scholarly journals Characterization of a Two-Component Regulatory System That Regulates Succinate-Mediated Catabolite Repression in Sinorhizobium meliloti

2010 ◽  
Vol 192 (21) ◽  
pp. 5725-5735 ◽  
Author(s):  
Preston P. Garcia ◽  
Ryan M. Bringhurst ◽  
Catalina Arango Pinedo ◽  
Daniel J. Gage
Keyword(s):  
Catabolite Repression ◽  
Sinorhizobium Meliloti ◽  
Response Regulator ◽  
Energy State ◽  
Carbon Sources ◽  
Regulatory System ◽  
Dicarboxylic Acids ◽  
Deletion Mutants ◽  
State Monitoring ◽  
Secondary Carbon

ABSTRACT When they are available, Sinorhizobium meliloti utilizes C4-dicarboxylic acids as preferred carbon sources for growth while suppressing the utilization of some secondary carbon sources such as α- and β-galactosides. The phenomenon of using succinate as the sole carbon source in the presence of secondary carbon sources is termed succinate-mediated catabolite repression (SMCR). Genetic screening identified the gene sma0113 as needed for strong SMCR when S. meliloti was grown in succinate plus lactose, maltose, or raffinose. sma0113 and the gene immediately downstream, sma0114, encode the proteins Sma0113, an HWE histidine kinase with five PAS domains, and Sma0114, a CheY-like response regulator lacking a DNA-binding domain. sma0113 in-frame deletion mutants show a relief of catabolite repression compared to the wild type. sma0114 in-frame deletion mutants overproduce polyhydroxybutyrate (PHB), and this overproduction requires sma0113. Sma0113 may use its five PAS domains for redox level or energy state monitoring and use that information to regulate catabolite repression and related responses.

scholarly journals Control of Inducer Accumulation Plays a Key Role in Succinate-Mediated Catabolite Repression in Sinorhizobiummeliloti

2002 ◽  
Vol 184 (19) ◽  
pp. 5385-5392 ◽  
Author(s):  
Ryan M. Bringhurst ◽  
Daniel J. Gage
Keyword(s):  
Catabolite Repression ◽  
Double Mutant ◽  
Minimal Medium ◽  
Carbon Sources ◽  
Constitutive Expression ◽  
Dicarboxylic Acids ◽  
Genetic Screen ◽  
Aberrant Expression ◽  
Inducer Exclusion ◽  
Secondary Carbon

ABSTRACT The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the α-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other α-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of β-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of α-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce β-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced β-galactoside transporters and cause the induction of α-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a β-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.

scholarly journals Genetic Analysis of Mutations Affecting pckA Regulation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1521-1531 ◽  
Author(s):  
Magne Østerås ◽  
Shelley A P O'Brien ◽  
Turlough M Finan
Keyword(s):  
Sinorhizobium Meliloti ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Root Nodules ◽  
Genetic Regulation ◽  
Carbon Sources ◽  
Dicarboxylic Acids ◽  
Phenotypic Analysis ◽  
Gene Encoding ◽  
Type Locus ◽  
Rapid Induction

Abstract The enzyme phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step in the gluconeogenic pathway in most organisms. We are examining the genetic regulation of the gene encoding Pck, pckA, in Rhizobium (now Sinorhizobium) meliloti. This bacterium forms N2-fixing root nodules on alfalfa, and the major energy sources supplied to the bacteria within these nodules are C4-dicarboxylic acids such as malate and succinate. R. meliloti cells growing in glucose minimal medium show very low pckA expression whereas addition of succinate to this medium results in a rapid induction of pckA transcription. We identified spontaneous mutations (rpk) that alter the regulation of pckA expression such that pckA is expressed in media containing the non-inducing carbon sources lactose and glucose. Genetic and phenotypic analysis allowed us to differentiate at least four rpk mutant classes that map to different locations on the R. meliloti chromosome. The wild-type locus corresponding to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk mutation were identified. The nucleotide sequence of this region revealed that both Tn5 insertions lay within a gene encoding a protein homologous to the Ga1R/LacI family of transcriptional regulators that are involved in metabolism.

