Gene expression analysis of wild type Pectobacteriumwasabie SCC3193 wildtype compared to a SCC3193 inactivation mutant of RsmA, inactivation mutant of ExpA and an ExpA RsmA double inactivation mutant.
Ontology highlight
ABSTRACT:
ORGANISM(S): Pectobacterium wasabiae
PROVIDER: GSE47545 | GEO | 2013/09/01
SECONDARY ACCESSION(S): PRJNA206433
REPOSITORIES: GEO
Similar Datasets
Project description:The phytopathogen Pectobacterium wasabiae SCC3193 is a model for research on virulence regulation in soft rot pathogens. Two proteins acting as global regulators of the expression on genes involved in virulence in SCC3193 are ExpA, a DNA binding transcriptional regulator, and RsmA, an RNA binding post transcriptional regulator. Studies of related enterobacterial phytopathogens suggest that homologues of ExpA (GacA) affect gene expression partly by modulating RsmA-like protein activity. However, the exact extent of this regulon overlap has not been mapped. To elucidate the role of RsmA in ExpA controlled gene expression goes through RsmA in P. wasabiae we performed a gene expression microarray analysis of the wild type strain, an expA mutant, an rsmA mutant, and an expA rsmA double mutant. The microarray-based data of various virulence related genes was confirmed through quantitative RT-PCR. Subsequently, assays were made to link observed transcriptomic differences to changes in growth rate, PCWDE production, and virulence in planta between the four strains. We could conclude that the majority of ExpA regulation seems routed via RsmA modulation. However we could identify genes involved in electron transport, oligogalacturonide transport and metabolism, which ExpA seems to influence independently of RsmA, directly or indirectly. Gene expression analysis from P. wasabie SCC3193 wildtype, SCC3193 rsmA mutant, SCC3193 expA mutant and SCC3193 expA rsmA double mutant was performed on RNA samples extraxcted from the strains at late-logarithmic (late-log) and early stationary (stat) growth phase. Strains were grown in liquid hrp inducing minimal medium cultures supplemented with 0.4% polygalacturonic acid at 200 rpm shaking and 28 degrees celsius. Three biological replicates were used from each strain at each growth phase point.
2013-09-01 | E-GEOD-47545 | biostudies-arrayexpress
Project description:Posttranscriptional regulator RsmA is part of a regulatory system that controls production of plant cell wall degrading enzymes (PCWDEs), motility and other virulence factors among the Pectobacterium spp of plant pathogens. We constructed the knockout mutation of rsmA gene in Pectobacterium wasabiae strain SCC3193. The rsmA- strain has three times lower growth rate but increased virulence when compared to wild type. To explain this phenotype we compared wild type and rsmA- mRNA levels using genome wide microarray. We found that the synthesis of all known virulence factors and flagella is upregulated, while cell division and peptidoglycan synthesis is down regulated in rsmA- strain. The TCA cycle is directed fully to energy production, indicating low energetic status of the cell that is a probable reason for the low growth rate of the mutant. Growth experiments showed that the enzymatic activities of extracellular virulence factors polygalacturonase and pectate lyase are very high throughout the growth curve even without induction by plant component. Wild type cells are rod shaped in liquid media, but elongated and hyperflagellated in swarm plate containing potato tuber extract and in potato tuber. The rsmA- strain exhibits the same conditional phenotype, but differentiation into swarmer cells is faster and cell elongation more pronounced. Virulence experiments in potato tuber showed that although rsmA- strain causes bigger tissue maceration, its ability to compete with the natural bacterial community in the host is decreased. We conclude that the lack of RsmA switches the cells to infective phenotype characterized by expression of virulence factors and swarming. The lack of control over these energy costly processes is probably the reason for decreased growth rate and fitness. Gene expression analysis from P. wasabie SCC3193 wildtype and SCC3193 rsmA mutant was performed on RNA samples extraxcted from the rsmA mutant (at 40 and 48 hours) and wildtype (at 24 hours). The strain were grown on 1.5% agar M9 minimal media plates supplemented with 10% potato extract. Three biological replicates were used from each strain at each time point.
