Analysis of the regulatory region of the heat-shock gene rpoH of Escherichia coli strains isolated from non-human hosts.

The regulatory region of the gene for sigma32, rpoH, of Escherichia coli strains isolated from non-human hosts and different geographic regions, was sequenced and compared with that of E. coli K12. The main nucleotide changes observed are localized to the right inverted octamer motif of the CytR box. The effect of these changes was evaluated using transcriptional fusions. The results presented could guide further studies on the transcription regulation of rpoH using E. coli K12 as a model.

Conserved regulatory elements of the promoter sequence of the gene rpoH of enteric bacteria.

The rpoH regulatory region of different members of the enteric bacteria family was sequenced or downloaded from GenBank and compared. In addition, the transcriptional start sites of rpoH of Yersinia frederiksenii and Proteus mirabilis, two distant members of this family, were determined. Sequences similar to the sigma(70) promoters P1, P4 and P5, to the sigma(E) promoter P3 and to boxes DnaA1, DnaA2, cAMP receptor protein (CRP) boxes CRP1, CRP2 and box CytR present in Escherichia coli K12, were identified in sequences of closely related bacteria such as: E.coli, Shigella flexneri, Salmonella enterica serovar Typhimurium, Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae. In more distant bacteria, Y.frederiksenii and P.mirabilis, the rpoH regulatory region has a distal P1-like sigma(70) promoter and two proximal promoters: a heat-induced sigma(E)-like promoter and a sigma(70) promoter. Sequences similar to the regulatory boxes were not identified in these bacteria. This study suggests that the general pattern of transcription of the rpoH gene in enteric bacteria includes a distal sigma(70) promoter, >200 nt upstream of the initiation codon, and two proximal promoters: a heat-induced sigma(E)-like promoter and a sigma(70) promoter. A second proximal sigma(70) promoter under catabolite-regulation is probably present only in bacteria closely related to E.coli.

[Genetic regulation of the heat-shock response in Escherichia coli]. / Regulación genética en la respuesta al estrés calórico en Escherichia coli.

Cells of almost any organism respond to a sudden up-shift of temperature and to several other stress conditions, by a transient increase in the cellular concentration of a set of proteins, the heat-shock proteins (HSPs). The main HSPs, chaperones and proteases, are constituents of the cellular machinery of protein folding, translocation, repair and degradation. The bacteria Escherichia coli has been a paradigm regarding heat shock gene expression in prokaryotes. In this bacterium, the expression of the HSPs is regulated at the transcriptional level. The approximately 40 genes that encode the HSPs define the heat-shock stimulon. Most of these genes, including the main chaperone and protease genes, are under the positive control of sigma32, encoded by rpoH, while approximately 10 genes, including rpoH and rpoE, are regulated by sigma(E) , encoded by rpoE. The cytoplasmic response to heat is regulated by sigma32, while that of the periplasm is regulated by sigma(E). The expression of both regulons is interconnected, since sigma(E) regulates the transcription of rpoH at high temperatures. The activity of these sigma factors, under non-stress and stress conditions, depends upon negative and positive regulatory mechanisms acting at different levels: transcription, translation, half-life and activity of the factors. Models for the regulation of the cytoplasmic and periplasmic response to heat in E. coli are presented.

Identification of sigma 32-like factors and ftsX-rpoH gene arrangements in enteric bacteria.

Western blot analyses using anti-Escherichia coli K-12 sigma 32 antibodies and Southern blot analyses using rpoH and ftsX DNA probes were performed using different enteric bacteria. Results show that the bacterial strains analysed have sigma 32-like transcription factors and ftsX and rpoH homologs in a similar map position. Although the presence of sigma 32-like factors seems to be extended to all Proteobacteria, rpoH and ftsX homologs seem to be present as neighbors in the genome only in the enteric bacteria.

In vivo effect of DNA relaxation on the transcription of gene rpoH in Escherichia coli.

The in vivo effect of Novobiocin, a gyrase inhibitor, on the transcription of gene rpoH which codes for sigma32, the main positive regulator of the heat-shock response, was studied. Novobiocin induced a three-fold increase and a slight decrease in the activity of the rpoH promoters P1 and P4, respectively. The Novobiocin-induced increase in the activity of promoter P1 correlates with an increase in the amount of proteins sigma32 and DnaK. These results suggest that the increase in expression of the heat-shock proteins induced by gyrase inhibitors is probably due to the increased activity of P1 on relaxed DNA.

