scholarly journals Different Spectra of Stationary-Phase Mutations in Early-Arising versus Late-Arising Mutants of Pseudomonas putida: Involvement of the DNA Repair Enzyme MutY and the Stationary-Phase Sigma Factor RpoS

2002 ◽  
Vol 184 (24) ◽  
pp. 6957-6965 ◽  
Author(s):  
Signe Saumaa ◽  
Andres Tover ◽  
Lagle Kasak ◽  
Maia Kivisaar
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Transcriptional Activation ◽  
Sigma Factor ◽  
Phenol Degradation ◽  
Test System ◽  
Growth Barriers ◽  
Mutational Processes ◽  
Sigma Factor Rpos ◽  
Selection Of

ABSTRACT Stationary-phase mutations occur in populations of stressed, nongrowing, and slowly growing cells and allow mutant bacteria to overcome growth barriers. Mutational processes in starving cells are different from those occurring in growing bacteria. Here, we present evidence that changes in mutational processes also take place during starvation of bacteria. Our test system for selection of mutants based on creation of functional promoters for the transcriptional activation of the phenol degradation genes pheBA in starving Pseudomonas putida enables us to study base substitutions (C-to-A or G-to-T transversions), deletions, and insertions. We observed changes in the spectrum of promoter-creating mutations during prolonged starvation of Pseudomonas putida on phenol minimal plates. One particular C-to-A transversion was the prevailing mutation in starving cells. However, with increasing time of starvation, the importance of this mutation decreased but the percentage of other types of mutations, such as 2- to 3-bp deletions, increased. The rate of transversions was markedly elevated in the P. putida MutY-defective strain. The occurrence of 2- to 3-bp deletions required the stationary-phase sigma factor RpoS, which indicates that some mutagenic pathway is positively controlled by RpoS in P. putida.

scholarly journals Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ςS-Dependent Transcription in Pseudomonas putida

2000 ◽  
Vol 182 (23) ◽  
pp. 6707-6713 ◽  
Author(s):  
Eve-Ly Ojangu ◽  
Andres Tover ◽  
Riho Teras ◽  
Maia Kivisaar
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Deficient Mutant ◽  
Wild Type ◽  
In Vivo Experiments ◽  
Silent Genes ◽  
Rna Polymerase Holoenzyme

ABSTRACT The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is ςS. In contrast to other minor sigma factors, RNA polymerase holoenzyme containing ςS (EςS) recognizes a number of promoters which are also recognized by that containing ς70 (Eς70). We have previously shown that transposon Tn4652 can activate silent genes in starvingPseudomonas putida cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the ς70-specific promoter consensus. The −10 hexameric sequence and the sequence downstream from the −10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and rpoS-deficient mutant P. putida, the effect of ςS on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the −10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the −10 element can affect ςS-dependent transcription. Combination of the −10 hexameric sequences and downstream sequences of different fusion promoters revealed that ςS-specific transcription from these promoters is not determined by the −10 hexameric sequence only. The results obtained in this study indicate that the sequence of the −10 element influences ςS-specific transcription in concert with the sequence downstream from the −10 box.

scholarly journals Characterization of a Stress-Induced Alternate Sigma Factor, RpoS, of Coxiella burnetii and Its Expression during the Development Cycle

2001 ◽  
Vol 69 (8) ◽  
pp. 4874-4883 ◽  
Author(s):  
Rekha Seshadri ◽  
James E. Samuel
Keyword(s):  
Stationary Phase ◽  
Coxiella Burnetii ◽  
Sigma Factor ◽  
Stationary Phases ◽  
Large Cell ◽  
Polyclonal Antiserum ◽  
Developmental Cycle ◽  
Growth Stages ◽  
Extracellular Environment ◽  
Sigma Factor Rpos

