Sequestration of a two-component response regulator by a riboswitch-regulated noncoding RNA

Abstract Background A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date. Results In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation. Conclusions Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction.

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Intra- and Interspecies Signaling betweenStreptococcus salivarius and Streptococcus pyogenes Mediated by SalA and SalA1 Lantibiotic Peptides

Journal of Bacteriology ◽

10.1128/jb.183.13.3931-3938.2001 ◽

2001 ◽

Vol 183 (13) ◽

pp. 3931-3938 ◽

Cited By ~ 99

Author(s):

M. Upton ◽

J. R. Tagg ◽

P. Wescombe ◽

H. F. Jenkinson

Keyword(s):

Streptococcus Pyogenes ◽

Response Regulator ◽

Genetic Locus ◽

Two Component System ◽

Peptide Modification ◽

Insertional Inactivation ◽

Two Component ◽

Genes Encoding ◽

Dose Dependent

ABSTRACT Streptococcus salivarius 20P3 produces a 22-amino-acid residue lantibiotic, designated salivaricin A (SalA), that inhibits the growth of a range of streptococci, including all strains ofStreptococcus pyogenes. Lantibiotic production is associated with the sal genetic locus comprisingsalA, the lantibiotic structural gene; salBCTXgenes encoding peptide modification and export machinery proteins; andsalYKR genes encoding a putative immunity protein and two-component sensor-regulator system. Insertional inactivation ofsalB in S. salivarius 20P3 resulted in abrogation of SalA peptide production, of immunity to SalA, and ofsalA transcription. Addition of exogenous SalA peptide tosalB mutant cultures induced dose-dependent expression ofsalA mRNA (0.2 kb), demonstrating that SalA production was normally autoregulated. Inactivation of salR encoding the response regulator of the SalKR two-component system led to reduced production of, and immunity to, SalA. The sal genetic locus was also present in S. pyogenes SF370 (M type 1), but because of a deletion across the salBCT genes, the corresponding lantibiotic peptide, designated SalA1, was not produced. However, in S. pyogenes T11 (M type 4) the sallocus gene complement was apparently complete, and active SalA1 peptide was synthesized. Exogenously added SalA1 peptide from S. pyogenes T11 induced salA1 transcription in S. pyogenes SF370 and in an isogenic S. pyogenes T11salB mutant and salA transcription in S. salivarius 20P3 salB. Thus, SalA and SalA1 are examples of streptococcal lantibiotics whose production is autoregulated. These peptides act as intra- and interspecies signaling molecules, modulating lantibiotic production and possibly influencing streptococcal population ecology in the oral cavity.

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

Microbiology ◽

10.1099/mic.0.26882-0 ◽

2004 ◽

Vol 150 (6) ◽

pp. 1851-1857 ◽

Cited By ~ 21

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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The Two-Component Regulatory System TCS08 Is Involved in Cellobiose Metabolism of Streptococcus pneumoniae R6

Journal of Bacteriology ◽

10.1128/jb.01170-06 ◽

2006 ◽

Vol 189 (4) ◽

pp. 1342-1350 ◽

Cited By ~ 46

Author(s):

Stuart J. McKessar ◽

Regine Hakenbeck

Keyword(s):

