The LysR-type transcriptional regulator CysB controls the repression of hslJ transcription in Escherichia coli

The LysR-type transcriptional regulator (LTTR) CysB is a transcription factor in Escherichia coli cells, where as a homotetramer it binds the target promoter regions and activates the genes involved in sulphur utilization and sulphonate-sulphur metabolism, while negatively autoregulating its own transcription. The hslJ gene was found to be negatively regulated by CysB and directly correlated with novobiocin resistance of the bacterium. cysB mutants showed upregulation of the hslJ : : lacZ gene fusion and exhibited increased novobiocin resistance. In this study the hslJ transcription start point and the corresponding putative σ 70 promoter were determined. The hslJ promoter region was defined by employing different hslJ–lacZ operon fusions, and transcription of the hslJ gene was shown to be subject to both repression imposed by the CysB regulator and direct or indirect autogenous negative control. These two regulations compete to some extent but they are not mutually exclusive. CysB acts as a direct repressor of hslJ transcription and binds the hslJ promoter region that carries the putative CysB repressor site. This CysB binding, apparently responsible for repression, is enhanced in the presence of the ligand N-acetylserine (NAS), hitherto considered to be a positive cofactor in CysB-mediated gene regulations. Interallelic complementation of characterized CysB mutants I33N and S277Ter partially restored the repression of hslJ transcription and the consequent novobiocin sensitivity, but did not complement the cysteine auxotrophy.

Download Full-text

Molecular Characterization of Cefoxitin-Resistant Escherichia coli from Canadian Hospitals

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.1.358-365.2005 ◽

2005 ◽

Vol 49 (1) ◽

pp. 358-365 ◽

Cited By ~ 96

Author(s):

Michael R. Mulvey ◽

Elizabeth Bryce ◽

David A. Boyd ◽

Marianna Ofner-Agostini ◽

Allison M. Land ◽

...

Keyword(s):

Escherichia Coli ◽

Sequence Analysis ◽

Promoter Region ◽

Hospital Setting ◽

Wild Type ◽

Promoter Regions ◽

Extended Spectrum Beta Lactamase ◽

Insertion Elements ◽

Baseline Information ◽

Clinically Significant

ABSTRACT A study designed to gain baseline information on strains of Escherichia coli displaying resistance to cefoxitin in Canada is described. A total of 29,323 E. coli isolates were screened at 12 participating hospital sites as part of an extended-spectrum beta-lactamase surveillance initiative. A total of 411 clinically significant, nonrepeat isolates displaying reduced susceptibilities to the NCCLS-recommended beta-lactams were submitted to a central laboratory over a 1-year period ending on 30 September 2000. Two hundred thirty-two isolates were identified as resistant to cefoxitin. All cefoxitin-resistant strains were subtyped by pulsed-field gel electrophoresis, and of these, 182 strains revealed a unique fingerprint and 1 strain was untypeable. PCR and sequence analysis of the ampC promoter region revealed 51 different promoter or attenuator variants and 14 wild-type promoters. Three promoter regions were interrupted by insertion elements, two contained IS10 elements, and one contained an IS911 variant. PCR and sequence analysis for the detection of acquired AmpC resistance (by the acquisition of ACT-1/MIR-1, CMY-2, or FOX) revealed that 25 strains contained CMY-2, including 7 of the strains found to have wild-type promoters. The considerable genetic variability in both the strain fingerprint and the promoter region suggests that AmpC-type resistance may emerge spontaneously by mutation of sensitive strains rather than by the spread of strains or plasmids in the hospital setting.

Download Full-text

Promoter Characterization and Constitutive Expression of the Escherichia coli gcvR Gene

Journal of Bacteriology ◽

10.1128/jb.180.7.1803-1807.1998 ◽

1998 ◽

Vol 180 (7) ◽

pp. 1803-1807 ◽

Cited By ~ 12

Author(s):

Angela C. Ghrist ◽

George V. Stauffer

Keyword(s):

