Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Gene expression profiling of constitutively competent comR and proQ mutants and RNase R (rnr) mutant of L. pneumophila

ABSTRACT: Bacterial competence is a genetically programmed process that is triggered as a response to environmental stimuli. DNA uptake from the extracellular milieu by competence requires the expression of a complex machinery composed of a competence pseudopilus and a DNA translocase. In L. pneumophila, it has been reported that microaerophilic conditions and genetic alteration of the competence repressors ComR and ProQ result in competence. We have analyzed the gene expression profiles of the proQ and comR mutants using the L. pneumophila Philadelphia-1 microarray. Many competence genes were found to be induced in the comR and proQ mutants and represent a defined, regulon. The gene expression profiling of the proQ and comR mutants was part of a study aimed at understanding the function of the processive 3'-5' exoribonuclease RNase R (rnr gene) in L. pneumophila. We found that the gene expression profile of the rnr mutant shares similarities with the gene expression profiles of the comR and proQ mutants and that loss of the exoribonuclease activity of RNase R was sufficient to induce competence development. RNase R is a processive 3'-5' exoribonuclease with a high degree of conservation in prokaryotes. Although some bacteria possess additional hydrolytic 3Â´-5-exoribonucleases such as RNase II, RNase R was found to be the only one predicted in the Legionella pneumophila genome. This provided a unique opportunity to study the role of RNase R in the absence of an additional RNase with similar enzymatic activity. We investigated the role of RNase R in the biology of Legionella pneumophila under various conditions and performed gene expression profiling using microarrays. At optimal growth temperature, loss of RNase R had no major consequence on bacterial growth and had a moderate impact on normal gene regulation. However, at lower temperatures loss of RNase R has a significant impact on bacterial growth and resulted in the accumulation of structured RNA degradation products. Concurrently, gene regulation was affected and specifically resulted in increased expression of the competence regulon. Loss of the exoribonuclease activity of RNase R was sufficient to induce competence development, a genetically-programmed process normally triggered as a response to environmental stimuli. Temperature-dependent expression of competence genes in the rnr mutant was found to be independent of the previously identified competence regulators comR and ProQ. comR: Three test samples and three reference samples were analyzed. The test sample being the strain KS79, a comR mutant of L. pneumophila strain JR32 which is used as a reference. proQ: Three test samples and three reference samples were analyzed. The test sample being the strain LELA3825, a proQ mutant of L. pneumophila strain JR32 which is used as a reference. rnr: Six test samples and six reference samples grown at 30Â°C or 37Â°C were analyzed. The test sample being the strain LELA559, a rnr mutant of L. pneumophila strain JR32 which is used as a reference.

ORGANISM(S): Legionella pneumophila

SUBMITTER: Xavier Charpentier

PROVIDER: E-GEOD-12796 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Loss of RNase R induces competence development in Legionella pneumophila.

Charpentier Xavier X Faucher Sébastien P SP Kalachikov Sergey S Shuman Howard A HA

Journal of bacteriology 20081010 24

RNase R is a processive 3'-5' exoribonuclease with a high degree of conservation in prokaryotes. Although some bacteria possess additional hydrolytic 3'-5' exoribonucleases such as RNase II, RNase R was found to be the only predicted one in the facultative intracellular pathogen Legionella pneumophila. This provided a unique opportunity to study the role of RNase R in the absence of an additional RNase with similar enzymatic activity. We investigated the role of RNase R in the biology of Legione ...[more]

PMID: 18849432

Similar Datasets

Project description:Bacterial competence is a genetically programmed process that is triggered as a response to environmental stimuli. DNA uptake from the extracellular milieu by competence requires the expression of a complex machinery composed of a competence pseudopilus and a DNA translocase. In L. pneumophila, it has been reported that microaerophilic conditions and genetic alteration of the competence repressors ComR and ProQ result in competence. We have analyzed the gene expression profiles of the proQ and comR mutants using the L. pneumophila Philadelphia-1 microarray. Many competence genes were found to be induced in the comR and proQ mutants and represent a defined, regulon. The gene expression profiling of the proQ and comR mutants was part of a study aimed at understanding the function of the processive 3'-5' exoribonuclease RNase R (rnr gene) in L. pneumophila. We found that the gene expression profile of the rnr mutant shares similarities with the gene expression profiles of the comR and proQ mutants and that loss of the exoribonuclease activity of RNase R was sufficient to induce competence development. RNase R is a processive 3'-5' exoribonuclease with a high degree of conservation in prokaryotes. Although some bacteria possess additional hydrolytic 3´-5-exoribonucleases such as RNase II, RNase R was found to be the only one predicted in the Legionella pneumophila genome. This provided a unique opportunity to study the role of RNase R in the absence of an additional RNase with similar enzymatic activity. We investigated the role of RNase R in the biology of Legionella pneumophila under various conditions and performed gene expression profiling using microarrays. At optimal growth temperature, loss of RNase R had no major consequence on bacterial growth and had a moderate impact on normal gene regulation. However, at lower temperatures loss of RNase R has a significant impact on bacterial growth and resulted in the accumulation of structured RNA degradation products. Concurrently, gene regulation was affected and specifically resulted in increased expression of the competence regulon. Loss of the exoribonuclease activity of RNase R was sufficient to induce competence development, a genetically-programmed process normally triggered as a response to environmental stimuli. Temperature-dependent expression of competence genes in the rnr mutant was found to be independent of the previously identified competence regulators comR and ProQ.