Streptococcus Pneumoniae ◽

Response Regulator ◽

Regulatory System ◽

Phosphotransferase System ◽

Two Component System ◽

Transcription Profiles ◽

Regulatory Systems ◽

Two Component ◽

Cognate Response Regulator ◽

Gram Positive Cocci

ABSTRACT The two-component system TCS08 is one of the regulatory systems that is important for virulence of Streptococcus pneumoniae. In order to investigate the TCS08 regulon, we have analyzed transcription profiles of mutants derived from S. pneumoniae R6 by microarray analysis. Since deletion mutants are often without a significant phenotype, we constructed a mutation in the histidine kinase HK08, T133P, in analogy to the phosphatase mutation T230P in the H box of the S. pneumoniae CiaH kinase described recently (D. Zähner, K. Kaminski, M. van der Linden, T. Mascher, M. Merai, and R. Hakenbeck, J. Mol. Microbiol. Biotechnol. 4:211-216, 2002). In addition, a deletion mutation was constructed in rr08, encoding the cognate response regulator. The most heavily suppressed genes in the hk08 mutant were spr0276 to spr0282, encoding a putative cellobiose phosphoenolpyruvate sugar phosphotransferase system (PTS). Whereas the R6 Smr parent strain and the Δrr08 mutant readily grew on cellobiose, the hk08 mutant and selected mutants with deletions in the PTS cluster did not, strongly suggesting that TCS08 is involved in the catabolism of cellobiose. Homologues of the TCS08 system were found in closely related streptococci and other gram-positive cocci. However, the genes spr0276 to spr0282, encoding the putative cellobiose PTS, represent a genomic island in S. pneumoniae and homologues were found in Streptococcus gordonii only, suggesting that this system might contribute to the pathogenicity potential of the pneumococcus.

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σE Regulates and Is Regulated by a Small RNA in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00020-07 ◽

2007 ◽

Vol 189 (11) ◽

pp. 4243-4256 ◽

Cited By ~ 100

Author(s):

Karl M. Thompson ◽

Virgil A. Rhodius ◽

Susan Gottesman

Keyword(s):

Escherichia Coli ◽

Small Rnas ◽

Noncoding Rna ◽

Response Regulator ◽

Heterologous Promoter ◽

Genomic Libraries ◽

Multicopy Suppressors ◽

Intergenic Regions ◽

Transposon Mutants

ABSTRACT RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia coli. Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB, negatively regulated the fusion. An insertion in the rep helicase and one upstream of dnaK decreased expression of the fusion. Multicopy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the gene for the response regulator OmpR; the second contained mipA, encoding a murein hydrolase. The involvement of MipA and OmpR in cell surface synthesis suggested that the rybB promoter might be dependent on σE. The sequence upstream of the +1 of rybB contains a consensus σE promoter. The activity of rybB-lacZ was increased in cells lacking the RseA anti-sigma factor and when σE was overproduced from a heterologous promoter. The activity of rybB-lacZ and the detection of RybB were totally abolished in an rpoE-null strain. In vitro, σE efficiently transcribes from this promoter. Both a rybB mutation and an hfq mutation significantly increased expression of both rybB-lacZ and rpoE-lacZ fusions, consistent with negative regulation of the σE response by RybB and other small RNAs. Based on the plasmid screens, NsrR, a repressor sensitive to nitric oxide, was also found to negatively regulate σE-dependent promoters in an RseA-independent fashion.

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Multiple Mechanisms of Action for Inhibitors of Histidine Protein Kinases from Bacterial Two-Component Systems

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.7.1693 ◽

1999 ◽

Vol 43 (7) ◽

pp. 1693-1699 ◽

Cited By ~ 130

Author(s):

Jamese J. Hilliard ◽

Raul M. Goldschmidt ◽

Lisa Licata ◽

Ellen Z. Baum ◽

Karen Bush

Keyword(s):

Pathogenic Bacteria ◽

Response Regulator ◽

Membrane Integrity ◽

Bacterial Killing ◽

Gram Positive Bacteria ◽

Component Systems ◽

Two Component ◽

Two Component Systems ◽

Bacterial Cell Membrane ◽

Cellular Incorporation

ABSTRACT Many pathogenic bacteria utilize two-component systems consisting of a histidine protein kinase (HPK) and a response regulator (RR) for signal transduction. During the search for novel inhibitors, several chemical series, including benzoxazines, benzimidazoles, bis-phenols, cyclohexenes, trityls, and salicylanilides, were identified that inhibited the purified HPK-RR pairs KinA-Spo0F and NRII-NRI, with 50% inhibitory concentrations (IC50s) ranging from 1.9 to >500 μM and MICs ranging from 0.5 to >16 μg/ml for gram-positive bacteria. However, additional observations suggested that mechanisms other than HPK inhibition might contribute to antibacterial activity. In the present work, representative compounds from the six different series of inhibitors were analyzed for their effects on membrane integrity and macromolecular synthesis. At 4× MIC, 17 of 24 compounds compromised the integrity of the bacterial cell membrane within 10 min, as measured by uptake of propidium iodide. In this set, compounds with lower IC50s tended to cause greater membrane disruption. Eleven of 12 compounds inhibited cellular incorporation of radiolabeled thymidine and uridine >97% in 5 min and amino acids >80% in 15 min. The HPK inhibitor that allowed >25% precursor incorporation had no measurable MIC (>16 μg/ml). Fifteen of 24 compounds also caused hemolysis of equine erythrocytes. Thus, the antibacterial HPK inhibitors caused a rapid decrease in cellular incorporation of RNA, DNA, and protein precursors, possibly as a result of the concomitant disruption of the cytoplasmic membrane. Bacterial killing by these HPK inhibitors may therefore be due to multiple mechanisms, independent of HPK inhibition.

