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

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).

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

Journal of Bacteriology ◽

10.1128/jb.01919-06 ◽

2007 ◽

Vol 189 (8) ◽

pp. 2976-2987 ◽

Cited By ~ 35

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.

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

Infection and Immunity ◽

10.1128/iai.68.10.5575-5580.2000 ◽

2000 ◽

Vol 68 (10) ◽

pp. 5575-5580 ◽

Cited By ~ 99

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.

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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

Journal of Bacteriology ◽

10.1128/jb.184.12.3167-3175.2002 ◽

2002 ◽

Vol 184 (12) ◽

pp. 3167-3175 ◽

Cited By ~ 13

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.

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Differential Suppression of priA2::kan Phenotypes in Escherichia coli K-12 by Mutations in priA, lexA, and dnaC

Genetics ◽

10.1093/genetics/143.1.5 ◽

1996 ◽

Vol 143 (1) ◽

pp. 5-13 ◽

Cited By ~ 21

Author(s):

Steven J Sandler ◽

Hardeep S Samra ◽

Alvin J Clark

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Mutant Allele ◽

Wild Type ◽

Suppressor Mutations ◽

Dnab Helicase ◽

K 12 ◽

Extragenic Suppressors ◽

Assembly Activity

Abstract First identified as an essential component of the ϕX174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec−, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.

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

Journal of Bacteriology ◽

10.1128/jb.182.23.6707-6713.2000 ◽

2000 ◽

Vol 182 (23) ◽

pp. 6707-6713 ◽

Cited By ~ 31

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.

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Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3468-3474.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3468-3474 ◽

Cited By ~ 19

Author(s):

Gyeong Tae Eom ◽

Jae Kwang Song ◽

Jung Hoon Ahn ◽

Yeon Soo Seo ◽

Joon Shick Rhee

Keyword(s):

Escherichia Coli ◽

Directed Evolution ◽

Abc Transporter ◽

Microtiter Plate ◽

Single Amino Acid ◽

Wild Type ◽

E Coli ◽

Single Amino Acid Change ◽

Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells

Journal of Bacteriology ◽

10.1128/jb.185.3.1097-1100.2003 ◽

2003 ◽

Vol 185 (3) ◽

pp. 1097-1100 ◽

Cited By ~ 27

Author(s):

Yazmid Reyes-Domínguez ◽

Gabriel Contreras-Ferrat ◽

Jesús Ramírez-Santos ◽

Jorge Membrillo-Hernández ◽

M. Carmen Gómez-Eichelmann

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Plasmid Dna ◽

Bimodal Distribution ◽

Dna Supercoiling ◽

Wild Type

ABSTRACT Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.

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exrB: amalB-linked gene inEscherichia coliB involved in sensitivity to radiation and filament formation

Genetics Research ◽

10.1017/s0016672300014798 ◽

1974 ◽

Vol 23 (2) ◽

pp. 175-184 ◽

Cited By ~ 25

Author(s):

Joseph Greenberg ◽

Leonard J. Berends ◽

John Donch ◽

Michael H. L. Green

Keyword(s):

Escherichia Coli ◽

Normal Growth ◽

Filament Formation ◽

Wild Type ◽

Sensitive Mutant ◽

Γ Radiation ◽

Linked Gene ◽

Derivatives Of

SUMMARYPAM 26, a radiation-sensitive mutant ofEscherichia colistrain B, is described. Its properties are attributable to a mutation in a gene,exrB, which is cotransducible withmalB. It differs fromuvrA(alsomalB-linked) derivatives of strain B in being sensitive to 1-methyl-3-nitro-1-nitroso-guanidine and γ-radiation, and in being able to reactivate UV-irradiated phage T3. It differs fromexrA(alsomalB-linked) derivatives of strain B in forming filaments during the course of normal growth as well as after irradiation. WhenexrBwas transduced into a K12 (lon+) strain, filaments did not form spontaneously. Three-point transductions established the order of markers asmet A malB exrB. Based on an analysis of the frequency of wild-type recombinants in a reciprocal transduction betweenexrAandexrBstrains, it was inferred that they are not isogenic and that the order of markers ismalB exrA exrB.

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Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

Acta Biochimica Polonica ◽

10.18388/abp.2003_3623 ◽

2003 ◽

Vol 50 (4) ◽

pp. 909-920 ◽

Cited By ~ 4

Author(s):

Iwona K Kolasa ◽

Tomasz Łoziński ◽

Kazimierz L Wierzchowski

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Transcription Initiation ◽

Specific Interaction ◽

Structural Data ◽

Structural Morphology ◽

Promoter Dna ◽

Sigma Subunit ◽

Kinetics Of

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

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Multiple Elements Controlling Adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa Cells

Infection and Immunity ◽

10.1128/iai.71.9.4985-4995.2003 ◽

2003 ◽

Vol 71 (9) ◽

pp. 4985-4995 ◽

Cited By ~ 108

Author(s):

Alfredo G. Torres ◽

James B. Kaper

Keyword(s):

Escherichia Coli ◽

Hela Cells ◽

Culture Conditions ◽

Enterohemorrhagic Escherichia Coli ◽

Differentially Expressed ◽

Wild Type ◽

Transposon Insertion ◽

Reduced Adherence ◽

Insertion Mutants

ABSTRACT Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated tcdA gene. An isogenic ompA mutant showed reduced adherence compared to the parent strain. Disruption of the ompA gene in the tdcA mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and tdcA mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.

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