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A caulobacter crescentus extracytoplasmic function sigma factor mediating the response to oxidative stress in stationary phase.

ABSTRACT: Alternative sigma factors of the extracytoplasmic function (ECF) subfamily are important regulators of stress responses in bacteria and have been implicated in the control of homeostasis of the extracytoplasmic compartment of the cell. This work describes the characterization of sigF, encoding 1 of the 13 members of this subfamily identified in Caulobacter crescentus. A sigF-null strain was obtained and shown to be severely impaired in resistance to oxidative stress, caused by hydrogen peroxide treatment, exclusively during the stationary phase. Although sigF mRNA levels decrease in stationary-phase cells, the amount of sigma(F) protein is greatly increased at this stage, indicating a posttranscriptional control. Data obtained indicate that the FtsH protease is either directly or indirectly involved in the control of sigma(F) levels, as cells lacking this enzyme present larger amounts of the sigma factor. Increased stability of sigma(F) protein in stationary-phase cells of the parental strain and in exponential-phase cells of the ftsH-null strain is also demonstrated. Transcriptome analysis of the sigF-null strain led to the identification of eight genes regulated by sigma(F) during the stationary phase, including sodA and msrA, which are known to be involved in oxidative stress response.

SUBMITTER: Alvarez-Martinez CE

PROVIDER: S-EPMC1426549 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

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A caulobacter crescentus extracytoplasmic function sigma factor mediating the response to oxidative stress in stationary phase.

Alvarez-Martinez Cristina E CE Baldini Regina L RL Gomes Suely L SL

Journal of bacteriology 20060301 5

Alternative sigma factors of the extracytoplasmic function (ECF) subfamily are important regulators of stress responses in bacteria and have been implicated in the control of homeostasis of the extracytoplasmic compartment of the cell. This work describes the characterization of sigF, encoding 1 of the 13 members of this subfamily identified in Caulobacter crescentus. A sigF-null strain was obtained and shown to be severely impaired in resistance to oxidative stress, caused by hydrogen peroxide ...[more]

PMID: 16484194

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Project description:BackgroundAs bacterial cells enter stationary phase, they adjust their growth rate to comply with nutrient restriction and acquire increased resistance to several stresses. These events are regulated by controlling gene expression at this phase, changing the mode of exponential growth into that of growth arrest, and increasing the expression of proteins involved in stress resistance. The two-component system SpdR/SpdS is required for the activation of transcription of the Caulobacter crescentus cspD gene at the onset of stationary phase.ResultsIn this work, we showed that both SpdR and SpdS are also induced upon entry into stationary phase, and this induction is partly mediated by ppGpp and it is not auto-regulated. Global transcriptional analysis at early stationary phase of a spdR null mutant strain compared to the wild type strain was carried out by DNA microarray. Twenty-three genes showed at least twofold decreased expression in the spdR deletion mutant strain relative to its parental strain, including cspD, while five genes showed increased expression in the mutant. The expression of a set of nine genes was evaluated by quantitative real time PCR, validating the microarray data, and indicating an important role for SpdR at stationary phase. Several of the differentially expressed genes can be involved in modulating gene expression, including four transcriptional regulators, and the RNA regulatory protein Hfq. The ribosomal proteins NusE and NusG, which also have additional regulatory functions in transcription and translation, were also downregulated in the spdR mutant, as well as the ParE1 toxin. The purified SpdR protein was shown to bind to the regulatory region of CC0517 by Electrophoretic Mobility Shift Assay, and the SpdR-regulated gene CC0731 was shown to be expressed at a lower level in the null cspD mutant, suggesting that at least part of the effect of SpdR on the expression of this gene is indirect.ConclusionsThe results indicate that SpdR regulates several genes encoding proteins of regulatory function, which in turn may be required for the expression of other genes important for the transition to stationary phase.