scholarly journals Sinorhizobium meliloti Mutants Lacking Phosphotransferase System Enzyme HPr or EIIA Are Altered in Diverse Processes, Including Carbon Metabolism, Cobalt Requirements, and Succinoglycan Production

2008 ◽  
Vol 190 (8) ◽  
pp. 2947-2956 ◽  
Author(s):  
Catalina Arango Pinedo ◽  
Ryan M. Bringhurst ◽  
Daniel J. Gage
Keyword(s):  
Carbon Metabolism ◽  
Sinorhizobium Meliloti ◽  
Carbon Sources ◽  
Deletion Mutants ◽  
Symbiotic Relationship ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Carbon Utilization ◽  
Carbon Regulation ◽  
Extreme Sensitivity

ABSTRACT Sinorhizobium meliloti is a member of the Alphaproteobacteria that fixes nitrogen when it is in a symbiotic relationship. Genes for an incomplete phosphotransferase system (PTS) have been found in the genome of S. meliloti. The genes present code for Hpr and ManX (an EIIAMan-type enzyme). HPr and EIIA regulate carbon utilization in other bacteria. hpr and manX in-frame deletion mutants exhibited altered carbon metabolism and other phenotypes. Loss of HPr resulted in partial relief of succinate-mediated catabolite repression, extreme sensitivity to cobalt limitation, rapid die-off during stationary phase, and altered succinoglycan production. Loss of ManX decreased expression of melA-agp and lac, the operons needed for utilization of α- and β-galactosides, slowed growth on diverse carbon sources, and enhanced accumulation of high-molecular-weight succinoglycan. A strain with both hpr and manX deletions exhibited phenotypes similar to those of the strain with a single hpr deletion. Despite these strong phenotypes, deletion mutants exhibited wild-type nodulation and nitrogen fixation when they were inoculated onto Medicago sativa. The results show that HPr and ManX (EIIAMan) are involved in more than carbon regulation in S. meliloti and suggest that the phenotypes observed occur due to activity of HPr or one of its phosphorylated forms.

scholarly journals Catabolite Repression of the Citrate Fermentation Genes in Klebsiella pneumoniae: Evidence for Involvement of the Cyclic AMP Receptor Protein

2001 ◽  
Vol 183 (18) ◽  
pp. 5248-5256 ◽  
Author(s):  
Margareta Meyer ◽  
Peter Dimroth ◽  
Michael Bott
Keyword(s):  
Cyclic Amp ◽  
Klebsiella Pneumoniae ◽  
Catabolite Repression ◽  
Response Regulator ◽  
Carbon Sources ◽  
Receptor Protein ◽  
Citrate Lyase ◽  
Citrate Fermentation ◽  
Camp Receptor ◽  
Transcriptional Start Sites

ABSTRACT Klebsiella pneumoniae is able to grow anaerobically with citrate as a sole carbon and energy source by a fermentative pathway involving the Na+-dependent citrate carrier CitS, citrate lyase, and oxaloacetate decarboxylase. The corresponding genes are organized in the divergent citC and citS operons, whose expression is strictly dependent on the citrate-sensing CitA-CitB two-component system. Evidence is provided here that the citrate fermentation genes are subject to catabolite repression, since anaerobic cultivation with a mixture of citrate and glucose or citrate and gluconate resulted in diauxic growth. Glucose, gluconate, and also glycerol decreased the expression of a chromosomalcitS-lacZ fusion by 60 to 75%, whereas a direct inhibition of the citrate fermentation enzymes was not observed. The purified cyclic AMP (cAMP) receptor protein (CRP) of K. pneumoniae bound to two sites in thecitC-citS intergenic region, which were centered at position −41.5 upstream of the citC andcitS transcriptional start sites. Binding was apparently stimulated by the response regulator CitB. These data indicate that catabolite repression of the citrate fermentation genes is exerted by CRP and that in the absence of repressing carbon sources the cAMP-CRP complex serves to enhance the basal, CitB-dependent transcription level.

