A dual-signal regulatory circuit activates transcription of a set of divergent operons in Salmonella typhimurium

Abstract

We present a molecular mechanism for signal transduction that activates transcription of the SlyA regulon in Salmonella typhimurium. We demonstrate that SlyA mediates transcriptional activation in response to guanosine tetraphosphate, ppGpp, according to the following observations: (i) in vivo transcription of SlyA-dependent genes is repressed when ppGpp is absent; this transcription can be restored by overproducing SlyA; (ii) in vivo dimerization and binding of SlyA to the target promoter are facilitated in the presence of ppGpp; and (iii) in vitro SlyA binding to the target promoter is enhanced when ppGpp is supplemented. Thus, ppGpp must be the cytoplasmic component that stimulates SlyA regulatory function by interacting directly with this regulator in Salmonella. This signaling domain, integrated by the PhoP/PhoQ 2-component system that activates slyA transcription by sensing Mg(2+), forms feedforward loops that regulate chromosomal loci identified through a motif search over the S. typhimurium genome. Many such loci are divergent operons, each formed by 2 neighboring genes in which transcription of these 2 loci proceeds in opposite directions. Both genes, however, are controlled by PhoP and SlyA through a single shared PhoP box and SlyA box present in their intergenic regions. A substitution in either box sequence causes a simultaneous cessation of transcription of a divergent operon, pagD-pagC, equivalent to the phenotype in a phoP or slyA mutant. We also identified several chromosomal loci that possess pagC-type genes without the cognate pagD-type genes. Therefore, our results provide a molecular basis for the understanding of SlyA-dependent phenotypes associated with Salmonella virulence.

Fig. 1.

Study of the regulatory function of the SlyA box in Salmonella. (A) Sequence alignment of the chromosomal regions from pagD-pagC and sifB-ugtL intergenic regions in Salmonella typhimurium. The arrowheads indicate the transcription direction. The right-pointing arrows correspond to the 5′ ends of cloned DNA fragments present in plasmids (pYS1033 and pYS1035 in C). The light and dark boxes correspond to the SlyA and PhoP boxes, respectively. (B) EMSA. A 20-nM DNA fragment with wild-type or substituted SlyA box sequence was incubated with 200 nM SlyA-His₆ protein, separated in a 4% polyacrylamide gel, and visualized with ethidium bromide. (C) β-Galactosidase activity was determined in wild type (14028s), a phoP mutant (YS11590), and a slyA mutant (YS11068) harboring pYS1033 (up-52-lacZ) or pYS1035 (up-1-lacZ).

Fig. 2.

A single SlyA box and a single PhoP box in the pagD-pagC intergenic region are essential for transcription of this divergent operon. (A) Substituted sequences (lowercase letters) in a half SlyA box (Left, SL⁻; Right, SR⁻), an entire SlyA box (SB⁻); and a PhoP box (PB⁻) of the pagD-pagC intergenic region. (B) β-Galactosidase activity from the pagD-lacZ and pagC-lacZ strains (see Table S2) with wild-type, chromosomal PhoP box (PB⁻), and SlyA box (SB⁻) mutated sequences. Expression in phoP mutant and slyA mutant was negative control. (C) β-Galactosidase activity was determined in wild-type (14028s) and in phoP (YS11590) and slyA (YS11068) mutant strains that harbor pYS1031 constructed with the intergenic region from the pagC direction and wild-type strains harboring a pYS1031-derived plasmid with substituted sequence at the SlyA box or PhoP box in A. Bacteria in B and C were grown for 4 h in N medium, 0.01 mM Mg²⁺.

Fig. 3.

ppGpp interacts with SlyA. (A) β-Galactosidase activity from pagC-lacZ and pagD-lacZ strains was determined in wild type (YS11865 and YS12000), the relA spoT mutants (YS12167a and YS12166a) harboring pYS1109 (pslyA), and relA spoT hns mutants (YS12167b and YS12166b). When required, 1 mM IPTG was supplemented. (B) Western blot analysis of SlyA-FLAG protein. Samples from strains in C were separated in 15% SDS/PAGE, and signals were monitored using M2 anti-FLAG antibodies. Relative protein amount (%) = (a given amount ÷ amount of d) × 100. Bacteria in A and B were grown in LB for 8 h. (C) In vivo SlyA binding to the pagD-pagC intergenic region was determined in YS11125 and YS11372b; wild-type 14028s was used as an untagged control. (D) EMSA. A DNA fragment with wild-type or substituted SlyA box sequence was incubated with SlyA-His₆ protein. Equal amounts of DNA were separated in a 4% polyacrylamide gel and visualized with ethidium bromide. ppGpp was supplemented when required. Relative bound DNA amount (%) = (a given amount ÷ amount of d) × 100. (E) Crosslinking of the SlyA dimer. Cell extracts prepared from slyA (YS11068) and slyA relA spoT mutants (YS14682) harboring pYS1109 (pslyA). Bacteria were grown for 8 h in LB. Anti-FLAG M2 antibodies (Sigma) were used for signal monitoring. The asterisk indicates the SlyA monomer; the double asterisk indicates the dimer. The arrowheads indicate a protein ladder marked with molecular weights.

Fig. 4.

SlyA and PhoP-activated chromosomal loci in Salmonella typhimurium. (A) Sequence alignment of the intergenic regions of SlyA and PhoP-dependent genes. White letters are sequences of the SlyA and PhoP boxes. Large bold letters are transcription starts (+1) demonstrated in Fig. S2. Numbering is the distance of a transcription start site to proximal SlyA box (for a pagD-type gene) and PhoP box (for pagC-type gene). (B) The mRNA level of transcripts was determined using RT-PCR analysis in wild type, phoP mutant, slyA mutant, and hns mutant. Constitutively transcribed rpoD gene indicated that similar amounts of total RNA were used. The DNA fragment was amplified using the primers (horizontal arrows in schematic diagram) listed in Table S3 and separated in an agarose gel. The dotted line indicates the intergenic regions. (C) In vivo binding of PhoP and SlyA to the intergenic regions was determined in strains harboring a chromosomal phoP-HA (YS11796) or slyA-FLAG (YS11125). Wild-type 14028s was used as an untagged control. Results from the immunoprecipitated DNA sample (IP) are shown. PCR was performed to amplify the intergenic regions (IG, dotted line in B), which were separated in an agarose gel. Amounts of input DNA were similar (data not shown). Bacteria were grown for 4 h in N medium (pH 7.4) containing 0.01 mM (L, in B and C) or 10 mM (H, in B) Mg²⁺. The PhoP-HA and SlyA-FLAG proteins shown in C and E were analyzed by Western blot using anti-HA and anti-FLAG M2 antibodies, respectively. (D) The mRNA level of transcripts was determined using RT-PCR analysis in wild type, relA spoT mutant, and slyA mutant. Signal was monitored as in B. (E) Western blot analysis of the amount of SlyA and PhoP in wild type (YS11125 and YS11796) and relA spoT mutant (YS11372b and YS14449). (F) In vivo SlyA binding to the intergenic regions (IG) was determined in wild type and relA spoT mutant as in C. Bacteria in D–F were grown for 8 h in LB medium.