Biomedical subjects

N Figueroa-Bossi

Publications and source records attributed to N Figueroa-Bossi.

12 recordsLinked to original sources

PubMed2001

Epitope tagging of chromosomal genes in Salmonella.

We have developed a simple and efficient procedure for adding an epitope-encoding tail to one or more genes of interest in the bacterial chromosome. The procedure is a modification of the gene replacement method of Datsenko and Wanner [Datsenko, K. A. & Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA 97, 6640-6645]. A DNA module that begins with the epitope-encoding sequence and includes a selectable marker is amplified by PCR with primers that carry extensions (as short as 36 nt) homologous to the last portion of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage lambda red functions yields recombinants carrying the targeted gene fused to the epitope-encoding sequence. The resulting C-terminal-tagged protein can be identified by standard immuno-detection techniques. In an initial application of the method, we have added the sequences encoding the FLAG and 3xFLAG and influenza virus hemagglutinin epitopes to various genes of Salmonella enterica serovar Typhimurium, including putative and established pathogenic determinants present in prophage genomes. Epitope fusion proteins were detected in bacteria growing in vitro, tissue culture cells, and infected mouse tissues. This work identified a prophage locus specifically expressed in bacteria growing intracellularly. The procedure described here should be applicable to a wide variety of Gram-negative bacteria and is particularly suited for the study of intracellular pathogens.

Amino Acid Sequence↗

PubMed2001

Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella.

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.

Animals↗

PubMed2001

Growth-dependent DNA breakage and cell death in a gyrase mutant of Salmonella.

A class of gyrase mutants of Salmonella enterica mimics the properties of bacteria exposed to quinolones. These mutants suffer spontaneous DNA breakage during normal growth and depend on recombinational repair for viability. Unlike quinolone-treated bacteria, however, they do not show accumulation of cleavable gyrase-DNA complexes. In recA or recB mutant backgrounds, the temperature-sensitive (ts) allele gyrA208 causes rapid cell death at 43 degrees. Here, we isolated "suppressor-of-death" mutations, that is, secondary changes that allow a gyrA208 recB double mutant to survive a prolonged exposure to 43 degrees and subsequently to form colonies at 28 degrees. In most isolates, the secondary change was itself a ts mutation. Three ts alleles were mapped in genes coding for amino acyl tRNA synthetases (alaS, glnS, and lysS). Allele alaS216 completely abolished DNA breakage in a gyrA208 recA double mutant. Likewise, treating this mutant with chloramphenicol prevented death and DNA damage at 43 degrees. Additional suppressors of gyrA208 lethality include rpoB mutations and, surprisingly, icd mutations inactivating isocitrate dehydrogenase. We postulate that the primary effect of the gyrase alteration is to hamper replication fork movement. Inhibiting DNA replication under conditions of continuing macromolecular synthesis ("unbalanced growth") activates a mechanism that causes DNA breakage and cell death, reminiscent of "thymineless" lethality.

Alleles↗

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↗

PubMed1998

Gene transfer between related bacteria by electrotransformation: mapping Salmonella typhi genes in Salmonella typhimurium.

Transfer of newly isolated mutations into a fresh background is an essential step of genetic analysis and strain construction. Gene transfer is hampered in Salmonella typhi and in other pathogenic bacteria by the lack of a generalized transduction system. We show here that this problem can be partially circumvented by using electrotransformation as a means for delivering S. typhi DNA into suitable S. typhi or Salmonella typhimurium recipients. Transferred DNA can recombine with the homologous region in the host chromosome. In one application of the method, mutations isolated in S. typhi were genetically mapped in S. typhimurium.

Chromosome Mapping↗

PubMed1997

Unsuspected prophage-like elements in Salmonella typhimurium.

We present evidence for the existence of two large (approximately 50 kb) excisable segments in the chromosome of Salmonella typhimurium. The two elements--designated Gifsy-1 and Gifsy-2--cover, respectively, the 57 units and the 24 units of the genetic map where they contribute indicative rare restriction sites. The two elements are closely interrelated and both contain a region of sequence similarity to the recE locus of the Rac prophage of Escherichia coli. Mutations within this region of Gifsy-1 yield the classical 'Sbc' phenotype: they suppress the recombination defect of recB mutants, apparently by activating a normally silent recE-like gene. At the same time, these 'sbcE' mutations activate a Xis-type function that promotes excision of one or other of the two elements. Predictably, curing of Gifsy-1 results in the loss of recB mutant suppression. Surprisingly, the suppressor phenotype is also lost in cells cured for Gifsy-2 even though the Gifsy-1-associated sbcE mutation is still present. Moreover, the excision frequency of Gifsy-1 drops dramatically in Gifsy-2-cured cells. Thus, both elements must co-operate in the activation of recombination and excision functions. Overall, the data presented here suggest that Gifsy-1 and Gifsy-2 are cryptic prophages. They are distinct from previously described Fels prophages. Unlike Fels, they are not specific to S. typhimurium strain LT2 since they are both also found in a virulent S. typhimurium isolate (ATCC 14028s).

Amino Acid Sequence↗

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↗

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

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↗