Lionello Bossi

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 λ 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.

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 Salmonellas 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.

Loss of Hfq activates the σE-dependent envelope stress response in Salmonella enterica

Ubiquitous RNA‐binding protein Hfq mediates the regulatory activity of many small RNAs (sRNAs) in bacteria. To identify potential targets for Hfq‐mediated regulation in Salmonella, we searched for lacZ translational fusions whose activity varied in the presence or absence of Hfq. Fusions downregulated by Hfq were more common than fusions showing the opposite response. Surprisingly, in a subset of isolates from the major class, the higher activity in the absence of Hfq was due to transcriptional activation by the alternative sigma factor RpoE (σE). Activation of the σE regulon normally results from envelope stress conditions that elicit proteolytic cleavage of the anti‐σE factor RseA. Using an epitope tagged variant of RseA, we found that RseA is cleaved at an increased rate in a strain lacking Hfq. This cleavage was dependent on the DegS protease and could be completely prevented upon expressing the hfq gene from an inducible promoter. Thus, loss of Hfq function appears to affect envelope biogenesis in a way that mimics a stress condition and thereby induces the σE response constitutively. In a RseA mutant, activation of the σE response causes Hfq‐dependent downregulation of outer membrane protein (OMP) genes including lamB, ompA, ompC and ompF. For ompA, downregulation results in part from σE‐dependent accumulation of MicA (SraD), a small RNA recently shown to downregulate ompA transcript levels in stationary phase. We show that the micA gene is under σE control, and that DegS‐mediated σE release is required for the accumulation of MicA RNA upon entry into stationary phase. A similar mechanism involving additional, still unidentified, sRNAs, might underlie the growth phase‐dependent regulation of other OMP mRNAs.

Prophage Contribution to Bacterial Population Dynamics

Cocultures of Salmonella strains carrying or lacking specific prophages undergo swift composition changes as a result of phage-mediated killing of sensitive bacteria and lysogenic conversion of survivors. Thus, spontaneous prophage induction in a few lysogenic cells enhances the competitive fitness of the lysogen population as a whole, setting a selection regime that forces maintenance and spread of viral DNA. This is likely to account for the profusion of prophage sequences in bacterial genomes and may contribute to the evolutionary success of certain phylogenetic lineages.

A small RNA downregulates LamB maltoporin in Salmonella

In Escherichia coli and Salmonella enterica, activation of σE‐dependent envelope stress response leads to the abrupt decline in the synthesis of all major outer membrane proteins (OMPs). Recent studies found that two σE‐controlled small RNAs (sRNAs), MicA and RybB, downregulate a number of OMPs. While RybB targets several different mRNAs, including ompC and ompD, MicA was up to date thought to act solely on ompA. Here we present evidence showing that MicA downregulates a second Salmonella OMP: LamB maltoporine. In strains overexpressing σE, MicA accumulation leads to a significant decrease in LamB protein and mRNA levels, as well as a reduction in β‐galactosidase activity in a strain carrying a lamB–lacZ translational fusion. The latter findings provided the basis for a genetic screen that allowed isolating point mutations in the micA gene and in its σE promoter. All alleles obtained displayed their altered regulatory phenotype from their natural chromosomal location. LamB downregulation by MicA requires a functional Hfq protein. Besides this role, confined to σE‐activated conditions, we show that loss of Hfq results in the accumulation of a lamB‐malM dimeric precursor and of malM mRNA during unchallenged growth. This suggests that Hfq normally intervenes in a mechanism that uncouples expression of the malK‐lamB‐malM operon, causing the distal portion of the transcript to be clipped off and degraded.

Unsuspected prophage‐like elements in Salmonella typhimurium

We present evidence for the existence of two large (≈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).

Recognition of heptameric seed sequence underlies multi‐target regulation by RybB small RNA in Salmonella enterica

Prokaryotic regulatory small RNAs act by a conserved mechanism and yet display a stunning structural variability. In the present study, we used mutational analysis to dissect the functional anatomy of RybB, a σE‐dependent sRNA that regulates the synthesis of major porins in Escherichia coli and Salmonella. Mutations in the chromosomal rybB locus that altered the expression of an ompC–lac fusion were identified. Some of the mutations cluster within a seven‐nucleotide segment at the 5′ end of the sRNA and affect its ability to pair with a sequence 40 nucleotides upstream from ompC translation start site. Other mutations map near the 3′ end of RybB, destabilizing the sRNA or altering its binding to Hfq. The 5′ end of RybB is also involved in ompD regulation. In this case, the sRNA can choose between two mutually exclusive pairing sites within the translated portion of the mRNA. Some of the RybB 5′ end mutations affect the choice between the two sites, resulting in regulatory responses that diverge from those observed in ompC. Further analysis of RybB target specificity identified chiP (ybfM), a gene encoding an inducible chitoporin, as an additional member of the RybB regulon. Altogether, our results indicate that an heptameric ‘seed’ sequence is sufficient to confer susceptibility to RybB regulation.

Differential accumulation of Salmonella[Cu, Zn] superoxide dismutases SodCI and SodCII in intracellular bacteria: correlation with their relative contribution to pathogenicity.

Most Salmonella enterica strains have two peri‐plasmic [Cu, Zn] superoxide dismutases, SodCI and SodCII, encoded by prophage and chromosomal genes respectively. Both enzymes are thought to play a role in Salmonella pathogenicity by intercepting reactive oxygen species produced by the hosts innate immune response. To examine the apparent redundancy, we have compared the levels of epitope‐tagged SodCI and SodCII proteins in bacteria growing in vitro, as well as inside tissue culture cells and in mouse tissues. Concomitantly, we have measured the abilities of mutants of either or both sodC genes to proliferate in infected mice in competition assays. Our results show a striking variation in the relative abundance of the two proteins in different environments. In vitro, both proteins accumulate when bacteria enter stationary phase; however, the increase is much sharper and conspicuous for SodCII than for SodCI. In contrast, SodCI vastly predominates in intracellular bacteria where SodCII levels are negligible. In agreement with these findings, most, if not all, of the contribution of [Cu, Zn] superoxide dismutase activity to murine salmonellosis can be ascribed to the SodCI protein. Overall the results of this work suggest that the duplicate sodC genes of Salmonella have evolved to respond to different sets of conditions encountered by bacteria inside the host and in the environment.

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 promoters 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.

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.