Project description:Bacteria have a variety of mechanisms for adapting to environmental perturbations. Changes in oxygen availability result in a switch between aerobic and anaerobic respiration, whereas iron limitation may lead to siderophore secretion. In addition to metabolic adaptations, many organisms respond by altering their cell shape. Caulobacter crescentus, when grown under phosphate-limiting conditions, dramatically elongates its polar stalk appendage. The stalk is hypothesized to facilitate phosphate uptake; however, the mechanistic details of stalk synthesis are not well characterized. We used a chemical mutagenesis approach to isolate and characterize stalk-deficient mutants, one of which had two mutations in the phosphomannose isomerase gene (manA) that were necessary and sufficient to inhibit stalk elongation. Transcription of the pho regulon was unaffected in the manA mutant; therefore, ManA plays a unique regulatory role in stalk synthesis. The mutant ManA had reduced enzymatic activity, resulting in a 5-fold increase in the intracellular fructose 6-phosphate/mannose 6-phosphate ratio. This metabolic imbalance impaired the synthesis of cellular envelope components derived from mannose 6-phosphate, namely, lipopolysaccharide O-antigen and exopolysaccharide. Furthermore, the manA mutations prevented C. crescentus cells from efficiently entering stationary phase. Deletion of the stationary-phase response regulator gene spdR inhibited stalk elongation in wild-type cells, while overproduction of the alarmone ppGpp, which triggers growth arrest and stationary-phase entry, increased stalk length in the manA mutant strain. These results demonstrate that sugar-phosphate metabolism regulates stalk elongation independently of phosphate starvation.IMPORTANCE Metabolic control of bacterial cell shape is an important mechanism for adapting to environmental perturbations. Caulobacter crescentus dramatically elongates its polar stalk appendage in response to phosphate starvation. To investigate the mechanism of this morphological adaptation, we isolated stalk-deficient mutants, one of which had mutations in the phosphomannose isomerase gene (manA) that blocked stalk elongation, despite normal activation of the phosphate starvation response. The mutant ManA resulted in an imbalance in sugar-phosphate concentrations, which had effects on the synthesis of cellular envelope components and entry into stationary phase. Due to the interconnectivity of metabolic pathways, our findings may suggest more generally that the modulation of bacterial cell shape involves the regulation of growth phase and the synthesis of cellular building blocks.

Project description:Expression of the catalase-peroxidase of Caulobacter crescentus, a gram-negative member of the alpha subdivision of the Proteobacteria, is 50-fold higher in stationary-phase cultures than in exponential cultures. To identify regulators of the starvation response, Tn5 insertion mutants were isolated with reduced expression of a katG::lacZ fusion on glucose starvation. One insertion interrupted an open reading frame encoding a protein with significant amino acid sequence identity to TipA, a helix-turn-helix transcriptional activator in the response of Streptomyces lividans to the peptide antibiotic thiostrepton, and lesser sequence similarity to other helix-turn-helix regulators in the MerR family. The C. crescentus orthologue of tipA was named skgA (stationary-phase regulation of katG). Stationary-phase expression of katG was reduced by 70% in the skgA::Tn5 mutant, and stationary-phase resistance to hydrogen peroxide decreased by a factor of 10. Like the wild type, the skgA mutant exhibited starvation-induced cross-resistance to heat and acid shock, entered into the helical morphology that occurs after 9 to 12 days in stationary phase, and during exponential growth induced katG in response to hydrogen peroxide challenge. Expression of skgA increased 5- to 10-fold in late exponential phase. skgA is the first regulator of a starvation-induced stress response identified in C. crescentus. SkgA is not a global regulator of the stationary-phase stress response; its action encompasses the oxidative stress-hydrogen peroxide response but not acid or heat responses. Moreover, SkgA is not an alternative sigma factor, like RpoS, which controls multiple aspects of starvation-induced cross-resistance to stress in enteric bacteria. These observations raise the possibility that regulation of stationary-phase gene expression in this member of the alpha subdivision of the Proteobacteria is different from that in Escherichia coli and other members of the gamma subdivision.

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A caulobacter crescentus extracytoplasmic function sigma factor mediating the response to oxidative stress in stationary phase.

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A caulobacter crescentus extracytoplasmic function sigma factor mediating the response to oxidative stress in stationary phase.

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