scholarly journals Autoregulation of Sinorhizobium meliloti exoR gene expression

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 2092-2101 ◽  
Author(s):  
Hai-Yang Lu ◽  
Hai-Ping Cheng
Keyword(s):  
Sinorhizobium Meliloti ◽  
Carbon Sources ◽  
Regulatory System ◽  
Leguminous Plant ◽  
Wild Type ◽  
Plant Host ◽  
Active Sensor ◽  
Bacterial Exopolysaccharide ◽  
Membrane Bound ◽  
And Control

The successful nitrogen-fixing symbiosis between the Gram-negative soil bacterium Sinorhizobium meliloti and its leguminous plant host alfalfa (Medicago sativa) requires the bacterial exopolysaccharide succinoglycan. Succinoglycan and flagellum production, along with the ability to metabolize more than 20 different carbon sources and control the expression of a large number of S. meliloti genes, is regulated by the ExoR–ExoS/ChvI signalling pathway. The ExoR protein interacts with and suppresses the sensing activities of ExoS, the membrane-bound sensor of the ExoS/ChvI two-component regulatory system. Here we show that exoR expression is clearly upregulated in the absence of any functional ExoR protein. This upregulation was suppressed by the presence of the wild-type ExoR protein but not by a mutated ExoR protein lacking signal peptide. The levels of exoR expression could be directly modified in real time by changing the levels of total ExoR protein. The expression of exoR was also upregulated by the constitutively active sensor mutation exoS96, and blocked by two single mutations, exoS* and exoSsupA , in the ExoS sensing domain. Presence of the wild-type ExoS protein further elevated the levels of exoR expression in the absence of functional ExoR protein, and reversed the effects of exoS96, exoS* and exoSsupA mutations. Altogether, these data suggest that ExoR protein autoregulates exoR expression through the ExoS/ChvI system, allowing S. meliloti cells to maintain the levels of exoR expression based on the amount of total ExoR protein.

scholarly journals The Response Regulator SprE (RssB) Modulates Polyadenylation and mRNA Stability in Escherichia coli

2009 ◽  
Vol 191 (22) ◽  
pp. 6812-6821 ◽  
Author(s):  
Valerie J. Carabetta ◽  
Bijoy K. Mohanty ◽  
Sidney R. Kushner ◽  
Thomas J. Silhavy
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Intracellular Localization ◽  
Carbon Sources ◽  
Adaptor Protein ◽  
Deletion Mutants ◽  
E Coli ◽  
Specific Mrnas ◽  
The Stability ◽  
Polymerase I

ABSTRACT In Escherichia coli, the adaptor protein SprE (RssB) controls the stability of the alternate sigma factor RpoS (σ38 and σS). When nutrients are abundant, SprE binds RpoS and delivers it to ClpXP for degradation, but when carbon sources are depleted, this process is inhibited. It also has been noted that overproduction of SprE is toxic. Here we show that null mutations in pcnB, encoding poly(A) polymerase I (PAP I), and in hfq, encoding the RNA chaperone Hfq, suppress this toxicity. Since PAP I, in conjunction with Hfq, is responsible for targeting RNAs, including mRNAs, for degradation by adding poly(A) tails onto their 3′ ends, these data indicate that SprE helps modulate the polyadenylation pathway in E. coli. Indeed, in exponentially growing cells, sprE deletion mutants exhibit significantly reduced levels of polyadenylation and increased stability of specific mRNAs, similar to what is observed in a PAP I-deficient strain. In stationary phase, we show that SprE changes the intracellular localization of PAP I. Taken together, we propose that SprE plays a multifunctional role in controlling the transcriptome, regulating what is made via its effects on RpoS, and modulating what is degraded via its effects on polyadenylation and turnover of specific mRNAs.

