Transcriptional control of the antimicrobial peptide resistance ugtL gene by the Salmonella PhoP and SlyA regulatory proteins.

The PhoP/PhoQ two-component system is a master regulator that governs the ability of Salmonella to cause a lethal infection in mice, the adaptation to low Mg(2+) environments, and resistance to a variety of antimicrobial peptides. We have recently established that the PhoP-activated ugtL gene is required for resistance to the antimicrobial peptides magainin 2 and polymyxin B. Here we report that ugtL transcription requires not only the PhoP protein but also the virulence regulatory protein SlyA. The PhoP protein footprinted two regions of the ugtL promoter, mutation of either one of which was sufficient to abolish ugtL transcription. Although the SlyA protein is a transcriptional activator of the ugtL gene, it footprinted the ugtL promoter at a region located downstream of the transcription start site. The PhoP protein footprinted the slyA promoter, indicating that it controls slyA transcription directly. The slyA mutant was hypersensitive to magainin 2 and polymyxin B, suggesting that the virulence attenuation exhibited by slyA mutants may be caused by hypersensitivity to antimicrobial peptides. We propose that the PhoP and SlyA proteins control ugtL transcription using a feed-forward loop design.

The PhoP/PhoQ two-component system is a master regulator that controls several physiological activities in Salmonella enterica serovar Typhimurium, including the ability to cause a lethal infection in mice (1)(2)(3), the adaptation to low Mg 2ϩ environments (4 -6), and resistance to a variety of antimicrobial peptides (2,7,8; for a review on the PhoP/PhoQ system, see Ref. 9). The sensor protein PhoQ responds to low Mg 2ϩ by promoting phosphorylation of the response regulator PhoP (10 -12), which binds to its target promoters with higher affinity than unphosphorylated PhoP (13), stimulating transcription of PhoP-activated genes (5,9). The purified PhoP protein has been shown to bind to a conserved hexanucleotide repeat separated by 5 nucleotides, termed PhoP box, present in the promoter region of many PhoP-activated genes in Salmonella (13) and Escherichia coli (14). However, several PhoP-activated genes seem to lack a PhoP box (13,15), suggesting that they are regulated by the PhoP protein only indirectly, by controlling the expression or activity of other regulatory proteins. Indeed, a subset of PhoP-activated genes is controlled via the PmrA/PmrB two-component system (16), which is activated by the PhoP-dependent PmrD protein at a post-transcriptional level (17).
The Salmonella SlyA protein is a transcriptional regulator of the MarR family that was originally identified based on its ability to induce expression of a cryptic hemolysin in E. coli K-12 (18,19). Salmonella slyA mutants exhibit some of the same phenotypes displayed by phoP and phoQ mutants, including virulence attenuation in mice and the inability to survive within macrophages (18). The PhoP/PhoQ system is required for transcription from one of the promoters of the slyA gene (20), suggesting that some of the phenotypes displayed phoP and phoQ mutants could be caused by their inability to express the SlyA protein and implying that SlyA participates in transcription of a subset of PhoP-regulated genes. Although the mechanism by which the PhoP/PhoQ system controls slyA transcription remains unknown, it has been suggested that the PhoP protein regulates slyA expression indirectly because a PhoP box could not be identified in the slyA promoter region (20).
We have demonstrated recently that the PhoP-activated ugtL gene is required for a modification of the lipid A mediating resistance to the antimicrobial peptides magainin 2 and polymyxin B (21). The ugtL gene is part of a Salmonella-specific gene cluster that includes the PhoP-activated pcgL gene (22) and the sifB gene, which is expressed in response to the low Mg 2ϩ conditions that normally activate the PhoP/PhoQ system (23). Here we report that transcription of the ugtL gene and resistance to the antimicrobial peptides magainin 2 and polymyxin B require both the phoP and slyA regulatory genes. We establish that the PhoP protein binds to the slyA promoter and that both the PhoP and SlyA proteins bind to the ugtL promoter, suggesting that these two proteins use a feed-forward loop to control ugtL expression. This is the first example of a gene that requires both PhoP and SlyA for transcription.