Escherichia Coli ◽

Transcription Start Site ◽

Promoter Region ◽

Start Codon ◽

Start Site ◽

Transcription Start ◽

Translation Start Codon ◽

Translation Start ◽

Glycine Cleavage ◽

Extension Analysis

ABSTRACT The Escherichia coli glycine cleavage repressor protein (GcvR) negatively regulates expression of the glycine cleavage operon (gcv). In this study, the gcvR translational start site was determined by N-terminal amino acid sequence analysis of a GcvR-LacZ fusion protein. Primer extension analysis of thegcvR promoter region identified a primary transcription start site 27 bp upstream of the UUG translation start site and a minor transcription start site approximately 100 bp upstream of the translation start codon. The -10 and -35 promoter regions upstream of the primary transcription start site were defined by mutational analysis. Expression of a gcvR-lacZ fusion was unaltered in the presence of glycine or inosine, molecules known to induce or repress expression of gcv, respectively. In addition, it was shown that gcvR-lacZ expression is neither regulated by the glycine cleavage activator protein (GcvA) nor autogenously regulated by GcvR. From DNA sequence analysis, it was predicted that the translation start codon of the downstream bcp gene overlaps the gcvR stop codon, suggesting that these genes may form an operon. However, a down mutation in the -10 promoter region of gcvR had no effect on the expression of a downstreambcp-lacZ fusion, and primer extension analysis of thebcp promoter region demonstrated that bcp has its own promoter within the gcvR coding sequence. These results show that gcvR and bcp do not form an operon. Furthermore, the deletion of bcp from the chromosome had no effect on gcv-lacZ expression.

Download Full-text

Molecular Characterization of the PhoP-PhoQ Two-Component System in Escherichia coli K-12: Identification of Extracellular Mg2+-Responsive Promoters

Journal of Bacteriology ◽

10.1128/jb.181.17.5516-5520.1999 ◽

1999 ◽

Vol 181 (17) ◽

pp. 5516-5520 ◽

Cited By ~ 117

Author(s):

Akinori Kato ◽

Hiroyuki Tanabe ◽

Ryutaro Utsumi

Keyword(s):

Escherichia Coli ◽

Direct Repeat ◽

Two Component System ◽

Transcription Start ◽

Promoter Regions ◽

S1 Nuclease ◽

Transcription Start Sites ◽

Two Component ◽

K 12

ABSTRACT We identified Mg2+-responsive promoters of thephoPQ, mgtA, and mgrB genes ofEscherichia coli K-12 by S1 nuclease analysis. Expression of these genes was induced by magnesium limitation and depended on PhoP and PhoQ. The transcription start sites were also determined, which allowed us to find a (T/G)GTTTA direct repeat in their corresponding promoter regions.

Download Full-text

NtcA-Regulated Heterocyst Differentiation Genes hetC and devB from Anabaena sp. Strain PCC 7120 Exhibit a Similar Tandem Promoter Arrangement

Journal of Bacteriology ◽

10.1128/jb.00616-09 ◽

2009 ◽

Vol 191 (18) ◽

pp. 5765-5774 ◽

Cited By ~ 16

Author(s):

Alicia M. Muro-Pastor ◽

Enrique Flores ◽

Antonia Herrero

Keyword(s):

Promoter Region ◽

Transcriptional Start Site ◽

Short Version ◽

Start Site ◽

Transcription Start ◽

Promoter Regions ◽

Heterocyst Differentiation ◽

Translational Start ◽

Pcc 7120 ◽

Diazotrophic Growth

ABSTRACT Transcription of the hetC gene, whose product is required for heterocyst differentiation, takes place from a long promoter region that includes the previously described HetR-independent, NtcA-activated promoter producing transcripts with a 5′ end corresponding to position −571 with respect to the translational start site of hetC. Northern blot analysis indicated that the accumulation of hetC transcripts depends on HetR, and a second transcriptional start site located at position −293 that leads to NtcA-dependent, HetR-dependent inducible transcription of hetC was identified. Upon nitrogen stepdown, expression of a P hetC ::gfp fusion was transiently induced in specific cells that were differentiating into heterocysts, both when the whole promoter region (containing transcription start points −571 and −293) or a short version (containing only the transcription start point −293) was used. Expression of hetC from the −293 position was delayed in a strain bearing a deleted promoter region lacking sequences upstream from position −570. Such a strain was still able to differentiate functional heterocysts and to grow diazotrophically, although diazotrophic growth was impaired under certain conditions. Similarly, a second, NtcA-dependent, HetR-dependent transcriptional start site was identified at position −454 in the promoter region upstream from the devBCA operon encoding an ABC transport system involved in heterocyst maturation, in which an NtcA-dependent promoter producing transcripts starting at position −704 had been previously noted. Thus, the hetC and devBCA promoter regions exhibit similar tandem promoter arrangements.

