Post-translational responses to elevated c-di-GMP levels in Pseudomonas aeruginosa
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Pseudomonas Aeruginosa Pao1
TISSUE(S): Cell Culture
SUBMITTER: 
Julius Ned Witte
LAB HEAD: Susanne Hauessler
PROVIDER: PXD027098 | Pride | 2022-07-25
REPOSITORIES: Pride
Similar Datasets
Project description:Pseudomonas aeruginosa is a Gram-negative bacterium able to survive and adapt in a multitude of niches as well as thriving within many different hosts. This versatility lies within a large genome of ca 6Mbp and a tight control in the expression of thousands of genes. Among the regulatory mechanisms widely spread in bacteria, cyclic-di-GMP signaling is one which influences all levels of control. It is made by diguanylate cyclases and broken by phosphodiesterases while the intracellular level of this molecule drives phenotypic responses. The signaling involve modification of enzymes or proteins function upon c-di-GMP binding, including modifying the activity of regulators which in turn will impact the transcriptome. In P. aeruginosa, there are ca 40 genes encoding putative DGC or PDE. The combined activity of those enzymes should reflect the c-di-GMP concentration while specific phenotypic output could be correlated to a given set of dgc/pde. This notion of specificity has been addressed in several studies and different strains of P. aeruginosa. Here, we engineered a mutant library for the 41 individual dgc/pde genes in P. aeruginosa PAO1. In most cases, we observed a significant to slight variation in the global c-di-GMP pool, with essentially an impact on biofilm phenotypes including initial attachment and maturation, while very little is observed on motility which differs from previous studies. We observe that minor changes in c-di-GMP level have a drastic phenotypic impact thus supporting the idea of local vs global c-di-GMP pool. Our RNA-seq analysis indicates that all PAO1 dgc/pde genes are expressed in both planktonic and biofilm growth conditions and our work suggests that c-di-GMP networks owe to be reconstructed in one single strain and cannot be built by cross-comparison with studies in other strains.
2022-05-23 | GSE203348 | GEO
Project description:Transcriptional profiles of Escherichia coli MG1655 in mixed culture with Pseudomonas aeruginosa PAO1 showed a number of E. coli genes to be upregulated including purA-F and other genes associated with purine synthesis. In contrast, genes associated with pyrimidine synthesis were unaffected. Competition experiments in both planktonic and biofilm cultures, using three purine synthesis mutants, purD, purH, and purT showed little difference in E. coli survival from the parent strain. As purines are components of the cell signals, cAMP and c-di-GMP, we conducted competition experiments with E. coli mutants lacking adenylate cyclase (cyaA), cAMP phosphodiesterase (cpdA), and the catabolite receptor protein (crp), as well as ydeH, an uncharacterized gene that has been associated with c-di-GMP synthesis. Survival of the cyaA and crp mutants during co-culture were significantly less than the parent strain. Supplementation of the media with 1mM cAMP could restore survival of the cyaA mutant but not the crp mutant. In contrast, survival of the cpdA mutant was similar to the parent strain. Survival of the ydeH mutant was moderately less than the parent, suggesting that cAMP has more impact on E. coli mixed culture growth than c-di-GMP. Addition of 1 mM indole restored the survival of both the cyaA and crp mutations. Mutants in genes for tryptophan synthesis (trpE) and indole production (tnaA) showed a loss of competition and recovery through indole supplementation, comparable to the cyaA and crp mutants. Overall, these results suggest indole and cAMP as major contributing factors to E. coli growth in mixed culture. E. coli MG1655 and P. aeruginosa PAO1 were grown in a minimal defined medium containing N-acetyl glucosamine, overnight at 37C to an OD of 1.5, and then inoculated in fresh media at an OD of 0.001 and grown in pure or mixed culture for 4-5 hours to a final OD of 0.5. RNA was extracted, purified, reverse transcribed to cDNA and then analyzed on E. coli and P. aeruginosa chips from Affymetrix. Expression profiles of mixed culture-grown organisms were compared to pure culture E. coli and P. aeruginosa grown on the same defined medium.