ABSTRACT Coxiella burnetii is an obligate intracellular bacterium that resides in an acidified phagolysosome and has a remarkable ability to persist in the extracellular environment.C. burnetii has evolved a developmental cycle that includes at least two morphologic forms, designated large cell variants (LCV) and small cell variants (SCV). Based on differential protein expression, distinct ultrastructures, and different metabolic activities, we speculated that LCV and SCV are similar to typical logarithmic- and stationary-phase growth stages. We hypothesized that the alternate sigma factor, RpoS, a global regulator of genes expressed under stationary-phase, starvation, and stress conditions in many bacteria, regulates differential expression in life cycle variants of C. burnetii. To test this hypothesis, we cloned and characterized the major sigma factor, encoded by an rpoD homologue, and the stress response sigma factor, encoded by an rpoS homologue. TherpoS gene was cloned by complementation of anEscherichia coli rpoS null mutant containing an RpoS-dependent lacZ fusion (osmY::lacZ). Expression ofC. burnetii rpoS was regulated by growth phase inE. coli (induced upon entry into stationary phase). A glutathione S-transferase–RpoS fusion protein was used to develop polyclonal antiserum against C. burnetii RpoS. Western blot analysis detected abundant RpoS in LCV but not in SCV. These results suggest that LCV and SCV are not comparable to logarithmic and stationary phases of growth and may represent a novel adaptation for survival in both the phagolysosome and the extracellular environment.

scholarly journals Regulation of Proteolysis of the Stationary-Phase Sigma Factor RpoS

1998 ◽  
Vol 180 (5) ◽  
pp. 1154-1158 ◽  
Author(s):  
Yanning Zhou ◽  
Susan Gottesman
Keyword(s):  
Gene Expression ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Exponential Phase ◽  
Specific Substrate ◽  
Response Regulators ◽  
Rapid Degradation ◽  
The Family ◽  
Regulation Of Activity ◽  
Sigma Factor Rpos

ABSTRACT RpoS, the stationary-phase sigma factor of Escherichia coli, is responsible for increased transcription of an array of genes when cells enter stationary phase and under certain stress conditions. RpoS is rapidly degraded during exponential phase and much more slowly during stationary phase; the resulting changes in RpoS accumulation play an important role in providing differential expression of RpoS-dependent gene expression. It has previously been shown that rapid degradation of RpoS during exponential growth depends on RssB (also called SprE and MviA), a protein with homology to the family of response regulators, and on the ClpXP protease. We find that RssB regulation of proteolysis does not extend to another ClpXP substrate, bacteriophage lambda O protein, suggesting that RssB acts on the specific substrate RpoS rather than on the protease. In addition, the activity of RpoS is down-regulated by RssB when degradation is blocked. In cells blocked for RpoS degradation by a mutation inclpP, cells devoid of RssB show a four- to fivefold-higher activity of an RpoS-dependent reporter fusion than cells overproducing RssB. Therefore, RssB allows specific environmental regulation of RpoS accumulation and may also modulate activity. The regulation of degradation provides an irreversible switch, while the regulation of activity may provide a second, presumably reversible level of control.

scholarly journals The genome-wide transcriptional response to varying RpoS levels inEscherichia coliK-12

2016 ◽  
Author(s):  
Garrett T. Wong ◽  
Richard P. Bonocora ◽  
Alicia N. Schep ◽  
Suzannah M. Beeler ◽  
Anna J. Lee Fong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Stress Responses ◽  
Sigma Factor ◽  
Core Promoter ◽  
Transcriptional Response ◽  
Transcriptional Responses ◽  
Cellular Processes ◽  
Functional Classes ◽  
Sigma Factor Rpos

AbstractThe alternative sigma factor RpoS is a central regulator of a many stress responses inEscherichia coli.The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We show that 23% of genes in theE. coligenome are regulated by RpoS level, and we identify many RpoS-transcribed genes and promoters. We observe three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS (“sensitive” genes), and genes whose expression changes very little with the production of a little RpoS (“insensitive”). We show that sequences outside the core promoter region determine whether a RpoS-regulated gene in sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes, and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase, and may also contribute to the diversity of stress responses directed by RpoS.ImportanceThe sigma factor RpoS is a global regulator that controls the response to many stresses inEscherichia coli.Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase, and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes in response to stresses.

