GcsR, a TyrR-Like Enhancer-Binding Protein, Regulates Expression of the Glycine Cleavage System in Pseudomonas aeruginosa PAO1

Sarwar, Zaara; Lundgren, Benjamin R.; Grassa, Michael T.; Wang, Michael X.; Gribble, Megan; Moffat, Jennifer F.; Nomura, Christopher T.

doi:10.1128/msphere.00020-16

Cited by 23 publications

(40 citation statements)

References 61 publications

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“…P. aeruginosa PAO1 was grown in minimal medium supplemented with 20 mM ethanolamine and 20 mM NH 4 Cl. At an OD 600 of 0.3, 0.5 ml of culture was treated with 1.0 ml of RNAprotect bacterial reagent (Qiagen), and RNA was then purified from the treated cells using the RNeasy kit (Qiagen) (25). Prior to cDNA synthesis, the purified RNA was checked for genomic DNA contamination by PCR designed to amplify the rplU gene (24,30,31).…”

Section: Methodsmentioning

confidence: 99%

“…Mapping the PA4022-eat-eutBC operon. The operon arrangement of the PA4022-eat-eutBC genes was examined using reverse transcriptase PCR (RT-PCR) (25). P. aeruginosa PAO1 was grown in minimal medium supplemented with 20 mM ethanolamine and 20 mM NH 4 Cl.…”

Section: Methodsmentioning

confidence: 99%

“…The P. aeruginosa and Escherichia coli strains used in this study are given in Table S1 in the supplemental material. The ⌬PA4021, ⌬PA4022, ⌬eat, and ⌬eutB deletion mutants of P. aeruginosa PAO1 were constructed using established methods that have been described (23)(24)(25). Bacteria were grown in Lennox broth (LB) or minimal medium (22 mM KH 2 PO 4 , 42 mM Na 2 HPO 4 , 8.6 mM NaCl, 1.0 mM MgSO 4 , 5.0 M FeSO 4 , and 2 mg liter Ϫ1 cyanocobalamin [pH 7.0]).…”

Section: Methodsmentioning

confidence: 99%

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Ethanolamine Catabolism in Pseudomonas aeruginosa PAO1 Is Regulated by the Enhancer-Binding Protein EatR (PA4021) and the Alternative Sigma Factor RpoN

et al. 2016

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Although genes encoding enzymes and proteins related to ethanolamine catabolism are widely distributed in the genomes of Pseudomonas spp., ethanolamine catabolism has received little attention among this metabolically versatile group of bacteria. In an attempt to shed light on this subject, this study focused on defining the key regulatory factors that govern the expression of the central ethanolamine catabolic pathway in Pseudomonas aeruginosa PAO1. This pathway is encoded by the PA4022-eateutBC operon and consists of a transport protein (Eat), an ethanolamine-ammonia lyase (EutBC), and an acetaldehyde dehydrogenase (PA4022). EutBC is an essential enzyme in ethanolamine catabolism because it hydrolyzes this amino alcohol into ammonia and acetaldehyde. The acetaldehyde intermediate is then converted into acetate in a reaction catalyzed by acetaldehyde dehydrogenase. Using a combination of growth analyses and ␤-galactosidase fusions, the enhancer-binding protein PA4021 and the sigma factor RpoN were shown to be positive regulators of the PA4022-eat-eutBC operon in P. aeruginosa PAO1. PA4021 and RpoN were required for growth on ethanolamine, and both of these regulatory proteins were essential for induction of the PA4022-eat-eutBC operon. Unexpectedly, the results indicate that acetaldehyde (and not ethanolamine) serves as the inducer molecule that is sensed by PA4021 and leads to the transcriptional activation of the PA4022-eat-eutBC operon. Due to its regulatory role in ethanolamine catabolism, PA4021 was given the name EatR. Both EatR and its target genes are conserved in several other Pseudomonas spp., suggesting that these bacteria share a mechanism for regulating ethanolamine catabolism. IMPORTANCEThe results of this study provide a basis for understanding ethanolamine catabolism and its regulation in Pseudomonas aeruginosa PAO1. Interestingly, expression of the ethanolamine-catabolic genes in this bacterium was found to be under the control of a positive-feedback regulatory loop in a manner dependent on the transcriptional regulator PA4021, the sigma factor RpoN, and the metabolite acetaldehyde. Previously characterized regulators of ethanolamine catabolism are known to sense and respond directly to ethanolamine. In contrast, PA4021 (EatR) appears to monitor the intracellular levels of free acetaldehyde and responds through transcriptional activation of the ethanolamine-catabolic genes. This regulatory mechanism is unique and represents an alternative strategy used by bacteria to govern the acquisition of ethanolamine from their surroundings. E thanolamine serves as a source of carbon and nitrogen for a variety of bacteria, including members of the Enterobacteriaceae, Pseudomonadaceae, and Firmicutes (1-5). From studies mostly centered on Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli, there is now a basic understanding of the catabolic steps involved in ethanolamine utilization. Extracellular ethanolamine enters the bacterial cell through simple diffusion or carrier-me...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Ethanolamine Catabolism in Pseudomonas aeruginosa PAO1 Is Regulated by the Enhancer-Binding Protein EatR (PA4021) and the Alternative Sigma Factor RpoN

et al. 2016

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“…, recently named gcsR for glycine cleavage system regulator, Sarwar et al. ), and small indels leading to putative loss‐of‐function mutations in two protease complexes that share similar protein targets and functions ( clpA from the ClpAP serine protease complex, and ftsH , hflC , and hflK from the FtsH/HflKC complex; Zhou and Jin ; Lundgren et al. ).…”

