J. Barbé

Evolution of cellular ATP concentration after UV-mediated induction of SOS system in Escherichiacoli

UV-irradiation of E. coli induces a two fold increase in ATP pool in the first 20 min. Afterwards, in RecA+ strains ATP level drops quickly below values of non irradiated cells. Mutants of E. coli defective in RecA protein or with either RecA protease activity deficient or protease resistant LexA repressor do not present this decrease, showing that it is due to cleavage of LexA repressor by RecA protease. The ATP increase produced in the first 20 min is dependent on RecBC exonuclease activity and it must be due to substrate level phosphorylation since an uncoupler such as dinitrophenol does not affect it.

Further characterization of SOS system induction in recBC mutants of Escherichia coli

Expression of several SOS functions such as induction of lambda prophage, inhibition of cell division and induction of both umuC and recA genes after UV-irradiation, nalidixic acid or mitomycin C addition was studied in an RecBC- mutant. UV-irradiation and mitomycin C induced all SOS functions studied in the RecBC- cells but at a lower level and delayed with respect to the wild-type strain. On the contrary, nalidixic acid was unable to trigger any of these SOS functions. In the RecBC- mutant, adenine only had a stimulating effect on the amplification of RecA protein synthesis following UV-irradiation. Nevertheless, in the wild-type strain the stimulating effect occurred in all SOS functions studied following UV-irradiation as well as in the amplification of RecA protein synthesis by nalidixic acid but not in the other SOS functions triggered by this compound. Furthermore, adenine produced a decrease in the mitomycin C-mediated induction of all SOS functions studied in both RecBC- and wild-type strains.

REGULATION OF DIVERGENT TRANSCRIPTION FROM THE UVRA-SSB PROMOTERS IN SINORHIZOBIUM MELILOTI

Abstract The Sinorhizobium meliloti uvrA gene was isolated by complementation of a Rhodobacter sphaeroides UvrA− mutant. DNA sequencing of the region upstream of the S. meliloti uvrA gene reveals the presence of the ssb gene in the opposite transcriptional orientation. PCR-mediated mutagenesis demonstrated that expression of these two genes is inducible by DNA damage, and depends, in both cases, on the direct repeat GTTCN7GTTC (cited according to the direction of uvrA transcription). Comparison of the sequences of recA and uvrA promoters from different bacterial species of the alpha group of the Proteobacteria has identified the direct repeat GTTCYYKTTTTGTTC as the SOS box in this phylogenetic group.

Changes in ATP Concentration in Escherichia coli during Induction of the SOS System by Mitomycin C and Bleomycin

Treatment of Escherichia coli with bleomycin induced a dramatic increase in ATP concentration in the first 30 min. Afterwards, in RecA+ strains, ATP dropped quickly to values similar to those of untreated cells. Mutants of E. coli defective in either RecA protein or RecA protease activity did not show this decrease, indicating that it was due to the action of RecA protease. The increase in ATP in the first 30 min was dependent on RecBC exonuclease activity and must have been due to substrate level phosphorylation, since an uncoupler such as dinitrophenol did not affect it. Nevertheless, mitomycin C did not induce any change in ATP pools of RecA+ strains, at least during 120 min following treatment. The implications of these findings are discussed in relation to the possible pathways of activation of RecA protease.

Effect of adenine, cytidine and guanosine on the expression of the SOS system in Escherichia coli.

Addition of cytidine or guanosine to UV-irradiated cells of a RecA+ strain of Escherichia coli did not produce any effect on the induction of two SOS functions: inhibition of cell division and expression of the umuC gene. Under the same conditions adenine gave a slight increase in the induction of these two responses. In a RecA441 mutant growing at 42 degrees C, both cytidine and guanosine inhibited these SOS functions, whereas adenine produced a large increase in their expression. Moreover, the ATP concentration of the RecA441 mutant at 42 degrees C showed a decrease which occurred earlier in the cells growing in the presence of cytidine or guanosine than in the absence of either compound. Adenine induced an increase of about three times the initial ATP concentration of this mutant at 42 degrees C which dropped quickly after 10 min. Neither cytidine nor guanosine increased the evolution of cellular ATP in UV-irradiated cells of the RecA+ strain, whereas adenine had only a slight positive effect. However, in UV-irradiated RecA+ cells with and without adenine, ATP levels dropped quickly to the initial value after 20 min. These data suggest that the influence of adenine, cytidine and guanosine on the expression of the RecA441 phenotype at 42 degrees C may be due to alteration of the cellular ATP concentration of this mutant.