EXPERIMENTAL PROCEDURES
Bacterial Strains, Plasmids, and Growth Conditions-Bacterial strains and plasmids used in this study are listed in Table I. All S. enterica serovar Typhimurium strains used in this study are derived from the wild-type strain 14028s. Phage P22-mediated transductions were performed as described previously (24). Bacteria were grown at 37°C in Luria-Bertani broth or in N minimal medium, pH 7.4 (25), supplemented with 0.1% casamino acids, 38 mM glycerol, 10 M or 10 mM MgCl 2 . When necessary, antibiotics were added at final concentrations of 50 g/ml for ampicillin, 20 g/ml for chloramphenicol, 50 g/ml for kanamycin, and 12.5 g/ml for tetracycline. E. coli DH5␣ was used as host for the preparation of plasmid DNA.
Construction of Strains with Chromosomal Mutations, Harboring lac Gene Fusions or Nucleotide Substitutions in Regulatory Sites-Strains harboring deletions were generated as described previously (26). Deletion of the slyA gene was carried out using primers 2143 (5Ј-ACTGAA-GCTACAGGTGCCAAGTGCGCAGCAAGCTAATTATGTGTAGGCTG-GAGCTGCTTC-3Ј) and 2144 (5Ј-TAAACCAGGCTTTACGTGTGGTCA-CATGGCCACACGTATGCATATGAATATCCTCCTTAG-3Ј) to amplify the Cm R cassette from plasmid pKD3 and integrate the resulting PCR product into the chromosome. Deletion of the slyA gene was confirmed by Southern blot analysis using as probe a PCR product generated with primers 2864 (5Ј-CTTCACAAGCAATGTTCCTTTGCGTC-3Ј) and 2865 (5Ј-ATGAATAAAACCCAGGGTGTGGAAC-3Ј). An analogous strategy was used to introduce a Cm R resistance gene upstream of the ugtL promoter, which allowed for mutagenesis of the PhoP binding sites.
A lac transcriptional fusion immediately downstream of the chromosomal copy of the slyA gene was constructed as described previously (27). First, the Cm R cassette was amplified using pKD3 plasmid DNA as template and primers 2813 (5Ј-ACACAATATTATGGAATTG-CACTCTCACGATTGAGGTGCAGTGTAGGCTGGAGCTGCTTC-3Ј) and 2814 (5Ј-CCAGGCTTTACGTGTGGTCACATGGCCACACGTATG-CCCCATATGAATATCCTCCTTAGT-3Ј), and the resulting PCR product was integrated 8 nucleotides behind the stop codon of the slyA gene. The insertion of the Cm R cassette at the expected location was confirmed by Southern blot analysis using as probe a PCR product generated with primers 2864 (5Ј-CTTCACAAGCAATGTTCCTTTGCG-TC-3Ј) and 2865 (5Ј-ATGAATAAAACCCAGGGTGTGGAAC-3Ј). The Cm R cassette was removed using plasmid pCP20 (26), and the lac transcriptional fusion plasmid pCE36 was integrated into the FLP recombination target sequence downstream of the slyA gene.