Section: Resultsmentioning

confidence: 99%

Epistatic interactions between ancestral genotype and beneficial mutations shape evolvability inPseudomonas aeruginosa

2016

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The idea that interactions between mutations influence adaptation by driving populations to low and high fitness peaks on adaptive landscapes is deeply ingrained in evolutionary theory. Here, we investigate the impact of epistasis on evolvability by challenging populations of two Pseudomonas aeruginosa clones bearing different initial mutations (in rpoB conferring rifampicin resistance, and the type IV pili gene network) to adaptation to a medium containing l-serine as the sole carbon source. Despite being initially indistinguishable in fitness, populations founded by the two ancestral genotypes reached different fitness following 300 generations of evolution. Genome sequencing revealed that the difference could not be explained by acquiring mutations in different targets of selection; the majority of clones from both ancestors converged on one of the following two strategies: (1) acquiring mutations in either PA2449 (gcsR, an l-serine-metabolism RpoN enhancer binding protein) or (2) protease genes. Additionally, populations from both ancestors converged on loss-of-function mutations in the type IV pili gene network, either due to ancestral or acquired mutations. No compensatory or reversion mutations were observed in RNA polymerase (RNAP) genes, in spite of the large fitness costs typically associated with mutations in rpoB. Although current theory points to sign epistasis as the dominant constraint on evolvability, these results suggest that the role of magnitude epistasis in constraining evolvability may be underappreciated. The contribution of magnitude epistasis is likely to be greatest under the biologically relevant mutation supply rates that make back mutations probabilistically unlikely.

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“…Significant changes in the level of transcripts coding for the GlyA1, GlyA2, GcvH2, GcvP2 and GcvT2 proteins of glycine cleavage system were noticed in the mutated strain (Table ). Glycine cleavage system is a metabolic pathway for the conversion of glycine into pyruvate, thus allowing the latter to enter the central metabolism (Sarwar et al ., ). Therefore, it may be speculated that the elevated level of glycine cleavage components is responsible for the increase in pyruvate, which is converted into phosphoenol pyruvate by PEP kinase and enters the very first step of phenazines biosynthesis increasing their production.…”

Section: Discussionmentioning

confidence: 97%

Nudix‐type RNA pyrophosphohydrolase provides homeostasis of virulence factor pyocyanin and functions as a global regulator in Pseudomonas aeruginosa

Kujawa

Lirski

Ziecina

et al. 2017

Molecular Microbiology

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The PA0336 protein from Pseudomonas aeruginosa belongs to the family of widely distributed Nudix pyrophosphohydrolases, which catalyze the hydrolysis of pyrophosphate bonds in a variety of nucleoside diphosphate derivatives. The amino acid sequence of the PA0336 protein is highly similar to that of the RppH Nudix RNA pyrophosphohydrolase from Escherichia coli, which removes pyrophosphate from 5'-end of triphosphorylated RNA transcripts. Trans-complementation experiments showed that the P. aeruginosa enzyme can functionally substitute for RppH in E. coli cells indicating that, similar to RppH, the Pseudomonas hydrolase mediates RNA turnover in vivo. In order to elucidate the biological significance of the PA0336 protein in Pseudomonas cells, a PA0336 mutant strain was constructed. The mutated strain considerably increased level of the virulence factor pyocyanin compared to wild type, suggesting that PA0336 could be involved in downregulation of P. aeruginosa pathogenicity. This phenotype was reversed by complementation with the wild type but not catalytically inactive PA0336, indicating that the catalytic activity was indispensable for its biological function. Pathogenesis tests in Caenorhabditis elegans showed that the PA0336 mutant of P. aeruginosa was significantly more virulent than the parental strain, confirming further that the P. aeruginosa RNA pyrophosphohydrolase PA0336 modulates bacterial pathogenesis by down-regulating production of virulence-associated factors. To study the role of PA0336 further, transcriptomes of the PA0336 mutant and the wild-type strain were compared using RNA sequencing. The level of 537 transcripts coding for proteins involved in a variety of cellular processes such as replication, transcription, translation, central metabolism and pathogenesis, was affected by the lack of PA0336. These results indicate that the PA0336 RNA pyrophosphohydrolase functions as a global regulator that influences many of transcripts including those involved in P. aeruginosa virulence.

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GcsR, a TyrR-Like Enhancer-Binding Protein, Regulates Expression of the Glycine Cleavage System in Pseudomonas aeruginosa PAO1

Cited by 23 publications

References 61 publications

Ethanolamine Catabolism in Pseudomonas aeruginosa PAO1 Is Regulated by the Enhancer-Binding Protein EatR (PA4021) and the Alternative Sigma Factor RpoN

Ethanolamine Catabolism in Pseudomonas aeruginosa PAO1 Is Regulated by the Enhancer-Binding Protein EatR (PA4021) and the Alternative Sigma Factor RpoN

Epistatic interactions between ancestral genotype and beneficial mutations shape evolvability inPseudomonas aeruginosa

Nudix‐type RNA pyrophosphohydrolase provides homeostasis of virulence factor pyocyanin and functions as a global regulator in Pseudomonas aeruginosa

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