2011-11-29 | E-GEOD-26932 | biostudies-arrayexpress
Project description:In the Pseudomonas aeruginosa type strain PA14, 40 genes are known to encode for diguanylate cyclases (DGCs) and/or phosphodiesterases (PDEs), which modulate the intracellular pool of the nucleotide second messenger c-di-GMP. While in general, high levels of c-di-GMP drive the switch from highly motile phenotypes towards a sessile lifestyle, the different c-di-GMP modulating enzymes are responsible for smaller and in parts non-overlapping phenotypes. In this study, we sought to utilize previously recorded P. aeruginosa gene expression datasets on 414 clinical isolates to uncover transcriptional changes as a result of a high expression of genes encoding diguanylate cyclases. We demonstrate that the selection of sub-groups of clinical isolates with high versus low expression of regulatory genes served the identification of their downstream regulons. Thus, analyzing transcriptional profiles of clinical isolates with variant expression of diguanylate cyclase genes might provide a unique opportunity to bypass the problem that for many c-di-GMP modulating enzymes it is not known under which conditions their expression is activated. However, because the high c-di-GMP associated phenotypes were rapidly lost in the clinical isolates, we were unable to confirm diguanylate specific gene regulation. It is possible that the elevated c-di-GMP concentrations observed in the clinical P. aeruginosa isolates were due to a memory response, in which the bacteria maintain a certain level of c-di-GMP even after being removed from the conditions that induced the production of c-di-GMP. Further studies would be needed to determine the specific mechanisms underlying this memory response in P. aeruginosa.
2023-08-21 | GSE233207 | GEO
Project description:Bacterial lifestyles are dictated by the conditions encountered during colonization of a specific ecological niche or host. A wide range of environmental stimuli triggers sensory modules, that modify activity of proteins, or/and initiate a transfer of information in complex regulatory networks, which modulate gene expression, and therefore adaptation. The transition between planktonic and biofilm growth is dependent on the homeostasis of the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclase (DGC) activity favor biofilm while low levels maintained by phosphodiesterase (PDE) enzymes encourage planktonic style or biofilm dispersal. In Pseudomonas aeruginosa over 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual enzymes possessing both canonical DGC and PDE motifs, i.e. GGDEF and EAL, respectively. It has been previously reported that the deletion of the PDE/DGC dual enzyme PA0285, one of 5 c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms and causes elevated c-di-GMP levels in P. aeruginosa. Here we confirm that this role is conserved in various P. aeruginosa (PAO1, PA14 and PAK) and Pseudomonas putida strains. Deletion of PA0285 has the highest impact during the early stage of surface colonization, and RNA-seq analysis in biofilm context indicates that it may be associated with upregulation of cupA fimbrial genes. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds the respective GTP (GGDEF) and c-di-GMP (EAL) substrates but only exhibits PDE activity in vitro. Both GGDEF and EAL domains are important for PA0285 PDE activity in vivo as suggested by site-directed mutants in each of the key motifs. Complementation of the PA0285 mutant strain with the C-terminal GGDEF/EAL portion in trans is not as effective as with the full-length gene suggesting that the transmembrane region and PAS domains contained in PA0285’s N-terminal portion influence its PDE activity in vivo. We speculate that the transmembrane portion or/and PAS domains of PA0285 impede its PDE activity in vivo through modulating the conformation of the enzyme, as shown for P. aeruginosa RbdA, in response to an unknown stimulus.
2023-07-01 | GSE223663 | GEO
Project description:C-di-GMP signaling can directly influence bacterial behavior by affecting the functionality of c-di-GMP-binding proteins. In addition, c-di-GMP can exert an indirect and more global effect on gene transcription or translation, e.g. via riboswitches or by binding to transcription factors. In this study, we investigated the effects of changes in intracellular c-di-GMP levels on gene expression and protein production in the opportunistic pathogen Pseudomonas aeruginosa. We induced c-di-GMP production via an ectopically introduced diguanylate cyclase and recorded the transcriptional, translational as well as proteomic profile of the cells. We demonstrate that rising levels of c-di-GMP in P. aeruginosa under growth conditions otherwise characterized by low c-di-GMP levels immediately cause a switch to a non-motile, auto-aggregative phenotype. This switch became apparent before any c-di-GMP-dependent role on transcription, translation, or protein abundance could be observed. Our results indicate that sudden rises in global c-di-GMP pools affect the P. aeruginosa phenotype via an alteration of protein functionality, rather than an impact on global gene transcription or translation.
