Fix+ and Fix- nodules – combined with a search for RpoN binding sequences in promoter regions of regulated genes – confirmed the expected control of σ54 on nitrogen fixation genes in nodules. The transcriptomic data also identified additional target genes, whose differential expression was able to explain the observed metabolite changes in a numerous cases. Moreover, the genes encoding the two-component regulatory system NtrBC were downregulated in root nodules induced by the rpoN mutant and contained a putative RpoN binding motif in their promoter region, suggesting direct regulation. The construction and characterization of an ntrB mutant strain revealed impaired nitrogen assimilation in free-living conditions, as well as a noticeable symbiotic phenotype by forming less but heavier nodules on P. vulgaris roots.

2018-06-06 | GSE111993 | GEO

Xylella fastidiosa

Project description:Role of σ54 in the regulation of genes involved in biogenesis of type I and type IV pili in Xylella fastidiosa

| PRJNA100951 | ENA

Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN

Project description:Alginate overproduction by P. aeruginosa, also known as mucoidy is associated with chronic endobronchial infections in cystic fibrosis (CF). Alginate biosynthesis in this bacterium is initiated by the extracytoplasmic function sigma factor (σ22, AlgU/T). In the wild type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered by the anti-sigma factor MucA that inhibits alginate production. However, the degradation of MucA by activated intramembrane proteases AlgW and/or MucP can lead to the conversion from nonmucoid strains to mucoid. Previously we reported that the absence of the sensor kinase KinB in PAO1 causes the initiation of AlgW-dependent proteolysis of MucA resulting in alginate overproduction. In the kinB mutant this activation requires alternate sigma factor RpoN (σ54). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant, RpoN controlled the expression of approximately 20% of the genome. Besides alginate biosynthesis and regulator genes such as AlgW, KinB, in concert with RpoN, also control a large number of genes including: those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, mice exhibited better survival when challenged with the kinB mutant than wt PAO1. Together, these data strongly suggest that KinB controls virulence factors important for acute pneumonia and conversion to mucoidy. 6 Samples total. Three with kinB mutant and three kinB wild type.

2012-03-01 | E-GEOD-35248 | biostudies-arrayexpress

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