Substitutions of the PhoP binding sites in the ugtL promoter were constructed as follows. First, we introduced a Cm R resistance cassette behind the predicted start codon of the divergently transcribed sifB, which is the gene located immediately upstream of ugtL, using a PCR product generated with primers 4050 (5Ј-ACATCTCACTCTTTAAAAATCCTCT-CCCGATAGTAATTGGGTGTAGGCTGGAGCTGCTTC-3Ј) and 4051 (5Ј-GTAATGAAGTATCATATAATCACTTGTGGTCTACATTATGATATGA-ATATCCTCCTTAGT-3Ј) and plasmid pKD3 as template (26). The resulting strain was termed sifB1 and displayed wild-type levels of ugtL transcription. Insertion of the Cm R cassette at the expected location was confirmed by sequencing a PCR product generated with primers 4050 and 3874 (5Ј-TCTTCATTTTGAGCCTCCTCGCAGG-3Ј). Second, the chromosomal DNA sample from the resulting sifB1 strain was used as template to produce the PCR-generated DNA fragments for mutagenesis of the PhoP binding sites in the ugtL promoter. Primers 4050 and 4052 (5Ј-CCTAATAATACTTTTAGTTTAACTTCTTATAAGACAATTTCTACA-CGCTTCACCAACTTATATCTTTCTCTAAAAATAATATAGTGCG-3Ј) were used to mutagenize the PhoP1 site, and primers 4050 and 4107 (5Ј-TCTGCCCTACCGCTAAACATCTCATTGTTGTTAGCCTAATAATT-GTTTTAGTAATTCTTCTTATAAGACAATTTCTAC-3Ј) were used to mutagenize the PhoP2 site. The PCR-generated DNA fragments were introduced into EG11250 competent cells carrying plasmid pKD46 as described previously (26). The substitution of the DNA sequence at the expected location was confirmed by sequencing PCR products generated with primers 3692 (5Ј-GGAATTCAATGTAGACCACAAGTGATTATATG-3Ј) and 3699 (5Ј-GGAATTCTTTGAGCCTCCTCGCAGGTTTTTATAAT-TTTATCGC-3Ј). S1 Nuclease Assay-The S1 nuclease protection assay was performed as described previously (4), with RNA harvested form mid exponential phase cultures (A 600 nm of 0.4 -0.6) grown in 50 ml of N-minimal medium, pH 7.7, containing 10 M MgCl 2 . Total RNA was isolated with TRIzol (Invitrogen) according to the manufacturer's specifications. A PCR product generated with primers 3724 (5Ј-CACGCCTCCTGGC-CATGAAATATGTC-3Ј) and 3725 (5Ј-CGCCCAACTGGAAACAAAGC-CGTCAG-3Ј) and Salmonella chromosomal DNA as template was used as probe. The probe was labeled at the 5Ј-end by phosphorylation with [␥-32 P]ATP using T 4 polynucleotide kinase (Invitrogen) as described previously (28).
Purification of the SlyA-FLAG and PhoP-His 6 Proteins-Salmonella wild-type strain (14028s) harboring plasmid pUHE21-2lacI q -slyA-FLAG (C terminus) was grown at 37°C shaking to A 600 nm 0.2 in 500 ml of N minimal medium, pH 7.4, containing 10 M MgCl 2 ; then isopropyl-1-thio-␤-D-galactopyranoside (final concentration, 0.5 mM) was added, and bacteria continued to be incubated for another 2 h. Cells were harvested, washed with phosphate-buffered saline once, resuspended in 10 ml of phosphate-buffered saline, and opened by sonication. The whole cell lysate was used for SlyA-FLAG purification by mixing with EZview Red Anti-FLAG M2 affinity Gel (Sigma) following the instructions from the manufacturer. Purification of the PhoP-His 6 protein was performed as described previously (28).
DNase I Footprinting-DNase I protection assays were carried out using DNA fragments amplified by PCR using Salmonella chromosomal DNA as template. Prior to the PCR, primers 3695 (5Ј-GAAATAT-GTCAGATAAAACGAATG-3Ј) and 3699 (see above) were labeled with T4 polynucleotide kinase and [␥-32 P]ATP. The ugtL promoter region was amplified with primers ␥-32 P-3695 and 3699 for the coding strand or with 3695 and ␥-32 P-3699 for the noncoding strand. Approximately 25 pmol of labeled DNA and 0, 15, 30, or 60 pmol of the PhoP-His 6 protein or 0, 5, 15, or 30 pmol of the SlyA-FLAG protein in a 100-l volume were incubated at room temperature for 15 min in a buffer of the following composition: 20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 50 mM KCl, 50 g/ml bovine serum albumin, 2.5 g/ml salmon sperm DNA, and 10% glycerol. DNase I (Invitrogen) (0.05 unit) was added, and the samples were incubated for 3 min at room temperature. The reaction was stopped by adding 10 l of 25 mM EDTA. DNA fragments were purified with the Wizard DNA cleanup system (Promega, Madison, WI) and dissolved in 30 l of H 2 O. Samples (6 l) were analyzed by denaturing 6% PAGE by comparison with a DNA sequence ladder generated with the appropriate primer by using a Maxam and Gilbert AϩG reaction.