Project description:Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co-incubation with Legionella hackeliae and L. pneumophila DeltadotA. One hundred and thirty-one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. The results provide the basis for a better understanding of the complex host-pathogen interactions and for further studies on the Dictyostelium response to Legionella infection. The bacterial strains used in this study were L. pneumophila Philadelphia I JR32, L. pneumophila Philadelphia I JR32 LELA 3118 (dotA3118:Tn903 DLL LacZ) and L. hackeliae (ATCC 35250). The Legionella strains were grown on buffered charcoal yeast extract agar (BCYE) at 37M-BM-0C with 5% CO2 atmosphere for 3 days. The D. discoideum wild-type strain AX2 was grown at 23M-BM-0C in 75 cm2 cell-culture flasks with 10 ml HL5 medium. For infection, Dictyostelium cells were harvested, resuspended in a 1:1 solution of HL5 medium and Soerensen buffer. Fifteen millilitres of a 1M-CM-^W10e6 cells/ml suspension were seeded into a 75 square-cm cell culture flask and the amoebae were inoculated with 10e7 bacteria/ml. Three different pairs of infection were compared: 1. AX2 infected with L. pneumophila JR32 versus uninfected cells; 19 microarrays of seven independent infections; 2. AX2 infected with L. pneumophila JR32 versus AX2 infected with L. pneumophila JR32 delta DotA; 4 microarrays of two independent infections; 3. AX2 infected with L. pneumophila JR32 versus AX2 infected with L. hackeliae; 4 microarrays of two independent infections. 24h post infection the RNA was isolated from 1.5M-CM-^W10e7 Dictyostelium cells and microarray analysis was performed as described (Farbrother et al., 2006).

Project description:Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co-incubation with Legionella hackeliae and L. pneumophila DeltadotA. One hundred and thirty-one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response, Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. The results provide the basis for a better understanding of the complex host-pathogen interactions and for further studies on the Dictyostelium response to Legionella infection. The bacterial strains used in this study were L. pneumophila Philadelphia I JR32, L. pneumophila Philadelphia I JR32 LELA 3118 (dotA3118:Tn903 DLL LacZ) and L. hackeliae (ATCC 35250). The Legionella strains were grown on buffered charcoal yeast extract agar (BCYE) at 37M-BM-0C with 5% CO2 atmosphere for 3 days. The D. discoideum wild-type strain AX2 was grown at 23M-BM-0C in 75 cm2 cell-culture flasks with 10 ml HL5 medium. For infection, Dictyostelium cells were harvested, resuspended in a 1:1 solution of HL5 medium and Soerensen buffer. Fifteen millilitres of a 1M-CM-^W10e6 cells/ml suspension were seeded into a 75 square-cm cell culture flask and the amoebae were inoculated with 10e7 bacteria/ml. Three different pairs of infection were compared: 1. AX2 infected with L. pneumophila JR32 versus uninfected cells; 19 microarrays of seven independent infections; 2. AX2 infected with L. pneumophila JR32 versus AX2 infected with L. pneumophila JR32 delta DotA; 4 microarrays of two independent infections; 3. AX2 infected with L. pneumophila JR32 versus AX2 infected with L. hackeliae; 4 microarrays of two independent infections. 24h post infection the RNA was isolated from 1.5M-CM-^W10e7 Dictyostelium cells and microarray analysis was performed as described (Farbrother et al., 2006).

Project description:In bacteria, phenotypic heterogeneity rescues the need for diversity in isogenic populations and allow concomitant multiple survival strategies when choosing only one is too risky. This powerful tactic is exploited for competence in streptococci and results in a bimodal activation, where only a subset of the community triggers the system. Deciphering the mechanisms underlying this puzzling behavior has remained challenging, especially since its study has been mainly achieved in S. mutans, where two different but interconnected regulation networks control competence. In this work, we sought to determine the origin of bimodality associated to the ComRS system thanks to the simplified S. salivarius model that harbors no supplemental signaling system. Using a single cell fluorescence reporter system together with the overexpression of the main actors of the regulation network, we showed that the ComR intracellular concentration is directly linked to the proportion of competent cells in the population. We report that this type of activation requires a functional positive feedback loop acting on comS through Opp and a putative ComS exporter, PptAB. To determine the origin of the heterogeneity amplified by the loop, we developed a mathematical model suggesting that either noise on ComS or ComR abundance could explain bimodality. Because ComR abundance is central for competence bimodal activation, we conducted an in silico identification and a systematic deletion of all the Two Component Systems (TCS) present in S. salivarius and identified CovRS, a well described virulence regulator in GAS and GBS, as a repressor of comR transcription. In vitro direct binding of CovR with the promoter of comR and transcriptomics confirmed those data. As CovRS integrates environmental stimuli that control ComR intracellular concentration, it represents the missing puzzle piece bridging environmental conditions and competence (bimodal) activation in salivarius streptococci.

Dataset Information

Gene expression profiling of constitutively competent comR and proQ mutants and RNase R (rnr) mutant of L. pneumophila

Publications

Loss of RNase R induces competence development in Legionella pneumophila.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets