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L Bossi

Publications and source records attributed to L Bossi.

At least 19 recordsLinked to original sources
PubMed1999

Inducible prophages contribute to Salmonella virulence in mice.

We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy-1 and Gifsy-2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy-2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy-2 carries the sodC gene for a periplasmic [Cu,Zn]-superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy-1 prophage to virulence - undetectable in the presence of Gifsy-2 as prophage - becomes significant in cells that lack Gifsy-2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy-2-encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy-1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy-2 genome.

Animals↗
PubMed1999

Histidine operon deattenuation in dnaA mutants of Salmonella typhimurium correlates with a decrease in the gene dosage ratio between tRNA(His) and histidine biosynthetic loci.

Expression of the histidine operon of Salmonella typhimurium is increased in dnaA(Ts) mutants at 37 degrees C. This effect requires an intact his attenuator and can be suppressed by increasing the gene copy number of the hisR locus, which encodes the tRNA(His). We present data which suggest that the his deattenuation defect in dnaA(Ts) mutants results from the loss of a gene dosage gradient between the hisR locus, close to oriC, and the his operon, far from oriC. Some of the conclusions drawn here may apply to other operons as well.

Bacterial Proteins↗
PubMed1998

The supercoiling sensitivity of a bacterial tRNA promoter parallels its responsiveness to stringent control.

In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase. Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro. Mutant analysis showed the sequence determinants of this dependence to lie in the region between the -10 box and the transcription start site. As with most promoters subject to stringent control, this portion of the hisR promoter is C-G-rich. Replacing a C/G bp with T/A at position -7 partially relieves the supercoiling response while changing the sequence between -5 and + 1 (-CCCCCG-) for -GTTAA- abolishes the response in vitro and in vivo. The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro. Studies in isoleucine-starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively. The data presented suggest that the hisR promoter's sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e. resistance to melting. We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix.

DNA, Superhelical↗
PubMed1996

Hyper-negative template DNA supercoiling during transcription of the tetracycline-resistance gene in topA mutants is largely constrained in vivo.

The excess linking deficit of plasmid DNA from topoisomerase I-defective bacteria (topA mutants) results mainly from transcription and is commonly ascribed to unbalanced relaxation of transcription-induced twin-supercoiled domains. This defect is aggravated in genes for membrane-binding proteins (such as the tet gene) where anchoring of the transcription complex to the bacterial membrane is thought to enhance twin-domain partitioning. Thus, it is often assumed that the 'hyper-negative' linking difference of plasmid DNA from topA mutants reflects unconstrained, hyper-negative DNA supercoiling inside the cell. We tested the validity of this assumption in the present study. A DNA sequence that undergoes a gradual B to Z transition under increasing negative superhelical tension was used as a sensor of unconstrained negative supercoiling. Z-DNA formation was probed at a site upstream from the inducible pTac promoter fused either to the tet gene or to the gene for cytosolic chloramphenicol acetyl transferase (cat). Although plasmid DNA linking deficit increased more extensively in topA mutants following tet activation than following cat activation, no significant differences were observed in the extents to which the B to Z DNA transition is stimulated in the two cases. We infer that the excess linking deficit of the tet-containing plasmid DNA reflects constrained negative DNA supercoiling inside the cell.

Chloramphenicol O-Acetyltransferase↗
PubMed1996

A class of gyrase mutants of Salmonella typhimurium show quinolone-like lethality and require rec functions for viability.