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Candida albicans Response Regulator Gene SSK1 Regulates a Subset of Genes Whose Functions Are Associated with Cell Wall Biosynthesis and Adaptation to Oxidative Stress

Eukaryotic Cell ◽

10.1128/ec.2.5.1018-1024.2003 ◽

2003 ◽

Vol 2 (5) ◽

pp. 1018-1024 ◽

Cited By ~ 115

Author(s):

Neeraj Chauhan ◽

Diane Inglis ◽

Elvira Roman ◽

Jesus Pla ◽

Dongmei Li ◽

...

Keyword(s):

Oxidative Stress ◽

Signal Transduction ◽

Hydrogen Peroxide ◽

Cell Wall ◽

Response Regulator ◽

Cell Wall Biosynthesis ◽

Regulator Protein ◽

Two Component ◽

Response Regulator Protein ◽

Mutant Cells

ABSTRACT Ssk1p of Candida albicans is a putative response regulator protein of the Hog1 two-component signal transduction system. In Saccharomyces cerevisiae, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown that C. albicans SSK1 does not complement the ssk1 mutant of S. cerevisiae and that the ssk1 mutant of C. albicans is not sensitive to sorbitol. In this study, we show that the C. albicans ssk1 mutant is sensitive to several oxidants, including hydrogen peroxide, t-butyl hydroperoxide, menadione, and potassium superoxide when each is incorporated in yeast extract-peptone-dextrose (YPD) agar medium. We used DNA microarrays to identify genes whose regulation is affected by the ssk1 mutation. RNA from mutant cells (strain CSSK21) grown in YPD medium for 3 h at 30°C was reverse transcribed and then compared with similarly prepared RNA from wild-type cells (CAF2). We observed seven genes from mutant cells that were consistently up regulated (three-fold or greater compared to CAF2). In S. cerevisiae, three (AHP1, HSP12, and PYC2) of the seven genes that were up regulated provide cells with an adaptation function in response to oxidative stress; another gene (GPH1) is regulated under stress conditions by Hog1p. Three other genes that are up regulated encode a cell surface protein (FLO1), a mannosyl transferase (MNN4-4), and a putative two-component histidine kinase (CHK1) that regulates cell wall biosynthesis in C. albicans. Of the down-regulated genes, ALS1 is a known cell adhesin in C. albicans. Verification of the microarray data was obtained by reverse transcription-PCR for HSP12, AHP1, CHK1, PYC2, GPH1, ALS1, MNN4-4, and FLO1. To further determine the function of Ssk1p in the Hog1p signal transduction pathway in C. albicans, we used Western blot analysis to measure phosphorylation of Hog1p in the ssk1 mutant of C. albicans when grown under either osmotic or oxidative stress. We observed that Hog1p was phosphorylated in the ssk1 mutant of C. albicans when grown in a hyperosmotic medium but was not phosphorylated in the ssk1 mutant when the latter was grown in the presence of hydrogen peroxide. These data indicate that C. albicans utilizes the Ssk1p response regulator protein to adapt cells to oxidative stress, while its role in the adaptation to osmotic stress is less certain. Further, SSK1 appears to have a regulatory function in some aspects of cell wall biosynthesis. Thus, the functions of C. albicans SSK1 differ from those of S. cerevisiae SSK1.

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