2012-08-24 | E-GEOD-40333 | biostudies-arrayexpress
Project description:Posttranscriptional regulator RsmA is part of a regulatory system that controls production of plant cell wall degrading enzymes (PCWDEs), motility and other virulence factors among the Pectobacterium spp of plant pathogens. We constructed the knockout mutation of rsmA gene in Pectobacterium wasabiae strain SCC3193. The rsmA- strain has three times lower growth rate but increased virulence when compared to wild type. To explain this phenotype we compared wild type and rsmA- mRNA levels using genome wide microarray. We found that the synthesis of all known virulence factors and flagella is upregulated, while cell division and peptidoglycan synthesis is down regulated in rsmA- strain. The TCA cycle is directed fully to energy production, indicating low energetic status of the cell that is a probable reason for the low growth rate of the mutant. Growth experiments showed that the enzymatic activities of extracellular virulence factors polygalacturonase and pectate lyase are very high throughout the growth curve even without induction by plant component. Wild type cells are rod shaped in liquid media, but elongated and hyperflagellated in swarm plate containing potato tuber extract and in potato tuber. The rsmA- strain exhibits the same conditional phenotype, but differentiation into swarmer cells is faster and cell elongation more pronounced. Virulence experiments in potato tuber showed that although rsmA- strain causes bigger tissue maceration, its ability to compete with the natural bacterial community in the host is decreased. We conclude that the lack of RsmA switches the cells to infective phenotype characterized by expression of virulence factors and swarming. The lack of control over these energy costly processes is probably the reason for decreased growth rate and fitness.
2012-08-24 | GSE40333 | GEO
Project description:Global expression profiling of airway epithelial cells infected with Pseudomonas aeruginosa and the rsmA mutant. Recent work in our laboratory investigated the impact of RsmA on a number of airway epithelial cell phenotypes including actin depolymerization, cytotoxicity and invasion. These cellular phenotypes were influenced by the positive effect of RsmA on the expression of the TTSS in P. aeruginosa (Mulcahy et al. 2006. Infect. Immun.). Pseudomonas aeruginosa is an important opportunistic pathogen which is capable of causing both acute and chronic infections in immunocompromised patients. Successful adaptation of the bacterium to its host environment relies on the ability of the organism to tightly regulate gene expression. RsmA, a small RNA-binding protein, controls the expression of a large number of virulence-related genes in P. aeruginosa, including those encoding the type III secretion system and associated effector proteins, with important consequences for epithelial cell morphology and cytotoxicity. In order to examine the influence of RsmA-regulated functions in the pathogen on gene expression in the host, we compared global expression profiles of airway epithelial cells in response to infection with P. aeruginosa PAO1 and an rsmA mutant. The RsmA-dependent response of host cells was characterized by significant changes in the global transcriptional pattern, including the increased expression of two Kruppel-like factors, KLF2 and KLF6. This increased expression was mediated by specific type III effector proteins. ExoS was required for the enhanced expression of KLF2, whereas both ExoS and ExoY were required for the enhanced expression of KLF6. Neither ExoT nor ExoU influenced the expression of the transcription factors. Additionally, the increased gene expression of KLF2 and KLF6 was associated with ExoS-mediated cytotoxicity. Therefore, this study identifies for the first time the human transcription factors KLF2 and KLF6 as targets of the P. aeruginosa type III exoenzymes S and Y, with potential importance in host cell death. Keywords: Expression profiling, Pseudomonas aeruginosa, microbial infection, airway epithelial cells, cystic fibrosis, exoenzymes, ExoS, ExoT, ExoU, ExoY, Kruppel-like factors, KLF2, KLF6
2006-09-15 | GSE4485 | GEO
Project description:In the opportunistic pathogen Pseudomonas aeruginosa RsmA is an RNA-binding protein that plays critical roles in the control of virulence, interbacterial interactions and biofilm formation. Although RsmA is thought to exert its regulatory effects by binding full-length transcripts, the extent to which RsmA binds nascent transcripts has not been addressed. Moreover, which transcripts are direct targets of this key post-transcriptional regulator is largely unknown. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, with cells grown in the presence and absence of the RNA polymerase inhibitor rifampicin, we identify hundreds of nascent transcripts that RsmA associates with in P. aeruginosa. We also find that the RNA chaperone Hfq targets a subset of the RsmA-associated nascent transcripts and that the two RNA-binding proteins can exert regulatory effects on common targets. Our findings establish that RsmA associates with many transcripts as they are being synthesized in P. aeruginosa, identify the direct targets of RsmA, and suggest that RsmA and Hfq may act in a combinatorial fashion on certain target transcripts. More broadly, our data suggest that the binding of post-transcriptional regulators to nascent transcripts may be commonplace in bacteria where distinct regulators can function alone or in concert to achieve control over the translation of transcripts as soon as they emerge from RNA polymerase.
