scholarly journals Physiological Effects of Crl in Salmonella Are Modulated by σS Level and Promoter Specificity

2007 ◽  
Vol 189 (8) ◽  
pp. 2976-2987 ◽  
Author(s):  
Véronique Robbe-Saule ◽  
Miguel Dias Lopes ◽  
Annie Kolb ◽  
Françoise Norel
Keyword(s):  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Transcription Initiation ◽  
Regulatory Protein ◽  
Acid Stress ◽  
Physiological Effects ◽  
Wild Type ◽  
Serovar Typhimurium

ABSTRACT The small regulatory protein Crl activates σS (RpoS), the stationary-phase and general stress response sigma factor. Crl has been reported to bind σS in vitro and to facilitate the formation of RNA polymerase holoenzyme. In Salmonella enterica serovar Typhimurium, Crl is required for the development of the rdar morphotype and transcription initiation of the σS-dependent genes csgD and adrA, involved in curli and cellulose production. Here, we examined the expression of other σS-dependent phenotypes and genes in a Δcrl mutant of Salmonella. Gene fusion analyses and in vitro transcription assays indicate that the magnitude of Crl activation differs between promoters and is highly dependent on σS levels. We replaced the wild-type rpoS allele in S. enterica serovar Typhimurium strain ATCC 14028 with the rpoS LT2 allele that shows reduced expression of σS; the result was an increased Crl activation ratio and larger physiological effects of Crl on oxidative, thermal, and acid stress resistance levels during stationary phase. We also found that crl, rpoS, and crl rpoS strains grew better on succinate than did the wild type and expressed the succinate dehydrogenase sdhCDBA operon more strongly. The crl and rpoS LT2 mutations also increased the competitive fitness of Salmonella in stationary phase. These results show that Crl contributes to negative regulation by σS, a finding consistent with a role for Crl in sigma factor competition via the facilitation of σS binding to core RNA polymerase.

scholarly journals In Vitro Properties of RpoS (σS) Mutants of Escherichia coli with Postulated N-Terminal Subregion 1.1 or C-Terminal Region 4 Deleted

2003 ◽  
Vol 185 (8) ◽  
pp. 2673-2679 ◽  
Author(s):  
J. Gowrishankar ◽  
Kaneyoshi Yamamoto ◽  
P. R. Subbarayan ◽  
Akira Ishihama
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Electrophoretic Mobility ◽  
Sigma Factor ◽  
Transcription Initiation ◽  
Terminal Region ◽  
Wild Type ◽  
Dna Templates ◽  
Derivatives Of

ABSTRACT Derivatives of the stationary-phase sigma factor σS of Escherichia coli lacking either of two conserved domains, the postulated N-terminal subregion 1.1 or the C-terminal region 4, were shown to be competent in vitro for transcription initiation from several σS-dependent promoters on supercoiled DNA templates. Unlike wild-type σS, however, the deletion derivatives were inactive on relaxed templates. The anomalous slow electrophoretic mobility of σS on denaturing gels was corrected by deletion of subregion 1.1, suggesting that this domain in σS may be structurally and functionally analogous to subregion 1.1 of σ70, substitutions in which have previously been shown to rectify the anomalous electrophoretic migration of σ70 (V. Gopal and D. Chatterji, Eur. J. Biochem. 244:614-618, 1997).

scholarly journals Construction and Characterization of aMycobacterium tuberculosis Mutant Lacking the Alternate Sigma Factor Gene, sigF

2000 ◽  
Vol 68 (10) ◽  
pp. 5575-5580 ◽  
Author(s):  
Ping Chen ◽  
Rafael E. Ruiz ◽  
Qing Li ◽  
Richard F. Silver ◽  
William R. Bishai
Keyword(s):  
Stationary Phase ◽  
Sigma Factor ◽  
Type Strain ◽  
Wild Type Strain ◽  
Axenic Culture ◽  
Wild Type ◽  
Intracellular Growth ◽  
Factor Gene ◽  
Time To Death

ABSTRACT The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain ofMycobacterium tuberculosis. The growth rate of the ΔsigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the ΔsigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the ΔsigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the ΔsigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.

scholarly journals Sigma Factor F Does Not Prevent Rifampin Inhibition of RNA Polymerase or Cause Rifampin Tolerance in Mycobacterium tuberculosis