Regulation of indoleacetic acid production inPseudomonas putidaGR12-2 by tryptophan and the stationary-phase sigma factor RpoS

2002 ◽  
Vol 48 (7) ◽  
pp. 635-642 ◽  
Author(s):  
Cheryl L Patten ◽  
Bernard R Glick
Keyword(s):  
Stationary Phase ◽  
Sigma Factor ◽  
Indoleacetic Acid ◽  
Recognition Sequence ◽  
Gene Encoding ◽  
Light Production ◽  
Key Enzyme ◽  
Plant Growth Promoting ◽  
Polymerase Chain ◽  
Sigma Factor Rpos

The phytohormone indole-3-acetic acid (IAA) accumulates in the culture medium of the plant growth-promoting bacterium Pseudomonas putida GR12-2 only when grown in the presence of exogenous tryptophan, suggesting that expression of indolepyruvate decarboxylase, a key enzyme in the IAA biosynthesis pathway in this bacterium, may be regulated by tryptophan. To test this hypothesis, we isolated the promoter region for the ipdc gene encoding indolepyruvate decarboxylase by inverse polymerase chain reaction (PCR) and inserted it upstream of the bioluminescent reporter gene luxAB on a plasmid in P. putida GR12-2. Activity of the ipdc promoter, measured by quantifying light production, increased fivefold in the presence of L-tryptophan, confirming that ipdc expression is induced by tryptophan. In addition, transcription of ipdc is regulated by the stationary phase sigma factor RpoS: the ipdc promoter contains a sequence similar to the RpoS recognition sequence, and transformation of P. putida GR12-2 with a plasmid carrying rpoS under the control of a constitutive promoter induced promoter activity before the onset of stationary phase when RpoS is not normally produced and prolonged a higher level of transcription at the later stages of the cell cycle.Key words: indoleacetic acid, indolepyruvate decarboxylase, regulation, tryptophan, RpoS.

scholarly journals Experimental Evolution ofEscherichia coliK-12 at High pH and with RpoS Induction

2018 ◽  
Vol 84 (15) ◽  
Author(s):  
Issam Hamdallah ◽  
Nadia Torok ◽  
Katarina M. Bischof ◽  
Nadim Majdalani ◽  
Sriya Chadalavada ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Experimental Evolution ◽  
Sigma Factor ◽  
Point Mutations ◽  
Envelope Proteins ◽  
Content Type ◽  
High Ph ◽  
K 12 ◽  
Sigma Factor Rpos

ABSTRACTExperimental evolution ofEscherichia coliK-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS1knockout ofphoB(encoding the positive regulator of the phosphate regulon). AphoB::kanRknockout increased growth at high pH.phoBmutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations inpcnB[poly(A) polymerase] also increased growth at high pH. Three out of four populations showed deletions oftorI, an inhibitor of TorR, which activates expression oftorCAD(trimethylamineN-oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma factor RpoS, either in the coding gene or in genes for regulators of RpoS expression. RpoS is required for survival at extremely high pH. In our microplate assay,rpoSdeletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadaptors that block degradation (IraM, IraD, and IraP). Other genes with mutations after high-pH evolution encode regulators, such as those encoded byyobG(mgrB) (PhoPQ regulator),rpoN(nitrogen starvation sigma factor),malI, andpurR, as well as envelope proteins, such as those encoded byompTandyahO. Overall,E. colievolution at high pH selects for mutations in key transcriptional regulators, includingphoBand the stationary-phase sigma factor RpoS.IMPORTANCEEscherichia coliin its native habitat encounters high-pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter the function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms.

scholarly journals Involvement of ςS in Starvation-Induced Transposition of Pseudomonas putidaTransposon Tn4652

2001 ◽  
Vol 183 (18) ◽  
pp. 5445-5448 ◽  
Author(s):  
Heili Ilves ◽  
Rita Hõrak ◽  
Maia Kivisaar
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Sigma Factor ◽  
Mobile Element ◽  
Environmental Stresses ◽  
Mobile Elements ◽  
Present Evidence ◽  
Transpositional Activity ◽  
Specific Regulation

ABSTRACT Transpositional activity of mobile elements can be induced by different environmental stresses. Here, we present evidence that transposition of Tn4652 is elevated in stationary-phasePseudomonas putida and suppressed in an isogenic ςS-defective strain. We demonstrate that transcription from the Tn4652 transposase promoter is controlled by the stationary-phase-specific sigma factor ςS. To our knowledge, this is the first example of direct stationary-phase-specific regulation of a mobile element transposase. Data presented in this report support the idea that activation of transposition under stressful conditions could be an inducible process.

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