2024-09-03 | GSE183394 | GEO
Project description:Sinorhizobium meliloti is a soil-dwelling symbiotic alphaproteobacterium. Cyclic di-GMP is an important second messenger controlling multiple functions in this microorganism. To understand transcriptional regulation by elevated c-di-GMP in S. meliloti, the transcriptome analysis was performed on the wild type strain S. meliloti Rm2011 carrying either an empty vector pWBT or diguanylate cyclase gene pleD overexpression plasmid pWBT-pleD.
2017-06-01 | E-MTAB-5410 | biostudies-arrayexpress
Project description:We analyzed a deletion mutant of the ECF M-OM-^C factor SigX and applied mRNA profiling to define the SigX dependent regulon in P. aeruginosa in response to low osmolarity medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and c-di-GMP signaling. In accordance, a sigX deletion mutant exhibited an altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative M-OM-^C factor SigX revealing its role in complex virulence-associated phenotypes in P. aeruginosa. Transcriptome profiling of the SigX deletion mutant and overexpressing wildtype complemented by ChIP-seq
2013-11-11 | E-GEOD-50937 | biostudies-arrayexpress
Project description:Transcriptional profiles of Escherichia coli MG1655 in mixed culture with Pseudomonas aeruginosa PAO1 showed a number of E. coli genes to be upregulated including purA-F and other genes associated with purine synthesis. In contrast, genes associated with pyrimidine synthesis were unaffected. Competition experiments in both planktonic and biofilm cultures, using three purine synthesis mutants, purD, purH, and purT showed little difference in E. coli survival from the parent strain. As purines are components of the cell signals, cAMP and c-di-GMP, we conducted competition experiments with E. coli mutants lacking adenylate cyclase (cyaA), cAMP phosphodiesterase (cpdA), and the catabolite receptor protein (crp), as well as ydeH, an uncharacterized gene that has been associated with c-di-GMP synthesis. Survival of the cyaA and crp mutants during co-culture were significantly less than the parent strain. Supplementation of the media with 1mM cAMP could restore survival of the cyaA mutant but not the crp mutant. In contrast, survival of the cpdA mutant was similar to the parent strain. Survival of the ydeH mutant was moderately less than the parent, suggesting that cAMP has more impact on E. coli mixed culture growth than c-di-GMP. Addition of 1 mM indole restored the survival of both the cyaA and crp mutations. Mutants in genes for tryptophan synthesis (trpE) and indole production (tnaA) showed a loss of competition and recovery through indole supplementation, comparable to the cyaA and crp mutants. Overall, these results suggest indole and cAMP as major contributing factors to E. coli growth in mixed culture. Two replicates of LB grown E. coli ZK126 and P. aeruginosa PAO1 were grown in pure culture at 37C to an OD of 0.3 (log phase) and then mixed 1:1 and incubated for an additional 45 min. RNA was extracted, purified, reverse transcribed to cDNA and then analyzed on E. coli and P. aeruginosa chips from Affymetrix. Expression profiles were compared to pure culture E. coli and P. aeruginosa grown on LB.
2011-11-09 | E-GEOD-26931 | biostudies-arrayexpress
Project description:We analyzed a deletion mutant of the ECF σ factor SigX and applied mRNA profiling to define the SigX dependent regulon in P. aeruginosa in response to low osmolarity medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and c-di-GMP signaling. In accordance, a sigX deletion mutant exhibited an altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative σ factor SigX revealing its role in complex virulence-associated phenotypes in P. aeruginosa.
2013-11-11 | GSE50937 | GEO
Project description:To determined the phosphorylation sites of MvaU purified from MPAO1, PAO1, PAO1/pHERD20T-fpkA, PAO1/pHERD20T-fpkB, PAO1/pHERD20T-fpkA-pfkB and PAO1/pHERD20T-fpkA-pfkB-pfpC
2024-07-20 | PXD035222 | Pride