2020-04-20 | GSE138338 | GEO
Project description:Investigation of whole genome gene expression level changes in Aspergillus nidulans OE::rsmA compared to wild-type RDIT9.32 (veA). A twelve array study using total RNA recovered from six separate cultures of Aspergillus nidulans wild-type RDIT9.32 (veA) and six separate cultures of Aspergillus nidulans overexpressing rsmA (restorer of secondary metabolism A), using custom-designed, four-plex arrays. The experiment was divided into two runs. In the first run, three biological replicates each of Aspergillus nidulans wild-type RDIT9.32 (veA) and Aspergillus nidulans carrying a plasmid overexpressing rsmA under the control of the gpdA promoter were assayed. In the second run, three biological replicates each of Aspergillus nidulans wild-type RDIT9.32 (veA) and Aspergillus nidulans overexpressing rsmA at the native locus under the control of the gpdA promoter were assayed.
2012-01-17 | E-GEOD-34220 | biostudies-arrayexpress
Project description:Investigation of whole genome gene expression level changes in Aspergillus nidulans OE::rsmA compared to wild-type RDIT9.32 (veA).
2012-01-17 | GSE34220 | GEO
Project description:Global expression profiling of airway epithelial cells infected with Pseudomonas aeruginosa and the rsmA mutant. Recent work in our laboratory investigated the impact of RsmA on a number of airway epithelial cell phenotypes including actin depolymerization, cytotoxicity and invasion. These cellular phenotypes were influenced by the positive effect of RsmA on the expression of the TTSS in P. aeruginosa (Mulcahy et al. 2006. Infect. Immun.). Pseudomonas aeruginosa is an important opportunistic pathogen which is capable of causing both acute and chronic infections in immunocompromised patients. Successful adaptation of the bacterium to its host environment relies on the ability of the organism to tightly regulate gene expression. RsmA, a small RNA-binding ; protein, controls the expression of a large number of virulence-related genes in P. aeruginosa, including those encoding the type III secretion system and associated effector proteins, with important consequences for epithelial cell morphology and cytotoxicity. In order to examine the influence of RsmA-regulated functions in ; the pathogen on gene expression in the host, we compared global expression profiles of airway epithelial cells in response to infection with P. aeruginosa PAO1 and an rsmA mutant. The RsmA-dependent response of host cells was characterized by significant changes in the global transcriptional pattern, including the increased expression of two Kruppel-like factors, KLF2 and KLF6. This increased expression was mediated by specific type III effector proteins. ExoS was required for the enhanced expression of KLF2, whereas both ExoS and ExoY were required for the enhanced expression of KLF6. Neither ExoT nor ExoU influenced the expression of the transcription factors. Additionally, the increased gene expression of KLF2 and KLF6 was associated with ExoS-mediated cytotoxicity. Therefore, this study identifies for the first time the human transcription factors ; KLF2 and KLF6 as targets of the P. aeruginosa type III exoenzymes S and Y, with potential importance in host cell death. Experiment Overall Design: RNA isolation and microarray sample preparation Experiment Overall Design: Biological samples from two independent infection experiments were used in two independent microarray experiments as outlined below. Total RNA was isolated from epithelial cells using a RNeasy Kit (Qiagen) according to manufacturer's instructions. Genomic DNA was removed using DNA-free (Ambion) and confirmed by PCR to be free of DNA, prior to cDNA synthesis. Isolated RNA was used in the preparation of fragmented cRNA for microarray hybridization according to