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SpoT-triggered stringent response controls usp gene expression in Pseudomonas aeruginosa.

ABSTRACT: The universal stress proteins (Usps) UspK (PA3309) and UspN (PA4352) of Pseudomonas aeruginosa are essential for surviving specific anaerobic energy stress conditions such as pyruvate fermentation and anaerobic stationary phase. Expression of the respective genes is under the control of the oxygen-sensing regulator Anr. In this study we investigated the regulation of uspN and three additional P. aeruginosa usp genes: uspL (PA1789), uspM (PA4328), and uspO (PA5027). Anr induces expression of these genes in response to anaerobic conditions. Using promoter-lacZ fusions, we showed that PuspL-lacZ, PuspM-lacZ, and PuspO-lacZ were also induced in stationary phase as described for PuspN-lacZ. However, stationary phase gene expression was abolished in the P. aeruginosa triple mutant Deltaanr DeltarelA DeltaspoT. The relA and spoT genes encode the regulatory components of the stringent response. We determined pppGpp and ppGpp levels using a thin-layer chromatography approach and detected the accumulation of ppGpp in the wild type and the DeltarelA mutant in stationary phase, indicating a SpoT-derived control of ppGpp accumulation. Additional investigation of stationary phase in LB medium revealed that alkaline pH values are involved in the regulatory process of ppGpp accumulation.

SUBMITTER: Boes N

PROVIDER: S-EPMC2580680 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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SpoT-triggered stringent response controls usp gene expression in Pseudomonas aeruginosa.

Boes Nelli N Schreiber Kerstin K Schobert Max M

Journal of bacteriology 20080905 21

The universal stress proteins (Usps) UspK (PA3309) and UspN (PA4352) of Pseudomonas aeruginosa are essential for surviving specific anaerobic energy stress conditions such as pyruvate fermentation and anaerobic stationary phase. Expression of the respective genes is under the control of the oxygen-sensing regulator Anr. In this study we investigated the regulation of uspN and three additional P. aeruginosa usp genes: uspL (PA1789), uspM (PA4328), and uspO (PA5027). Anr induces expression of thes ...[more]

PMID: 18776018

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Project description:The bacterial stringent stress response, mediated by the signaling molecule guanosine tetraphosphate, ppGpp, has recently gained attention as being important during normal cellular growth and as a potential new therapeutic target, which warrants detailed mechanistic understanding. Here, we used intracellular protein tracking in Pseudomonas aeruginosa PAO1, which indicated that RelA was bound to the ribosome, while SpoT localized at the cell poles. Transcriptome sequencing (RNA-Seq) was used to investigate the transcriptome of a ppGpp-deficient strain under nonstressful, nutrient-rich broth conditions where the mutant grew at the same rate as the parent strain. In the exponential growth phase, the lack of ppGpp led to >1,600 transcriptional changes (fold change cutoff of ±1.5), providing further novel insights into the normal physiological role of ppGpp. The stringent response was linked to gene expression of various proteases and secretion systems, including aprA, PA0277, impA, and clpP2 The previously observed reduction in cytotoxicity toward red blood cells in a stringent response mutant appeared to be due to aprA Investigation of an aprA mutant in a murine skin infection model showed increased survival rates of mice infected with the aprA mutant, consistent with previous observations that stringent response mutants have reduced virulence. In addition, the overexpression of relA, but not induction of ppGpp with serine hydroxamate, dysregulated global transcriptional regulators as well as >30% of the regulatory networks controlled by AlgR, OxyR, LasR, and AmrZ. Together, these data expand our knowledge about ppGpp and its regulatory network and role in environmental adaptation. It also confirms its important role throughout the normal growth cycle of bacteria.IMPORTANCE Microorganisms need to adapt rapidly to survive harsh environmental changes. Here, we showed the broad influence of the highly studied bacterial stringent stress response under nonstressful conditions that indicate its general physiological importance and might reflect the readiness of bacteria to respond to and activate acute stress responses. Using RNA-Seq to investigate the transcriptional network of Pseudomonas aeruginosa cells revealed that >30% of all genes changed expression in a stringent response mutant under optimal growth conditions. This included genes regulated by global transcriptional regulators and novel downstream effectors. Our results help to understand the importance of this stress regulator in bacterial lifestyle under relatively unstressed conditions. As such, it draws attention to the consequences of targeting this ubiquitous bacterial signaling molecule.

