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Biomedical subjects

R D'Ari

Publications and source records attributed to R D'Ari.

83 records · Page 5Linked to original sources
PubMed1979

Suppression of tif-mediated induction of SOS functions in Escherichia coli by an altered dnaB protein.

The tif-1 mutation in the Escherichia coli recA gene is known to cause induction of the various "SOS" functions at high temperature, including massive synthesis of the recA protein, lethal filamentation, elevated mutagenesis, and, in lambda lysogens, induction of prophage. It is shown here that the deoxyribonucleic acid initiation mutation dnaB252 suppresses all these manifestations of tif expression. Induction of lambda by ultraviolet irradiation, however, is not affected by the dnaB252 mutation. No similar suppression of tif is observed with other dnaB mutations affecting deoxyribonucleic acid elongation or with other deoxyribonucleic acid initiation mutations at the dnaA and dnaC loci. The fact that an alteration of the dnaB protein specifically suppresses tif-mediated SOS induction implies a role of the replication apparatus in this process, as has been suggested for ultraviolet induction. The induction of lambda is known to proceed via repressor cleavage, presumably promoted by an activated (protease) form of the recA protein. Since lambda induction is normal after ultraviolet irradiation of the tif-1 dnaB252(lambda) strain, tif-mediated induction in this strain may be blocked in a tif-specific step leading to activation of the recA (tif) protein. It is possible that the recA (tif) mutant protein may be directly involved in the replication complex in processes leading to this activation.

Bacterial Proteins↗
PubMed1979

Prophage substitution and prophage loss from superinfected Escherichia coli recA(P1) lysogens.

It is shown that the plasmid prophage P1 can be displaced by a superinfecting P1 phage in Escherichia coli recA(P1) lysogens. Six widely separated phage markers were used to distinguish between residual recombination and total substitution. It is further shown that superinfection of recA lysogens can lead to loss of both phage (curing). These two phenomena, previously reported in Rec+ strains, are thus independent of host recombination and may result from perturbations of some function involved in plasmid maintenance.

Chloramphenicol↗
PubMed1977

Effects of mutations in the immunity system of bacteriophage P1.

A mutant of bacteriophage P1 that made an altered c1 repressor is described. The mutant c1 product had two configurations: in lysogens, at high temperatures, it permitted constitutive expression of the normally repressed DNA replication function ban and was insensitive to the action of ant, a product expressed by the virulent mutant P1virs and by the heteroimmune phage P7 (formerly phiamp+) and normally able to overcome c1 repression; in mutant lysogens at low temperatures, the mutant repressor was apparently normal (able to repress ban and sensitive to ant action). Genetic studies of this mutant led to the isolation of a derivative that formed unstable lysogens. These studies suggested that the ban product was normally under c1 control; they further showed that ant overcame c1 repression by inactivating c1 rather than by creating a bypass of repressor activity.

Coliphages↗
PubMed1977

Maintenance of bacteriophage P1 plasmid.

Three mutants of bacteriophage P1 affected in their ability to maintain the lysogenic state stably are described here. These mutants were normal in lytic growth, but lysogenic derivatives segregated nonlysogens at abnormally high rates (1 to 30% per division). Cells harboring these mutant prophages were elongated or filamentous. The mutations responsible for this prophage instability fell into two classes on the bases of their genetic location, their effect on the ability to lysogenize recA bacteria, and their suppressibility by ant mutations eliminating antirepressor activity. The two mutants that were able to form recA lysogens showed the same prophage instability and partial inhibition of cell division in recA as in rec+ lysogens. The fact that plasmid-linked mutations can cause prophage instability suggests that P1 codes for at least some of the functions determining its own autonomy and segregation.

Chloramphenicol↗
PubMedDate not supplied

DNA replication and indirect induction of the SOS response in Escherichia coli.

The SOS response can be induced indirectly in Escherichia coli by infection with UV irradiated bacteriophage P1, lambda or M13. Induction, monitored quantitatively by means of a sfiA::lac operon fusion, was stronger with the plasmid phage P1 than with lambda, but the kinetics were similar, showing that plasmid and non-plasmid phages are not fundamentally different in their ability to produce indirect induction. In the absence of lambda DNA replication the level of induction was strongly reduced, indicating that the attempt to replicate damaged DNA results in induction of the SOS response. The slight residual induction observed in the absence of DNA replication suggests the existence of a second pathway leading from DNA lesions to induction of the SOS response.

Coliphages↗
PubMedDate not supplied

How Escherichia coli sets different basal levels in SOS operons.