We have identified a new class of DNA gyrase mutants of Salmonella typhimurium that show chronic derepression of the SOS regulon. Thus, these mutants mimic the response of wild-type cells to gyrase inhibitors of the quinolone family. SOS induction by conditional lethal mutations gyrA208 or gyrB652, like that mediated by quinolones, is completely dependent on the function of the recB gene product. Introduction of recA or recB null mutations into these strains exacerbates their temperature-sensitive phenotype and prevents growth at the otherwise permissive temperature of 37 degrees C. Selection of suppressors that concomitantly restore growth at 37 degrees C and SOS induction in a recB- background yielded mutations that relleve the RecB requirement for homologous recombination; namely, sbcB mutations as well as mutations at a new locus that was named sbcE. Such mutations also restore SOS induction in quinolone-treated gyr+ recB- strains. These findings indicate that Rec functions are needed for growth of the gyrase mutants at 37 degrees C and suggest that recombinational repair intermediates constitute the SOS-inducing signal in the mutants as well as in quinolone-treated wild-type bacteria. Unlike quinolones, however, the gyr mutations described in this study do not cause detectable accumulation of "cleavable' gyrase-DNA complexes in plasmid or chromosomal DNA. Yet gyrA208 (the only allele tested) was found to trigger RecB-mediated reckless degradation of chromosomal DNA in recA-cells at restrictive temperatures. Indirect evidence suggests that double-stranded DNA ends, entry sites for the RecBCD enzyme, are generated in the gyr mutants by the breakage of DNA-replication forks. We discuss how this could occur and how recombinational rescue of collapsed replication forks could account for cell survival (and SOS induction) in the gyr mutants as well as in quinolone-treated bacteria.

4-Quinolones↗
PubMed1996

Long-distance effect of downstream transcription on activity of the supercoiling-sensitive leu-500 promoter in a topA mutant of Salmonella typhimurium.

Expression of the lacZ gene from the supercoiling-sensitive leu-500 promoter on a plasmid in topA mutant cells was stimulated by activating a divergently oriented Tac promoter, 400 bp upstream from leu-500. The stimulation was approximately threefold regardless of whether the Tac promoter drove the expression of the tet gene, whose product is membrane bound, or of the cat gene, whose product is cytosolic. Putting a second copy of the Tac promoter downstream from lacZ, approximately 3,000 bp from leu-500 in the same orientation as the latter, resulted in 30-fold increase in lacZ expression upon isopropyl-beta-D-thiogalactopyranoside induction. Again, these effects were independent of the nature of the gene upstream from leu-500 (tet or cat). With both tet- and cat-harboring constructs, activation of the two Tac promoter copies caused plasmid DNA to become hypernegatively supercoiled in topA mutant cells. Thus, neither leu-500 activation nor hypernegative plasmid DNA supercoiling appears to require membrane anchoring of DNA in this system. Replacing the downstream copy of Tac with a constitutive promoter resulted in high-level lacZ expression even when the upstream copy was repressed. Under these conditions, no hypernegative DNA supercoiling was observed, indicating that the activity of plasmid-borne leu-500 in topA mutant cells does not necessarily correlate with the linking deficit of plasmid DNA. The response of the leu-500-lacZ fusion to downstream transcription provides a sensitive assay for transcriptional supercoiling in bacteria.

DNA Topoisomerases, Type I↗
PubMed1995

RNA polymerase (rpoB) mutants selected for increased resistance to gyrase inhibitors in Salmonella typhimurium.

Some rifampicin-resistance (RifR) mutations make bacteria slightly resistant to the gyrase inhibitors novobiocin (Nov) and nalidixic acid (Nal). This suggested that it might be possible to isolate rpoB mutants using either drug for positive selection. In an initial test, we confirmed the presence of Rif-resistant isolates among clones selected for Nov resistance. These mutants are also more resistant to Nal. In a subsequent experiment, we found that mutants selected for low-level resistance to Nal include isolates harboring mutations genetically linked to the rpoB locus; of two such mutants studied, one is temperature-sensitive for growth. These two mutants, which are only marginally affected in their response to Nov, are normally sensitive to Rif and thus might be representative of a new class of rpoB alleles. The Rif-resistant and Rif-sensitive rpoB alleles that increase resistance to gyrase inhibitors have one property in common: they all suppress, to varying degrees, the defect in his operon regulation (transcriptional deattenuation) caused by a gyrase defect or inhibition by novobiocin. To further analyse the transcription-supercoiling relationships in these mutants, we examined the ability of RNA polymerase to recruit gyrase activity during transcription. This was done by two independent approaches: (i) observing transcription-induced accumulation of hyper-negatively supercoiled plasmid DNA in a topA mutant background and (ii) measuring transcription-induced plasmid DNA cleavage in the presence of oxolinic acid. Results indicate that the rpoB alleles described in this study diminish the recruitment of gyrase activity by the transcription process. This property correlates with a decrease in the rate of transcription initiation.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Gyrase↗
PubMed1994

GABA and phosphatidylserine in human photosensitivity: a pilot study.

