M. Carmen Gómez-Eichelmann

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

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.

FATTY ACID PROFILE OF Escherichia coli DURING THE HEAT-SHOCK RESPONSE

The possible changes in the fatty acid profile of Escherichia coli during heat‐shock have been investigated. Bacteria growing in steady‐state at 30°C were subjected to an abrupt temperature upshift to 45°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°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°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.

Methylated cytosine at Dcm (CC T A GG) sites in Escherichia coli: Possible function and evolutionary implications

The frequency and distribution of methylated cytosine (5-MeC) at CCTAGG (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.

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.

Role of single-strand DNA 3′-5′ exonuclease ExoI and nuclease SbcCD in stationary-phase mutation in Escherichia coli K-12

In RecBCD+ cells, a mutated single-strand DNA 3′-5′ exonuclease ExoI (SbcB15) induced an increase in stationary-phase mutation. In sbcB15 cells, as in wild-type cells, these mutations partially required RecA, RecB, RecF, and expression of the LexA regulon. The absence of nuclease SbcCD in sbcB15 cells decreased stationary-phase mutation and induced an increase in the number of cell filaments. The absence of ExoI (Δxon) in wild-type or sbcC cells did not change significantly the stationary-phase mutation. Differences between the sbcB15 and ΔxonA cells suggest a correlation between level of SOS induction and the generation of stationary-phase mutations.

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 σ32. 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.

Molecular rearrangements between two plasmids of the FII incompatibility group in different recombination—Deficient Escherichia Coli strains

The frequencies and types of plasmid molecular rearrangements generated in different recombinant mutants which carried two plasmids of the FII incompatibility group were studied. The wild-type cells generated molecular rearrangements mainly by interplasmidic recombination with a frequency of 2.4 x 10(-6) per cell per cell doubling. Cells in which RecF was the principal recombination pathway generated different types of molecular rearrangements that involved either both plasmids or one of the plasmids and the chromosome. The frequencies of molecular rearrangements for these cells were 50-fold greater than those of wild-type cells. The recA- cells, even when the RecE pathway was derepressed, generated rearrangements only between one of the plasmids and the chromosome, at very low frequencies (10(-9]. In wild-type cells and in RecF cells, interplasmidic recombination generated mainly cointegrates carrying DNA deletions. These cointegrates were stable in recA- or recA- RecE+ cells, but unstable in wild-type or RecF+ cells. In the latter, the cointegrates generated smaller plasmids with different molecular structures at relatively low frequencies.

Genetic expression and gyrase dependence of methylated and undermethylated DNA in Escherichia coli

The genetic expression and dependence on gyrase of plasmid pBR322 were studied in dam-3, dcm-6, and dam-3 dcm-6 derivatives of a minicell-producing Escherichia coli strain. The results obtained with both methylated and undermethylated plasmid DNA were similar.