Project description:Cystic fibrosis (CF) is a genetic disease that affects mucin-producing body organs such as the lungs. Characteristic of CF is the production of thick, viscous mucus, containing the glycoprotein mucin, that can lead to progressive airway obstruction. Recently, we demonstrated that the presence of mucin induced a rapid surface adaptation in motile bacteria termed surfing motility, which data presented here indicates is very different from swarming motility. Pseudomonas aeruginosa, the main colonizing pathogen in CF, employs several stress coping mechanisms to survive the highly viscous environment of the CF lung. We used motility-based assays and RNA-Seq to study the stringent stress response in the hypervirulent CF isolate LESB58 (Liverpool Epidemic Strain). Motility experiments revealed that an LESB58 stringent response mutant (?relA?spoT) was unable to surf. Transcriptional profiling of ?relA?spoT mutant cells from surfing agar plates, when compared to wild-type cells from the surfing edge, revealed 2,584 dysregulated genes. Gene Ontology and KEGG enrichment analysis revealed effects of the stringent response on amino acid, nucleic acid and fatty acid metabolism, TCA cycle and glycolysis, type VI secretion, as well as chemotaxis, cell communication, iron transport, nitrogen metabolic processes and cyclic-di-GMP signalling. Screening of the ordered PA14 transposon library revealed 224 mutants unable to surf and very limited overlap with genes required for swarming. Mutants affecting surfing included two downstream effector genes of the stringent stress response, the copper regulator cueR and the quinolone synthase pqsH. Both the cueR and pqsH cloned genes complemented the surfing deficiency of ?relA?spoT. Our study revealed insights into stringent stress dependency in LESB58 and showed that surfing motility is stringently-controlled via the expression of cueR and pqsH. Downstream factors of the stringent stress response are important to investigate in order to fully understand its ability to colonize and persist in the CF lung.

Project description:BackgroundThe bacterium Pseudomonas aeruginosa is capable of three types of motilities: swimming, twitching and swarming. The latter is characterized by a fast and coordinated group movement over a semi-solid surface resulting from intercellular interactions and morphological differentiation. A striking feature of swarming motility is the complex fractal-like patterns displayed by migrating bacteria while they move away from their inoculation point. This type of group behaviour is still poorly understood and its characterization provides important information on bacterial structured communities such as biofilms. Using GeneChip® Affymetrix microarrays, we obtained the transcriptomic profiles of both bacterial populations located at the tip of migrating tendrils and swarm center of swarming colonies and compared these profiles to that of a bacterial control population grown on the same media but solidified to not allow swarming motility.ResultsMicroarray raw data were corrected for background noise with the RMA algorithm and quantile normalized. Differentially expressed genes between the three conditions were selected using a threshold of 1.5 log2-fold, which gave a total of 378 selected genes (6.3% of the predicted open reading frames of strain PA14). Major shifts in gene expression patterns are observed in each growth conditions, highlighting the presence of distinct bacterial subpopulations within a swarming colony (tendril tips vs. swarm center). Unexpectedly, microarrays expression data reveal that a minority of genes are up-regulated in tendril tip populations. Among them, we found energy metabolism, ribosomal protein and transport of small molecules related genes. On the other hand, many well-known virulence factors genes were globally repressed in tendril tip cells. Swarm center cells are distinct and appear to be under oxidative and copper stress responses.ConclusionsResults reported in this study show that, as opposed to swarm center cells, tendril tip populations of a swarming colony displays general down-regulation of genes associated with virulence and up-regulation of genes involved in energy metabolism. These results allow us to propose a model where tendril tip cells function as «scouts» whose main purpose is to rapidly spread on uncolonized surfaces while swarm center population are in a state allowing a permanent settlement of the colonized area (biofilm-like).

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SpoT-triggered stringent response controls usp gene expression in Pseudomonas aeruginosa.

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SpoT-triggered stringent response controls usp gene expression in Pseudomonas aeruginosa.

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