Bacterial Killing Assays-Bacterial killing assays by antimicrobial peptides were carried out in duplicate, and the survival percentage was determined as described previously (21).
␤-Galactosidase Assays-␤-Galactosidase assays were carried out in triplicate, and the activity was determined as described previously (29).

RESULTS
The Regulatory Gene slyA Is Essential for Transcription of the PhoP-activated ugtL Gene-We analyzed the 536-bp region upstream of the ugtL coding region but did not find an obvious PhoP box sequence resembling the (T/G)GTTTA-N 5 -(T/ G)GTTTA motif at an equivalent position as the PhoP box present in the promoter region of other PhoP-activated genes characterized in E. coli (14,15,30) and Salmonella (13). This raised the possibility of other regulatory proteins being involved in ugtL transcription. Thus, we compared transcription of the ugtL gene in a strain harboring a chromosomal ugtL-lac fusion with that exhibited by a set of isogenic strains with mutations in the regulatory genes phoP, pmrA, or slyA. The rationale for choosing the pmrA and slyA mutants was that they exhibit increased susceptibility to polymyxin B (6) and virulence attenuation (18), respectively, like a phoP mutant (1)(2)(3)31).
Mutation of the slyA gene abolished ugtL transcription under all tested conditions (i.e. Luria-Bertani broth and N minimal medium with 10 M or 10 mM Mg 2ϩ ) ( Fig. 1A and data not shown), whereas wild-type levels of ugtL transcription were detected in the pmrA mutant (data not shown). Consistent with our previous results (22), the low Mg 2ϩ activation of the ugtL gene was PhoP-dependent (Fig. 1A). The phenotype of the slyA mutant is solely the result of a lack of slyA gene function because ugtL transcription could be restored to wild-type levels by a plasmid containing a wild-type copy of the slyA gene (Fig.  1B). We determined that the transcription start site of the ugtL gene is located 176 bp upstream of the putative start codon of ugtL and requires functional phoP and slyA genes ( Fig. 2A). These experiments demonstrate that the regulatory genes phoP and slyA are required for ugtL transcription.
Expression of the slyA Gene from a Heterologous Promoter Fails to Rescue ugtL Transcription in a phoP Mutant-Because PhoP has been implicated in transcription of one of the promoters of the slyA gene (20), we hypothesized that slyA expression from a heterogeneous promoter would restore ugtL transcription in a phoP mutant. Unexpectedly, plasmid pslyA, harboring a wild-type copy of the slyA gene, could not recover ugtL expression in the phoP mutant despite restoring wild-type levels of ugtL transcription to the slyA mutant (Fig. 1B). These results argue that the role of the PhoP protein in ugtL expression is not limited to promoting transcription of the slyA gene.
We explored the possibility that the PhoP protein may also control the levels of the SlyA protein at a post-transcriptional level by expressing a SlyA-FLAG protein from a heterologous promoter in wild-type and phoP strains. (The slyA-FLAG gene restored ugtL transcription to the slyA mutant but not to the phoP mutant, like the wild-type slyA gene (data not shown).) However, the phoP mutant harbored amounts of SlyA-FLAG protein similar to that of the wild-type strain (Fig. 1C), suggesting that the PhoP protein may modify the activity of the SlyA protein and/or act in conjunction with SlyA to promote ugtL transcription.