Application of Phenotype Microarray for Profiling Carbon Sources Utilization between Biofilm and Non-Biofilm of Pseudomonas aeruginosa from Clinical Isolates

2020 ◽  
Vol 21 (14) ◽  
pp. 1539-1550
Author(s):  
Nur S. Ismail ◽  
Suresh K. Subbiah ◽  
Niazlin M. Taib
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Carbon Sources ◽  
Anaerobic Respiration ◽  
Regulatory System ◽  
Negative Control ◽  
High Morbidity ◽  
Metabolic Profiles ◽  
Phenotype Microarray ◽  
Diverse Environment ◽  
Phenotype Microarrays

Background: This is the fastest work in obtaining the metabolic profiles of Pseudomonas aeruginosa in order to combat the infection diseases which leads to high morbidity and mortality rates. Pseudomonas aeruginosa is a high versatility of gram-negative bacteria that can undergo aerobic and anaerobic respiration. Capabilities in deploying different carbon sources, energy metabolism and regulatory system, ensure the survival of this microorganism in the diverse environment condition. Determination of differences in carbon sources utilization among biofilm and non-biofilm of Pseudomonas aeruginosa provides a platform in understanding the metabolic activity of the microorganism. Methods: The study was carried out from September 2017 to February 2019. Four archive isolates forming strong and intermediate biofilm and non-biofilms producer were subcultured from archive isolates. ATCC 27853 P. aeruginosa was used as a negative control or non-biofilm producing microorganism. Biofilm formation was confirmed by Crystal Violet Assay (CVA) and Congo Red Agar (CRA). Metabolic profiles of the biofilm and non-biofilms isolates were determined by phenotype microarrays (Biolog Omnilog). Results and Discussion: In this study, Pseudomonas aeruginosa biofilm isolates utilized uridine, L-threonine and L-serine while non-biofilm utilized adenosine, inosine, monomethyl, sorbic acid and succinamic acid. Conclusion: The outcome of this result will be used for future studies to improve detection or inhibit the growth of P. aeruginosa biofilm and non-biofilm respectively.

scholarly journals Signal Transduction and Regulatory Mechanisms Involved in Control of the σS (RpoS) Subunit of RNA Polymerase

2002 ◽  
Vol 66 (3) ◽  
pp. 373-395 ◽  
Author(s):  
Regine Hengge-Aronis
Keyword(s):  
Rna Polymerase ◽  
Translational Control ◽  
Current Knowledge ◽  
Response Regulator ◽  
Regulatory System ◽  
Stress Conditions ◽  
Acidic Ph ◽  
High Osmolarity ◽  
Multiple Signal ◽  
Rpos Mrna

SUMMARY The σS (RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and related bacteria. While rapidly growing cells contain very little σS, exposure to many different stress conditions results in rapid and strong σS induction. Consequently, transcription of numerous σS-dependent genes is activated, many of which encode gene products with stress-protective functions. Multiple signal integration in the control of the cellular σS level is achieved by rpoS transcriptional and translational control as well as by regulated σS proteolysis, with various stress conditions differentially affecting these levels of σS control. Thus, a reduced growth rate results in increased rpoS transcription whereas high osmolarity, low temperature, acidic pH, and some late-log-phase signals stimulate the translation of already present rpoS mRNA. In addition, carbon starvation, high osmolarity, acidic pH, and high temperature result in stabilization of σS, which, under nonstress conditions, is degraded with a half-life of one to several minutes. Important cis-regulatory determinants as well as trans-acting regulatory factors involved at all levels of σS regulation have been identified. rpoS translation is controlled by several proteins (Hfq and HU) and small regulatory RNAs that probably affect the secondary structure of rpoS mRNA. For σS proteolysis, the response regulator RssB is essential. RssB is a specific direct σS recognition factor, whose affinity for σS is modulated by phosphorylation of its receiver domain. RssB delivers σS to the ClpXP protease, where σS is unfolded and completely degraded. This review summarizes our current knowledge about the molecular functions and interactions of these components and tries to establish a framework for further research on the mode of multiple signal input into this complex regulatory system.