the manufacturer's protocol (Affymetrix). Briefly, 8.5ug of RNA was converted to double stranded cDNA by reverse transcription, using a Superscript cDNA synthesis kit (Invitrogen), with an oligo dT primer containing a T7 RNA polymerase site added 3' of the poly (T) (Affymetrix). After second strand synthesis, cDNA was converted to biotin-labelled cRNA by in vitro transcription (Enzo). The labelled cRNA was purified using Genechip clean up module (Qiagen). Total cRNA quality and yield was measured using a GeneQuant spectrophotometer and 20ug of cRNA was fragmented at 94ºC for 35 mins in fragmentation buffer (40 mM tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate). Full length and fragmented cRNA sample integrity was assessed by electrophoresis on a 1% agarose-TBE gel visualized by staining with SyBr green. Fragmented cRNA was sent to MRC geneservice (Cambridge, UK) for assessment of RNA quality using a Bioanalyzer (Agilent) before hybridization and scanning. The arrays were washed and stained in a fluidics station and scanned according to the manufacturer's protocols (Affymetrix). Experiment Overall Design: Microarray data analysis Experiment Overall Design: Raw image analysis was performed using Microarray Suite v5.0 software (Affymetrix). Downstream data analysis was performed using GeneSight software version 2.7 (Biodiscovery). For all analyses, the signal intensities from two independent infection and microarray experiments were used. Statistically significant changes in gene expression were identified by firstly performing analysis of variance (ANOVA), for comparison between multiple groups, with a P value of < 0.05. The list of transcripts generated using ANOVA was then subjected to a Student's t-test, for comparison between two groups, with a P value of < 0.05. To identify different levels of gene expression compared to baseline, a fold change cut-off of greater than or equal to 1.5 was applied to this data.
2007-10-27 | E-GEOD-4485 | biostudies-arrayexpress
Project description:This RNA-seq analysis examined the global effect of the RNA binding RsmA proteins in P. syringae under the T3SS-inducing condition and in KB medium. The results showed differential expression of more than 50% genes in the genome, which include significant down-regulation of T3SS genes.
2021-03-16 | GSE162091 | GEO
Project description:Erwinia carotovora subsp. atroseptica (Eca) is an enterobacterial phytopathogen causing economically-significant soft rot disease. Pathogenesis is mediated by multiple secreted virulence factors, many of which are secreted by the Type II (Out) secretion system. DsbA catalyses the introduction of disulphide bonds into periplasmic and secreted proteins. In this study, the extracellular proteome (secretome) of wild type Eca SCRI1043 and dsbA and out mutants was analysed by spectral counting mass spectrometry. This revealed that dsbA inactivation had a huge impact on the secretome and identified diverse DsbA- and Out-dependent secreted proteins, representing known, predicted and novel candidate virulence factors. Further characterisation of the dsbA mutant showed that secreted enzyme activities, motility, production of the quorum sensing signal and virulence were absent or greatly reduced. The impact of DsbA on secreted virulence factor production was mediated at multiple levels, including impacting on the Out secretion system and the virulence gene regulatory network. Transcriptome analyses revealed that the abundance of a broad, but defined, set of transcripts, including many virulence factors, was altered in the dsbA mutant, identifying a new virulence regulon responsive to extracytoplasmic conditions. In conclusion, DsbA plays a crucial, multi-faceted role in the pathogenesis of Eca. Keywords: Mutant comparison
2008-08-26 | GSE10647 | GEO