Previous experimental studies have shown that the simultaneous administration of gamma-aminobutyric acid (GABA) and phosphatidylserine (PS) can exert an anticonvulsant activity in different seizure models; moreover, a preliminary trial showed some effect of the association GABA-PS in patients with absence seizures. The aim of this study was to investigate the antiepileptic properties of GABA-PS in the model of human photosensitivity. Nine patients with epilepsy associated with an EEG pattern of photoconvulsive response at intermittent photic stimulation entered a 3-day study. The photosensitivity range (PSR) was determined at hourly intervals both in basal conditions and after the administration of a single oral dose of GABA (3000 mg) and PS (600 or 1200 mg). The administration of GABA-PS was not associated with any systematic changes of PSR, nor with any significant differences of time course profiles on each daily session. No correlation was found between PSR percent deviations from baseline and GABA serum levels. These results indicate that a single acute administration of GABA-PS has no effect in the human photosensitivity model, and suggest that the efficacy of GABA-PS in human epilepsy, as shown by a preliminary investigation, may possibly require chronic administration.

Adolescent↗
PubMed1994

The relative contributions of transcription and translation to plasmid DNA supercoiling in Salmonella typhimurium.

Mutations affecting DNA topoisomerase I (topA) in Salmonella typhimurium were isolated and graded on the basis of their ability to reverse the effects of gyrB mutations on his operon expression. Different topA and gyrB alleles (in otherwise isogenic strains) were used to gather insights into the transcription-dependent variability of plasmid DNA-linking deficit in growing bacteria. This study showed that modulation of DNA supercoiling by transcription results from the action of two components: one is highly dependent on the coupling of translation to RNA-chain elongation; and the other is unrelated to protein synthesis and entirely dependent on promoter determinants. The former greatly predominates in DNA topoisomerase I mutants (topA and topA gyrB) while the latter is the sole contributor to plasmid DNA-linking deficit in wild-type cells. Altogether, these data suggest that whereas translation acts by enhancing the formation of twin supercoiled domains during elongation, the promoter-dependent effects bear no relation to the twin-supercoiled-domain model and are better explained by a mechanism which responds to the binding/unwinding of template DNA by RNA polymerase.

Alleles↗
PubMed1994

Phenotypic suppression of DNA gyrase deficiencies by a deletion lowering the gene dosage of a major tRNA in Salmonella typhimurium.

One of the pleiotropic phenotypes of mutations affecting DNA gyrase activity in Salmonella typhimurium is the constitutive deattenuation of the histidine operon. In the present work, we isolated and characterized a suppressor mutation which restores his attenuation in the presence of a defective gyrase. Such a suppressor, initially named sgdA1 (for suppressor gyrase deficiency), was found to correct additional phenotypes associated with defective gyrase function. These include the aberrant nucleoid partitioning of a gyrB mutant and the conditional lethality of a gyrA mutation. Furthermore, the sgdA1 mutation was found to confer low-level resistance to nalidixic acid. The last phenotype permitted isolation of a number of additional sgdA mutants. Genetic analysis established the recessive character of these alleles as well as the position of the sgdA locus at 57 U on the Salmonella genetic map. All of the sgdA mutants result from the same molecular event: a deletion removing three of the four tandemly repeated copies of argV, the gene which specifies tRNA(2Arg), the major arginine isoacceptor tRNA. These findings, combined with the observation of some Sgd-like phenotypes in a tRNA modification mutant (hisT mutant), lead us to propose that protein synthesis contributes, directly or indirectly, to the pathology of gyrase alterations in growing bacteria. We discuss plausible mechanisms which may be responsible for these effects.

Base Sequence↗
PubMed1993

Functional tRNAs with altered 3' ends.