scholarly journals Evolutionary rewiring: a modified prokaryotic gene-regulatory pathway in chloroplasts

2013 ◽  
Vol 368 (1622) ◽  
pp. 20120260 ◽  
Author(s):  
Sujith Puthiyaveetil ◽  
Iskander M. Ibrahim ◽  
John F. Allen
Keyword(s):  
Green Algae ◽  
Gene Transcription ◽  
Response Regulator ◽  
Regulatory System ◽  
Photosynthetic Electron Transport ◽  
Sensor Kinase ◽  
Ps I ◽  
Component Systems ◽  
Two Component Systems ◽  
Gene Regulatory

Photosynthetic electron transport regulates chloroplast gene transcription through the action of a bacterial-type sensor kinase known as chloroplast sensor kinase (CSK). CSK represses photosystem I (PS I) gene transcription in PS I light and thus initiates photosystem stoichiometry adjustment. In cyanobacteria and in non-green algae, CSK homologues co-exist with their response regulator partners in canonical bacterial two-component systems. In green algae and plants, however, no response regulator partner of CSK is found. Yeast two-hybrid analysis has revealed interaction of CSK with sigma factor 1 (SIG1) of chloroplast RNA polymerase. Here we present further evidence for the interaction between CSK and SIG1. We also show that CSK interacts with quinone. Arabidopsis SIG1 becomes phosphorylated in PS I light, which then specifically represses transcription of PS I genes. In view of the identical signalling properties of CSK and SIG1 and of their interactions, we suggest that CSK is a SIG1 kinase. We propose that the selective repression of PS I genes arises from the operation of a gene-regulatory phosphoswitch in SIG1. The CSK-SIG1 system represents a novel, rewired chloroplast-signalling pathway created by evolutionary tinkering. This regulatory system supports a proposal for the selection pressure behind the evolutionary stasis of chloroplast genes.

AgmR controls transcription of a regulon with several operons essential for ethanol oxidation in Pseudomonas aeruginosa ATCC 17933

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1851-1857 ◽  
Author(s):  
Nicole Gliese ◽  
Viola Khodaverdi ◽  
Max Schobert ◽  
Helmut Görisch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ethanol Oxidation ◽  
Response Regulator ◽  
Regulatory System ◽  
Pyrroloquinoline Quinone ◽  
Kanamycin Resistance ◽  
Kanamycin Resistance Cassette ◽  
Two Component ◽  
Ethanol Dehydrogenase ◽  
Oxidation System

The response regulator AgmR was identified to be involved in the regulation of the quinoprotein ethanol oxidation system of Pseudomonas aeruginosa ATCC 17933. Interruption of the agmR gene by insertion of a kanamycin-resistance cassette resulted in mutant NG3, unable to grow on ethanol. After complementation with the intact agmR gene, growth on ethanol was restored. Transcriptional lacZ fusions were used to identify four operons which are regulated by the AgmR protein: the exaA operon encodes the pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the exaBC operon encodes a soluble cytochrome c 550 and an aldehyde dehydrogenase, the pqqABCDE operon carries the PQQ biosynthetic genes, and operon exaDE encodes a two-component regulatory system which controls transcription of the exaA operon. Transcription of exaA was restored by transformation of NG3 with a pUCP20T derivative carrying the exaDE genes under lac-promoter control. These data indicate that the AgmR response regulator and the exaDE two-component regulatory system are organized in a hierarchical manner. Gene PA1977, which appears to form an operon with the agmR gene, was found to be non-essential for growth on ethanol.

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