The SlyA and PhoP Proteins Bind to the ugtL Promoter-We conducted gel shift assays using the purified SlyA-FLAG and PhoP-His 6 proteins and a 536-bp DNA fragment corresponding to the intergenic region between the ugtL gene and the upstream sifB gene. We determined that these proteins could individually gel shift the DNA fragment (data not shown), indicating the presence of binding sites for SlyA and PhoP in the ugtL promoter region. Then, we conducted DNase I footprinting assays and found that the PhoP protein binds to two regions of the ugtL promoter: an upstream region harboring an imperfect hexanucleotide repeat resembling a PhoP box but located in the opposite strand, and a ϩ1-proximal region with no resemblance to the PhoP box and overlapping the potential Ϫ10 of the ugtL promoter.
The region protected by the SlyA protein extends beyond the transcription start site for the ugtL gene (Fig. 2C), which was unexpected given that slyA functions as an activator of ugtL transcription (Fig. 1A). The region protected by SlyA includes the imperfect palindrome TTTAG-N 7 -CTTAA, which is similar to the SlyA binding sequence TTAG-N 4 -CTAA described for the slyA promoter (32), the only SlyA-regulated promoter investigated in molecular detail. These results demonstrate that the PhoP and SlyA proteins bind to the ugtL promoter.
The A, S1 nuclease protection assay of RNAs extracted from bacteria grown in N medium, pH 7.4, with 10 M Mg 2ϩ . Lane AG corresponds to Maxam and Gilbert AϩG reaction. An S1 product is observed in wild-type Salmonella (14028s) but not in the phoP (MS7953s) or slyA (EG14078) mutants. B, DNase I footprinting analysis of the ugtL promoter performed with probes for the coding and noncoding strands (see "Experimental Procedures") and increasing amounts of PhoP (15, 30, and 60 pmol) or SlyA (5, 15, and 30 pmol) proteins. Solid vertical lines correspond to regions protected by the PhoP or SlyA proteins. C, DNA sequence of the sifB-ugtL intergenic region. The red and green underlines correspond to the PhoP1 and PhoP2 sites, respectively. The gray boxes correspond to sequences resembling the PhoP box, the black underline to the region protected by the SlyA protein, and the arrow to the transcription start site determined as described in A. Numbering is from the putative start codon of ugtL.
role of the PhoP binding sites in the ugtL promoter, we generated a set of isogenic strains harboring wild-type sequences or nucleotide substitutions at either the upstream or downstream PhoP binding sites in the ugtL promoter. (These three strains also harbored a Cm R cassette within the sifB open reading frame, the presence of which did not alter ugtL transcription (data not shown).) First, we carried out DNase I footprinting assays using the ugtL promoter fragments bearing mutations in the PhoP1 or PhoP2 sites: we demonstrated that the PhoP protein bound to the PhoP1 site even when the PhoP2 site was mutated and vice versa (Fig. 3B). Then, we investigated ugtL transcription and determined that strains harboring mutations in either the PhoP1 site or the PhoP2 site exhibited very low levels of ugtL transcription (Fig. 3A), similar to those displayed by the phoP mutant. These results demonstrate that binding of the PhoP protein to both PhoP binding sites in the ugtL promoter is required for ugtL transcription.
The PhoP Protein Binds to the Promoter of the slyA Gene-Investigation of PhoP-controlled slyA transcription has been carried out using plasmid-borne constructs harboring the slyA promoter region (20). Thus, we reevaluated slyA transcription using isogenic wild-type and phoP strains harboring a lac gene fusion immediately downstream of the chromosomal copy of the slyA gene. When the slyA ϩ -lac bacteria were grown under PhoP-inducing conditions (i.e. 10 M Mg 2ϩ ), the ␤-galactosidase activity was dependent on a functional PhoP/PhoQ system (Fig.  4A). As expected for a PhoP-activated gene, there was no slyA transcription when bacteria were grown under PhoP-repressing conditions (i.e. 10 mM Mg 2ϩ ), regardless of the presence of a functional phoP gene (Fig. 4A).