The recA and sfiA genes of Escherichia coli are SOS operons regulated negatively by the LexA repressor. The steady state level of expression of recA is 10-fold higher than that of sfiA, as measured by means of recA::lac and sfiA::lac operon fusions. To study the molecular basis of this difference, we have compared the expression of these two operons in strains in which the concentration of LexA repressor was normal (lexA+), zero (spr amber mutation) or higher than normal (plasmid pJL45, carrying the lexA gene linked to the lac promoter). The results indicate (i) that the recA promoter is about 4 times stronger than the sfiA promoter (as measured in the spr strains), (ii) that neither operon has a physiologically significant level of lexA-independent expression (pJL45 strains), and (iii) that the recA operator has about 2.5 times lower affinity than the sfiA operator for LexA repressor (comparison of lex+ and spr strains). Considering our previous results that the sfiA operon (high operator affinity of LexA) is derepressed very rapidly after inducing treatments and that the recA operon (low operator affinity) is repressed very rapidly when induction is stopped, we conclude that differences in operator affinity do not affect inducibility but serve only to set the basal levels of the different SOS functions.

Bacterial Proteins↗
PubMedDate not supplied

The SOS chromotest: direct assay of the expression of gene sfiA as a measure of genotoxicity of chemicals.

We used a gene fusion, placing the lacZ gene encoding beta-galactosidase under the control of the sfiA promoter, to construct a new tester strain for genotoxic agents. The assay is performed in a few hours and involves simple enzymatic assays. The dose response curves contain a linear portion which enables to define the SOS Inducing Potency (SOSIP) of compounds. For the compounds tested SOSIPs extend over 7 decades and correlate generally well with the mutagenic potency assayed in the Salmonella/microsome assay (Mutatest) and in a phage induction assay (Inductest). Sensitivities (lowest amount detected) are comparable in the SOS Chromotest and Mutatest but lower in the Inductest. Our results suggest that at least part of the response in the Mutatest depends on the induction of an SOS function, and that most of the genotoxins are inducer of the SOS system -i.e. can lead to activation of the RecA protease.

4-Nitroquinoline-1-oxide↗
PubMedDate not supplied

Regulation of chromosome segregation in Escherichia coli.

Cell division is tightly coupled to DNA replication in Escherichia coli, as evidenced by the rarity of anucleate cells in steady state cultures. When DNA synthesis is arrested, cell division also comes to a halt and filamentous growth ensues, again with little formation of anucleate cells. To test the precise role of the SfiA division inhibitor during filamentous growth, we compared sfiA+ and sfiA- strains in their response to thymine starvation. More residual division was observed in the sfiA mutant culture, and autoradiographic analysis revealed that 13% of the final population consisted of cells of normal size containing no DNA compared to 0.9% in the thymine-starved sfiA+ culture. The SfiA division inhibitor is known to be synthesized massively during thymine starvation as part of the inducible SOS response. We conclude that it prevents aberrant division and formation of anucleate cells, thus assuring proper segregation when DNA synthesis is perturbed. The SfiC division inhibition mechanism, also associated with the SOS response, does not affect cell division during thymine starvation. On the other hand, an SOS-independent mechanism of division arrest clearly comes into play during thymine starvation of a sfiA sfiC mutant: although considerable aberrant division took place, the majority of the cells formed long filaments with 1 or 2 masses of DNA. Thus, E. coli assures proper chromosome segregation by two systems when DNA replication is perturbed: the rapid, efficient SfiA division inhibitor and a less stringent SOS-independent mechanism.

Cell Division↗
PubMedDate not supplied

Preliminary physiologic characterization and genetic analysis of a new Escherichia coli mutant, lov, resistant to mecillinam.

A mutant of Escherichia coli resistant to mecillinam was selected. The target of this antibiotic is penicillin-binding protein 2 (PBP2), which is active in cell wall elongation and responsible for the rod-like shape of the organism. Several observations indicate that PBP2 remains active in the mutant. For example, cells are small and rod-shaped in the absence of mecillinam but become spherical in its presence; the formation of filaments in the presence of furazlocillin shows that cell wall elongation still takes place. The mutant also has a low growth rate and small cell volume, even in rich medium. In these respects, the mutant resembles cya or crp strains (in which the cyclic adenosine monophosphate-catabolite activator protein [cAMP-CAP] complex is inactive), although its catabolic capacities are unaffected. The mutation, called lov, lies at 41 minutes on the E. coli genetic map. The phenotype is partially suppressed by the P1-mediated transduction of an rpsL ribosomal mutation conferring streptomycin resistance.

Amdinocillin↗