The CCA trinucleotide is a universally conserved feature of the 3' end of tRNAs, where it serves as the site of amino acid attachment. Despite this extreme conservation, we have isolated functional mutants of tRNA(His) and tRNA(Val1) with altered CCA ends. A mutant that leads to de-repression of the histidine biosynthetic operon in Salmonella typhimurium has been characterized and found to have the CCA end of the sole tRNA(His) species mutated to UCA. However, constructed mutants of tRNA(His) with ACA or GCA ends appeared to be nonfunctional in vivo. Mutants of Escherichia coli tRNA(Val1) with GCA or ACA ends were isolated on the basis of their ability to promote frameshifting at a specific sequence. These same tRNA(Val1) mutants also caused read-through of stop codons that were one, or in some instances two, codons downstream of the valine codon decoded by the mutant tRNA. A startling implication of these data is that disruption of interactions between the CCA end of the tRNA and the large ribosomal subunit promotes these aberrant codon-anticodon interactions on the small ribosomal subunit.

Amino Acid Sequence↗
PubMed1991

Common sequence determinants of the response of a prokaryotic promoter to DNA bending and supercoiling.

Inhibiting the activity of DNA gyrase by mutation or by drugs in S. typhimurium causes the loss of transcription attenuation in the histidine operon. We show that gyrase activity is needed to maintain high-level expression of the tRNA(His) gene (hisR), a prerequisite for proper functioning of the attenuation mechanism. A point mutation in the promoter of the tRNA gene cluster which includes the hisR gene, specifically relieves the promoter response to negative supercoiling thereby restoring full hisR transcription (and, in turn, his attenuation) in the presence of a defective gyrase. The very same mutation, a single base-pair substitution between the -10 box and the transcription start site (-7), was found independently among suppressors of the transcriptional deficiency caused by disruption of DNA curvature upstream from the hisR promoter. We show that the -7 change does not lead to a generalized increase of promoter strength; the effects of the mutation are seen only when gyrase is inhibited or the upstream curvature is altered. This suggests that the upstream curvature intervenes in the same initiation step which is sensitive to superhelical tension. Three additional promoter mutations correcting (or alleviating) the effects of the upstream alteration (including a change at -8) are described and discussed.

Base Sequence↗
PubMed1990

[Diagnostic and therapeutic of human hydatidosis. Study of a hospital case series].

A case-study on 95 inpatients with hydatid disease admitted to a community hospital in Milan is presented. Clinical records from January 1975 through December 1985 were reviewed. Data were analyzed according to: individual characteristics of the subject; previous history of hydatid disease and present signs and symptoms; tests and instrumental examinations performed to ascertain the disease; type of treatment and outcome. Liver (56%) and lung (18%) were the most commonly involved organs, but lung location was, relatively to age, more frequent in children and teenagers than in adults. Intradermal (Casoni) or serological (Ghedini) tests and scintigraphy for the hepatic localization, and lung stratigraphy or histologic section for the pulmonary one, were the most frequent examinations performed. Seventy-three patients (77%) underwent surgical treatment.

Adolescent↗
PubMed1989

[Acute and chronic cerebrovascular diseases. A therapeutic approach].

The effectiveness of a specific treatment for ictus in its acute phase is highly controversial. A series of variables is identified in 1503 cases that would appear to have a particular effect on the reduction of mortality. On these, specific treatment with Buflomedil coincides with a reduction in lethal outcome of 4% over the two-year period out of 380 cases. In their turn chronic postictal and cerebral ischaemia states (a total of 105 cases) respond positively to prolonged treatment with Buflomedil in a percentage varying from 57% in postictal chronic states to 70-73% in chronic cerebral ischemias.

Brain↗
PubMed1988

Transcription induces gyration of the DNA template in Escherichia coli.

We show that transcription modulation of a plasmid sequence in exponentially growing Escherichia coli cells leads to a rapid change in the linking number of plasmid DNA. Activation of transcription is accompanied by an increase in the plasmid's level of negative supercoiling. The added superhelical turns, whose number is proportional to the strength of the promoter and to the length of the transcript, are promptly removed when transcription is turned off. The transcription-induced increase of template supercoiling can still be detected in the presence of an inhibitor of ATP-dependent DNA gyrase [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3]. Altogether, our results indicate that, in addition to being under a general control, DNA superhelicity can be modulated locally in response to the topological perturbations associated with DNA tracking processes. We discuss a model in which supercoiling changes are produced by differential swiveling activities on the opposite sides of a transcriptional flow during transcriptional modulation.

DNA Topoisomerases, Type II↗