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.

Transcription level of operon ftsYEX and activity of promoter P1 of rpoH during the cell cycle in Escherichia coli.

In Escherichia coli, the genes of the main heat-shock proteins are under the control of the product of gene rpoH, protein sigma 32. The distal promoter P1 of rpoH is located within the terminator of the division operon ftsYEX which could imply some coupling between ftsYEX transcription termination, P1 transcription and cell division. To study the possibility of this coupling, the level of transcription of ftsYEX and the activity of promoter P1 of rpoH were examined in synchronous cultures. Results indicate that transcription of ftsYEX and of rpoH from P1 is continuous, suggesting that ftsYEX transcription termination and P1 activity are not coupled to the cell cycle.

Escherichia coli membrane fluidity as detected by excimerization of dipyrenylpropane: sensitivity to the bacterial fatty acid profile.

A coordinated study of membrane fluidity and fatty acid composition has been carried out in Escherichia coli W3110. The lipid acyl chain profile of the bacteria, altered by growing cells in steady state at 30, 37, 42, or 45 degrees C, was determined by gas chromatography of the fatty acid methyl esters. In parallel experiments, total membranes obtained from cells of the above-mentioned cultures were labeled with dipyrenylpropane and their relative fluidity was measured on the basis of the excimer to monomer fluorescence intensity ratio of the fluorophore. It has been found that, at constant assay temperature, fluidity determined with dipyrenylpropane decreases gradually with the growth temperature increment, from 30 to 45 degrees C. Interestingly, when fatty acid composition is taken into account, fluidity increases linearly in the range under study, with the proportion of unsaturated fatty acyl chains, both variables being highly correlated (0.924

Fatty acid profile of Escherichia coli during the heat-shock response.

The possible changes in the fatty acid profile of Escharichia coli during heat-shock have been investigated. Bacteria growing in steady-state at 30 degrees C were subjected to an abrupt temperature upshift to 45 degrees C and held at the high temperature for various periods of time in order to elicit the heat-shock response. Fatty acid compositions of lipids extracted from samples taken at different times after the temperature upshift, as well as from cultures in steady-state at 30 and 45 degrees C, were determined by gas-chromatography. It has been found that the total unsaturates to total saturates ratio decreases gradually during heat-shock and that 30 min after the temperature jump, the reduction is equivalent to 57% of the difference between ratios corresponding to steady-state cultures at 30 and 45 degrees C. Consistent with this remodeling of lipid acyl chains, there is a decrease in the excimerization rate of the fluidity probe dipyrenylpropane incorporated into sonicated E. coli lipid extracts. Such modifications occur within the time-span of the heat-shock response, as judged from our previous measurements of the kinetics of change in heat-shock proteins induction ratio. Together, these results indicate that the control of membrane fluidity during the heat-shock response can be accounted for, at least in part, by an important change in the fatty acid composition of Escherichia coli lipids.

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.

Membrane fluidity of Escherichia coli during heat-shock.

The excimer-forming fluorophore dipyrenylpropane has been used to measure the relative fluidity of total membranes isolated from Escherichia coli grown at 30 or 45 degrees C, or exposed to a heat-shock from 30 to 45 degrees C for various periods of time. Parallel experiments were performed using [35S]methionine pulse-labeling of cells, to study the induction of heat-shock proteins (HSPs) at different times after the sudden change in E. coli growth-temperature from 30 to 45 degrees C. Results suggest that upon an abrupt temperature upshift from 30 to 45 degrees C, membrane fluidity adjustment to the steady-state level at the high temperature, takes place during the E. coli heat-shock response.

[DNA supercoiling and topoisomerases in Escherichia coli]. / Superenrollamiento del DNA y topoisomerasas de Escherichia coli.

The chromosomal DNA of all cells is under helical tension or supercoiling. There are two classes of DNA supercoiling: plectonemic and toroidal. Plectonemic supercoiling is generated by the action of DNA topoisomerases, while toroidal supercoiling is generated by DNA-protein interactions and by topoisomerase activitities. DNA supercoiling plays an important role in replication, repair, recombination, transposition and transcription. DNA topoisomerases type I are ATP-independent enzymes that cut one DNA strand and relax supercoiled molecules. DNA topoisomerases type II requiere ATP, cut both DNA strands and supercoil relaxed molecules. All organisms have more than one topoisomerase of each, type I and type II. Escherichia coli has two topoisomerases type I: topoisomerase I and topoisomerase III and two topoisomerases type II: topoisomerase II or gyrase and topoisomerase IV. In this review we discuss the concept of DNA supercoiling and present current knowledge on E. coli DNA topoisomerases.

[The bacterial nucleoid]. / El nucleoide bacteriano.

The bacterial genome is present in the cell within a complex structure, the nucleoid. The nucleoid contains the genomic DNA, and molecules of RNA and proteins. The main proteins of the nucleoid are: RNA polymerase, topoisomerases and the histone-like proteins: HU, H-NS (H1), H, HLP1, IHF and FIS. The DNA molecule in the nucleoid is under helical tension or supercoiling and is organized into 43 +/- 10 topodomains. DNA supercoiling is generated by the activity of the topoisomerases and by DNA-protein interactions. In this review, we analize current knowledge in Escherichia coli about genome organization and proteins of the nucleoid.

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.

Thermally-induced cell lysis in Escherichia coli K12.

Escherichia coli cells exposed to high temperatures exhibit a progressive loss of viability. We observed two mechanisms of cell death induced by lethal temperatures: with and without lysis. The number of cells lysed by heat decreased at later stages of the growth curve, when cells were pre-treated at lower temperatures for 10 minutes and when cells were pre-treated with novobiocin, nalidixic acid and cadmium chloride. Cell lysis was similar in wild type, rpoH, groE and dnaK mutant cells as well as in cells which overproduce heat shock proteins GroE or DnaK. Results using cells aligned for cell division and cells growing at 42 degrees C, 45 degrees C and 47 degrees C suggest that cells near division are more sensitive to lysis and that a high concentration of heat-shock proteins increases their resistance to lysis.

Escherichia coli cells with mutations in the gene for adenylate cyclase (cya) exhibit a heat shock response.

Adenylate cyclase mutants of Escherichia coli showed the heat-shock response. The heat-shock response was studied in two different mutants and in different growth media, including rich and minimal media. These results are in disagreement with the proposal that the cya gene regulates the expression of the heat-shock genes.

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.

Stability of ColE1-like and pBR322-like plasmids in Escherichia coli.

The average copy number, the level of ampicillin resistance conferred by one plasmid, and the degree of plasmid multimerization were determined for several ColE1-like and pBR322-like plasmids. From the results obtained, the variance of the units of partition corresponding to each plasmid studied was calculated. Experimentally determined plasmid stability was compared with that calculated using the variance of the units of partition and the ratio between the generation times of plasmid-free and of plasmid-carrying cells, assuming that the units of partition are distributed randomly between daughter cells. Stability of the pBR322-like plasmids present mainly as monomers in the bacterial host was consistent with random partitioning, whereas pBR322-like plasmids, present mainly as dimers, and the ColE1-like plasmid showed greater stability than that predicted with random partitioning at cell division.

Effect of DNA supercoiling and catabolite repression on the expression of the tetA genes in Escherichia coli.

The effect of novobiocin, a gyrase inhibitor, and of catabolite repression on the expression of different classes of tetA genes in Escherichia coli was studied. The results obtained showed that the complete induction of the tetA genes corresponding to classes A, B, and D depends on DNA supercoiling, and that the expression of the tetA genes corresponding to classes A and C is not under catabolite repression.