Topoisomerase activity during the heat shock response in Escherichia coli K-12.

During the upshift of temperature from 30 to 42, 45, 47, or 50 degrees C, an increase in the level of supercoiling of a reporter plasmid was observed. This increase was present in groE and dnaK mutants but was inhibited in cells treated with chloramphenicol and novobiocin. The intracellular [ATP]/[ADP] ratio increased rapidly after an upshift in temperature from 30 to 47 degrees C and then decreased to reach a level above that observed at 30 degrees C. These results suggest that gyrase and proteins synthesized during heat shock are responsible for the changes seen in plasmid supercoiling. Proteins GroE and DnaK are probably not involved in this phenomenon.

Methylated cytosine at Dcm (CCATGG) sites in Escherichia coli: possible function and evolutionary implications.

The frequency and distribution of methylated cytosine (5-MeC) at CCATGG (Dcm sites) in 49 E. coli DNA loci (207,530 bp) were determined. Principal observations of this analysis were: (1) Dcm frequency was higher than expected from random occurrence but lower than calculated with Markov chain analysis; (2) CCTGG sites were found more frequently in coding than in noncoding regions, while the opposite was true for CCAGG sites; (3) Dcm site distribution does not exhibit any identifiably regular pattern on the chromosome; (4) Dcm sites at oriC are probably not important for accurate initiation of DNA replication; (5) 5-MeC in codons was more frequently found in first than in second and third positions; (6) there are probably few genes in which the mutation rate is determined mainly by DNA methylation. It is proposed that the function of Dcm methylase is to protect chromosomal DNA from restriction-enzyme EcoRII. The Dcm methylation contribution to determine frequency of oligonucleotides, mutation rate, and recombination level, and thus evolution of the E. coli genome, could be interpreted as a consequence of the acquisition of this methylation.

Distribution of insertion sequence IS1 in multiple-antibiotic resistant clinical Enterobacteriaceae strains.

The presence of insertion sequence IS1 in 70 multiple-antibiotic resistant clinical strains was determined. This 70-strain collection comprised 46 Escherichia coli, 18 Salmonella and 6 Shigella strains. The presence of IS1 was detected in the chromosome and plasmids of 73% and 63% of the strains, respectively, and 51% of the strains carried IS1 in both. The frequency of IS1 was higher in Salmonella than in E. coli and Shigella strains. A total of 31 strains carried large plasmids with IS1; 10 of these strains (32.3%) were able to transfer all or some of the antibiotic resistance markers to E. coli K12 or S. typhimurium recipient strains. Resistance markers of all clinical strains were maintained stably after several generations of growth. The presence of IS1 in a relatively high percentage of plasmids of multiple-antibiotic resistant clinical isolates, suggests a role for this sequence in the dissemination of genes which code for antibiotic resistance.

Presence of 5-methylcytosine in CC(A/T)GG sequences (Dcm methylation) in DNAs from different bacteria.

The presence of CC(A/T)GG sequences with methylated internal cytosine (Dcm methylation) was determined in DNA from different genera of eubacteria. This methylation was studied by using restriction enzymes EcoRII and BstNI, which cleave unmethylated or methylated CC(A/T)GG sequences. Dcm methylation was only detected in genera of the family Enterobacteriaceae closely related to Escherichia: Shigella, Citrobacter, Salmonella, and Klebsiella.

Influence of the Uvr repair system on the mutagenicity of antiparasitic drugs.

One amebicide (chloroquine diphosphate) and 2 anthelmintic compounds (niclosamide and pyrvinium pamoate) were found to be mutagenic for Salmonella typhimurium TA1537, TA1538, TA100 and TA98 Uvr- strains respectively. Drugs tested on homologous Uvr+ strains (TA1977, TA1978, UTH8414 and UTH8413) showed decreased mutagenic activity of the compounds. This indicates that premutational damage induced by the drugs was totally or partially repaired. Furthermore, results obtained in the present study suggest that niclosamide and pyrvinium pamoate induce premutational lesions by adduct formation, and that chloroquine diphosphate, known as an intercalating agent, behaves as an adduct-forming compound as regards its effects on Uvr- and Uvr+ S. typhimurium strains.