2010 ◽  
Vol 192 (20) ◽  
pp. 5472-5479 ◽  
Author(s):  
Ruben C. Hartkoorn ◽  
Claudia Sala ◽  
Sophie J. Magnet ◽  
Jeffrey M. Chen ◽  
Florence Pojer ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Potent Inhibitor ◽  
Sigma Factors ◽  
Antituberculosis Drugs ◽  
Axenic Cultures

ABSTRACT The tolerance of Mycobacterium tuberculosis to antituberculosis drugs is a major reason for the lengthy therapy needed to treat a tuberculosis infection. Rifampin is a potent inhibitor of RNA polymerase (RNAP) in vivo but has been shown to be less effective against stationary-phase bacteria. Sigma factor F is associated with bacteria entering stationary phase and has been proposed to impact rifampin activity. Here we investigate whether RNAP containing SigF is more resistant to rifampin inhibition in vitro and whether overexpression of sigF renders M. tuberculosis more tolerant to rifampin. Real-time and radiometric in vitro transcription assays revealed that rifampin equally inhibits transcription by RNAP containing sigma factors SigA and SigF, therefore ruling out the hypothesis that SigF may be responsible for increased resistance of the enzyme to rifampin in vitro. In addition, overexpression or deletion of sigF did not alter rifampin susceptibility in axenic cultures of M. tuberculosis, indicating that SigF does not affect rifampin tolerance in vivo.

scholarly journals ςBldN, an Extracytoplasmic Function RNA Polymerase Sigma Factor Required for Aerial Mycelium Formation in Streptomyces coelicolor A3(2)

2000 ◽  
Vol 182 (16) ◽  
pp. 4606-4616 ◽  
Author(s):  
Maureen J. Bibb ◽  
Virginie Molle ◽  
Mark J. Buttner
Keyword(s):  
Rna Polymerase ◽  
Sigma Factor ◽  
Streptomyces Coelicolor ◽  
Aerial Mycelium ◽  
Sigma Factors ◽  
Colony Development ◽  
Null Mutant ◽  
Wild Type

ABSTRACT Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci. whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N′-nitro-N-nitrosoguanidine)-inducedwhi strains (N. J. Ryding et al., J. Bacteriol. 181:5419–5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed thatwhiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed whiN null mutant failed to form aerial mycelium (the “bald” phenotype) and, as a consequence, whiN was renamed bldN. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S. coelicolor bld mutants. Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficientbld strains. bldN was transcribed from a single promoter, bldNp. bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC,bldF, bldK, or bldJ or onbldN itself. Transcription from the p1 promoter of the response-regulator gene bldM depended onbldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for ςBldN holoenzyme in vitro.

scholarly journals Effect of Inactivation of degS on Salmonella enterica Serovar Typhimurium In Vitro and In Vivo

2005 ◽  
Vol 73 (1) ◽  
pp. 459-463 ◽  
Author(s):  
Gary Rowley ◽  
Andrew Stevenson ◽  
Jan Kormanec ◽  
Mark Roberts
Keyword(s):  
Sigma Factor ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Wild Type ◽  
Alternative Pathways ◽  
E Coli ◽  
Mammalian Tissues ◽  
Serovar Typhimurium

ABSTRACT The alternative sigma factor (RpoE σE) enables Salmonella enterica serovar Typhimurium to adapt to stressful conditions, such as oxidative stress, nutrient deprivation, and growth in mammalian tissues. Infection of mice by Salmonella serovar Typhimurium also requires σE. In Escherichia coli, activation of the σE pathway is dependent on proteolysis of the anti-sigma factor RseA and is initiated by DegS. DegS is also important in order for E. coli to cause extraintestinal infection in mice. We constructed a degS mutant of the serovar Typhimurium strain SL1344 and compared its behavior in vitro and in vivo with those of its wild-type (WT) parent and an isogenic rpoE mutant. Unlike E. coli degS strains, the Salmonella serovar Typhimurium degS strain grew as well as the WT strain at 42°C. The degS mutant survived very poorly in murine macrophages in vitro and was highly attenuated compared with the WT strain for both the oral and parenteral routes of infection in mice. However, the degS mutant was not as attenuated as the serovar Typhimurium rpoE mutant: 100- to 1,000-fold more degS bacteria than rpoE bacteria were present in the livers and spleens of mice 24 h after intraperitoneal challenge. In most assays, the rpoE mutant was more severely affected than the degS mutant and a σE-dependent reporter gene was more active in the degS mutant than the rpoE strain. These findings indicate that degS is important for activation of the σE pathway in serovar Typhimurium but that alternative pathways for σE activation probably exist.