Expression of recA-gene dependent SOS functions in Salmonella typhimurium

Thymine starvation of RecA+ Thy- strains of Salmonella typhimurium does not induce the inhibition of cellular respiration, one of the recA-gene dependent SOS functions. Nevertheless, thymine deprivation is able to produce a normal induction of prophage and thymineless death in these same strains. However, when these mutants are treated, in the presence of thymine, with UV-irradiation or bleomycin, they show a normal inhibition of cellular respiration and other SOS functions. Thus, one injurious treatment (thymine deprivation) may trigger prophage induction but not cessation of respiration, whereas another agent (UV-irradiation) may induce both. Together, these results suggest a possible discrimination in the pathways and conditions of expression of various SOS functions.

Sulfide and phosphine ligands in carboplatin analogs

Abstract Three new carboplatin analogs containing P and S ligands are reported. A new synthesis of the intermediate cis -dichlorobis{tris(hydroxymethyl)phosphine}platinum(II) is described. In vitro tests show damage to bacterial DNA and participation of RecA protein in the repair system. Results from a preliminary study in vivo using Leukemia L 1210° cells on mice BDF 1 show that these compounds are potentially active.

Increase in plasmid transformation efficiency in SOS-induced Escherichia coli cells.

SummaryUV irradiation of competent cells of Escherichia coli K12 produced an increase in the efficiency of transformation with plasmid DNA. This phenomenon has been called IPTE (increase in plasmid transformation efficiency) and is dependent on the activated state of the RecA protein. IPTE is independent of the lexA, recB recC, and recF genes. It is not related to the size or the replicon type of the plasmid. Furthermore, it is also induced in cells which have been previously treated with other SOS system-inducing agents such as bleomycin, mitomycin C, or nalidixic acid. IPTE is therefore similar to other repair (SOS) functions inducible by DNA damage since all of them are dependent upon activation of the RecA protein. IPTE differs from other SOS functions in the absence of a direct control by the LexA repressor.

Characterization of SE1, a new general transducing phage of Salmonella typhimurium.

A transducing phage, SE1, which is able to infect Salmonella typhimurium was isolated from a Salmonella enteritidis strain. SE1 is a temperate phage which is heteroimmune with respect to phages P22, L, KB1 and ES18. It is similar in morphology and size to phages P22, L and KB1 and is serologically related to phages P22 and L but not to KB1. Efficiencies of generalized transduction effected by phage SE1 are similar to those for P22HT (int7), a mutant which mediates a high frequency of chromosomal gene transduction. The lengths of chromosomal DNA transduced by SE1 and P22HT (int7) are similar. Furthermore, the SE1 prophage does not exclude the transducing particles from cells it has lysogenized; consequently it is possible to use both SE1 lysogens and non-lysogenic strains as recipients in SE1-mediated transduction experiments, and obtain similar transduction efficiencies. However, the SE1 prophage gives rise to a lysogenic conversion that decreases the rate of adsorption of SE1 and L phages by about 50%, but does not affect adsorption of P22. Altogether these results suggest that phage SE1 may be a useful tool in the genetic manipulation of S. typhimurium.

Further characterization of the expression of SOS functions in recA430 mutants of Escherichia coli

recA-dependent inhibition of cell division and cessation of cell respiration are not expressed in recA430 (formerly lexB) mutants of Escherichia coli after ultraviolet irradiation. Our results suggest that, to be induced in UV-treated cells, inhibition of division as well as cessation of respiration require the protease activity of the RecA protein.