Download Full-text

Molecular Characterization of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00791-15 ◽

2016 ◽

Vol 198 (10) ◽

pp. 1487-1498 ◽

Cited By ~ 15

Author(s):

Katie B. Nichols ◽

Makrina Totsika ◽

Danilo G. Moriel ◽

Alvin W. Lo ◽

Ji Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Promoter Region ◽

Core Body Temperature ◽

Systemic Infection ◽

Transcriptional Regulator ◽

Fold Increase ◽

Mobility Shift ◽

Genomic Locus ◽

Content Type ◽

Tract Infections

ABSTRACTThe vacuolating autotransporter toxin (Vat) contributes to uropathogenicEscherichia coli(UPEC) fitness during systemic infection. Here, we characterized Vat and investigated its regulation in UPEC. We assessed the prevalence ofvatin a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing thevatgene corresponded to three majorE. colisequence types (ST12, ST73, and ST95), and these strains secreted the Vat protein. Further analysis of thevatgenomic locus identified a conserved gene located directly downstream ofvatthat encodes a putative MarR-like transcriptional regulator; we termed this genevatX. Thevat-vatXgenes were present in the UPEC reference strain CFT073, and reverse transcriptase PCR (RT-PCR) revealed that the two genes are cotranscribed. Overexpression ofvatXin CFT073 led to a 3-fold increase invatgene transcription. Thevatpromoter region contained three putative nucleation sites for the global transcriptional regulator histone-like nucleoid structuring protein (H-NS); thus, thehnsgene was mutated in CFT073 (to generate CFT073hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073hnscompared to that in wild-type CFT073. Direct H-NS binding to thevatpromoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected byvat-containing UPEC strains, demonstrating that Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic serine protease autotransporter protein ofEnterobacteriaceae(SPATE) secreted by UPEC during infection.IMPORTANCEUropathogenicEscherichia coli(UPEC) is the major cause of hospital- and community-acquired urinary tract infections. The vacuolating autotransporter toxin (Vat) is a cytotoxin known to contribute to UPEC fitness during murine sepsis infection. In this study, Vat was found to be highly conserved and prevalent among a collection of urosepsis clinical isolates and was expressed at human core body temperature. Regulation ofvatwas demonstrated to be directly repressed by the global transcriptional regulator H-NS and upregulated by the downstream genevatX(encoding a new MarR-type transcriptional regulator). Additionally, increased Vat-specific IgG titers were detected in plasma from corresponding urosepsis patients infected withvat-positive isolates. Hence, Vat is a highly conserved and tightly regulated urosepsis-associated virulence factor.

Download Full-text

The curli biosynthesis regulator CsgD co-ordinates the expression of both positive and negative determinants for biofilm formation in Escherichia coli

Microbiology ◽

10.1099/mic.0.26306-0 ◽

2003 ◽

Vol 149 (10) ◽

pp. 2847-2857 ◽

Cited By ~ 111

Author(s):

Eva Brombacher ◽

Corinne Dorel ◽

Alexander J. B. Zehnder ◽

Paolo Landini

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Regulatory System ◽

Multicopy Plasmid ◽

Transcription Activator ◽

Transcription Start ◽

Promoter Regions ◽

E Coli ◽

Two Component ◽

Extracellular Structures

Production of curli, extracellular structures important for biofilm formation, is positively regulated by OmpR, which constitutes with the EnvZ protein an osmolarity-sensing two-component regulatory system. The expression of curli is cryptic in most Escherichia coli laboratory strains such as MG1655, due to the lack of csgD expression. The csgD gene encodes a transcription activator of the curli-subunit-encoding csgBA operon. The ompR234 up-mutation can restore csgD expression, resulting in curli production and increased biofilm formation. In this report, it is shown that ompR234-dependent csgD expression, in addition to csgBA activation during stationary phase of growth, stimulates expression of the yaiC gene and negatively regulates at least two other genes, pepD and yagS. The promoter regions of these four genes share a conserved 11 bp sequence (CGGGKGAKNKA), necessary for csgBA and yaiC regulation by CsgD. While at both the csgBA and yaiC promoters the sequence is located upstream of the promoter elements, in both yagS and pepD it overlaps either the putative −10 sequence or the transcription start point, suggesting that CsgD can function as both an activator and a repressor. Adhesion experiments show that csgD-independent expression of both yagS and pepD from a multicopy plasmid negatively affects biofilm formation, which, in contrast, is stimulated by yaiC expression. Thus it is proposed that CsgD stimulates biofilm formation in E. coli by contemporary activation of adhesion positive determinants (the curli-encoding csg operons and the product of the yaiC gene) and repression of negative effectors such as yagS and pepD.

Download Full-text