scholarly journals Effect of Growth Temperature on Crl-Dependent Regulation of σS Activity in Salmonella enterica Serovar Typhimurium

2008 ◽  
Vol 190 (13) ◽  
pp. 4453-4459 ◽  
Author(s):  
Véronique Robbe-Saule ◽  
Ingrid Carreira ◽  
Annie Kolb ◽  
Françoise Norel
Keyword(s):  
Stationary Phase ◽  
Salmonella Enterica ◽  
Regulatory Protein ◽  
Effect Of Temperature ◽  
Resistance Level ◽  
Serovar Typhimurium ◽  
Negative Effect ◽  
Transcription In Vitro ◽  
Physiological Impact

ABSTRACT The small regulatory protein Crl favors association of the stationary-phase sigma factor σS (RpoS) with the core enzyme polymerase and thereby increases σS activity. Crl has a major physiological impact at low levels of σS. Here, we report that the Crl effects on σS-dependent gene expression, the H2O2 resistance of Salmonella enterica serovar Typhimurium, and the resistance of this organism to acidic pH are greater at 28°C than at 37°C. Immunoblot experiments revealed a negative correlation between σS and Crl levels; the production of Crl was slightly greater at 28°C than at 37°C, whereas the σS levels were about twofold lower at 28°C than at 37°C. At both temperatures, Crl was present in excess of σS, and increasing the Crl level further did not increase the H2O2 resistance level of Salmonella and the expression of the σS-dependent gene katE encoding the stationary-phase catalase. In contrast, increasing the σS level rendered Salmonella more resistant to H2O2 at 28°C, increased the expression of katE, and reduced the magnitude of Crl activation. In addition, the effect of Crl on katE transcription in vitro was not dependent on temperature. These results suggest that the effect of temperature on Crl-dependent regulation of the katE gene and H2O2 resistance are mediated mainly via an effect on σS levels. In addition, our results revealed that σS exerts a negative effect on the production of Crl in stationary phase when the cells contain high levels of σS.

scholarly journals An N-Terminally Truncated RpoS (σS) Protein in Escherichia coli Is Active In Vivo and Exhibits Normal Environmental Regulation Even in the Absence of rpoS Transcriptional and Translational Control Signals

2002 ◽  
Vol 184 (12) ◽  
pp. 3167-3175 ◽  
Author(s):  
K. Rajkumari ◽  
J. Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Translational Control ◽  
Sigma Factor ◽  
Receptor Protein ◽  
Wild Type ◽  
Truncated Protein

ABSTRACT RpoS (σS) in Escherichia coli is a stationary-phase-specific primary sigma factor of RNA polymerase which is 330 amino acids long and belongs to the eubacterial σ70 family of proteins. Conserved domain 1.1 at the N-terminal end of σ70 has been shown to be essential for RNA polymerase function, and its deletion has been shown to result in a dominant-lethal phenotype. We now report that a σS variant with a deletion of its N-terminal 50 amino acids (σSΔ1-50), when expressed in vivo either from a chromosomal rpoS::IS10 allele (in rho mutant strains) or from a plasmid-borne arabinose-inducible promoter, is as proficient as the wild type in directing transcription from the proU P1 promoter; at three other σS-dependent promoters that were tested (osmY, katE, and csiD), the truncated protein exhibited a three- to sevenfold reduced range of activities. Catabolite repression at the csiD promoter (which requires both σS and cyclic AMP [cAMP]-cAMP receptor protein for its activity) was also preserved in the strain expressing σSΔ1-50. The intracellular content of σSΔ1-50 was regulated by culture variables such as growth phase, osmolarity, and temperature in the same manner as that described earlier for σS, even when the truncated protein was expressed from a template that possessed neither the transcriptional nor the translational control elements of wild-type rpoS. Our results indicate that, unlike that in σ70, the N-terminal domain in σS may not be essential for the protein to function as a sigma factor in vivo. Furthermore, our results suggest that the induction of σS-specific promoters in stationary phase and during growth under conditions of high osmolarity or low temperature is mediated primarily through the regulation of σS protein degradation.

scholarly journals Leucine-Responsive Regulatory Protein (Lrp) Acts as a Virulence Repressor in Salmonella enterica Serovar Typhimurium

2008 ◽  
Vol 191 (4) ◽  
pp. 1278-1292 ◽  
Author(s):  
Chang-Ho Baek ◽  
Shifeng Wang ◽  
Kenneth L. Roland ◽  
Roy Curtiss
Keyword(s):  
Wild Type Strain ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Regulatory Protein ◽  
Constitutive Expression ◽  
Wild Type ◽  
Promoter Regions ◽  
Serovar Typhimurium ◽  
Regulator Genes ◽  
Virulence Regulator

ABSTRACT Leucine-responsive regulatory protein (Lrp) is a global gene regulator that influences expression of a large number of genes including virulence-related genes in Escherichia coli and Salmonella. No systematic studies examining the regulation of virulence genes by Lrp have been reported in Salmonella. We report here that constitutive expression of Lrp [lrp(Con)] dramatically attenuates Salmonella virulence while an lrp deletion (Δlrp) mutation enhances virulence. The lrp(Con) mutant caused pleiotropic effects that include defects in invasion, cytotoxicity, and colonization, whereas the Δlrp mutant was more proficient at these activities than the wild-type strain. We present evidence that Lrp represses transcription of key virulence regulator genes—hilA, invF, and ssrA—in Salmonella pathogenicity island 1 (SPI-1) and 2 (SPI-2), by binding directly to their promoter regions, P hilA , P invF , and P ssrA . In addition, Western blot analysis showed that the expression of the SPI-1 effector SipA was reduced in the lrp(Con) mutant and enhanced in the Δlrp mutant. Computational analysis revealed putative Lrp-binding consensus DNA motifs located in P hilA , P invF , and P ssrA . These results suggest that Lrp binds to the consensus motifs and modulates expression of the linked genes. The presence of leucine enhanced Lrp binding to P invF in vitro and the addition of leucine to growth medium decreased the level of invF transcription. However, leucine had no effect on expression of hilA and ssrA or on cellular levels of Lrp. In addition, Lrp appears to be an antivirulence gene, since the deletion mutant showed enhanced cell invasion, cytotoxicity, and hypervirulence in BALB/c mice.

scholarly journals ArgR-Independent Induction and ArgR-Dependent Superinduction of the astCADBE Operon in Escherichia coli

2002 ◽  
Vol 184 (11) ◽  
pp. 2940-2950 ◽  
Author(s):  
Alexandros K. Kiupakis ◽  
Larry Reitzer
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Nitrogen Source ◽  
Nitrogen Limitation ◽  
Nitrogen Sources ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Nitrogen Regulator I

ABSTRACT For Escherichia coli, growth in the absence of ammonia is termed nitrogen limited and results in the induction of genes that assimilate other nitrogen sources, a response mediated by σ54 and nitrogen regulator I (NRI, also called NtrC). The astCADBE operon, which is required for growth with arginine as the sole nitrogen source, is moderately expressed during general nitrogen limitation and maximally expressed in the presence of arginine. The operon is also induced in stationary phase. Primer extension analysis of E. coli revealed the presence of a σ54-dependent promoter utilized in exponential phase during nitrogen limitation and a σS-dependent promoter active during stationary phase. We used an ast-lacZ fusion to show that arginine stimulates expression, that ArgR, the arginine repressor, enhances expression from both promoters but is not essential, and that transcription by the two forms of the RNA polymerase is competitive and mutually exclusive. We demonstrated the binding of RNA polymerase holoenzymes, NRI, and ArgR to the promoter region in vitro. We also reconstituted transcription from both promoters with purified components, which confirmed the accessory role of ArgR for the σ54-dependent promoter. Thus, the ast operon exhibits nitrogen source-specific induction that is unique for an NRI-dependent gene. The transcriptional regulation of the ast operon in E. coli differs from that in Salmonella enterica serovar Typhimurium, in which ArgR is required for ast operon expression.

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