It has been suggested that the PhoP protein regulates the slyA gene indirectly because a PhoP box could not be identified in the slyA promoter (20). However, DNase I footprinting analysis of a 345-bp fragment comprising the slyA promoter revealed that the PhoP protein protected two regions (Fig. 4B). One of these regions resembled one of the hexamers that make up a PhoP box, but it was located on the opposite strand (Fig.  4C), as found in the ugtL promoter (Fig. 2B). The two regions protected by the PhoP protein are located within a 101-bp fragment shown previously to be necessary and sufficient for PhoP-mediated transcription of a plasmid-borne slyA promoter  (20). These results indicate that the PhoP protein controls slyA transcription directly.

Feed-forward Loop by PhoP and SlyA Proteins
SlyA Is Required for Resistance to Antimicrobial Peptides Magainin 2 and Polymyxin B-We hypothesized that the slyA mutant would be susceptible to the antimicrobial peptides magainin 2 and polymyxin B because the slyA gene is necessary for transcription of the ugtL gene (Fig. 1A) and because the ugtL gene is required for resistance to these antimicrobial peptides (21). Indeed, the slyA mutant was as susceptible as the ugtL mutant to polymyxin B (Fig. 5A). The hypersusceptibility of the slyA mutant is due solely to the absence of ugtL expression because transcription of the ugtL gene from a heterologous promoter restored wild-type levels of polymyxin B resistance to the slyA mutant (Fig. 5B). On the other hand, the slyA mutant was more susceptible to magainin 2 than the ugtL mutant (Fig. 5C), and ugtL transcription from a heterologous promoter recovered magainin 2 resistance only partially in the slyA mutant (Fig. 5D). These results suggest that SlyA controls expression of magainin 2 resistance determinants in addition to the ugtL gene, which appears to be the sole SlyA-regulated gene mediating polymyxin B resistance. DISCUSSION We have identified the first example of a regulatory circuit involving the virulence regulatory proteins PhoP and SlyA of S. enterica. We have established that transcription of the magainin 2 and polymyxin B resistance gene ugtL is controlled by the PhoP and SlyA proteins in what appears to be a feedforward loop design (Fig. 6). This model is supported by the following data. (i) ugtL transcription was abolished in phoP and slyA mutants when bacteria were grown under PhoP-inducing conditions (Fig. 1A). (ii) The PhoP and SlyA proteins footprinted the ugtL promoter (Fig. 2C). (iii) Mutation of the PhoP binding sites in the ugtL promoter reduced ugtL transcription to the levels observed in a phoP mutant (Fig. 3A). (iv) slyA transcription was abolished in a phoP mutant in bacteria grown under PhoP-inducing conditions (Fig. 4A). (v) The PhoP protein footprinted the slyA promoter (Fig. 4B). And (vi) the slyA mutant was hypersensitive to the antimicrobial peptides polymyxin B (Fig. 5, A and B) and magainin 2 (Fig. 5, C and D), like a ugtL mutant.
Transcriptional Activation of the ugtL Promoter-The majority of PhoP-activated promoters described to date harbor a conserved hexanucleotide repeat separated by 5 nucleotides, termed the PhoP box, located 12 bp upstream of the Ϫ10 region (14). The ugtL promoter is atypical in that it contains a PhoP box further upstream of the Ϫ10 and located in the opposite strand (i.e. the PhoP1 site). There is a second PhoP binding site that overlaps the Ϫ10 region of the ugtL promoter and does not resemble a PhoP box (i.e. the PhoP2 site.). Mutation of the PhoP1 or PhoP2 sites abolished ugtL transcription (Fig. 3A), possibly because it prevented binding of the PhoP protein to the ugtL promoter as suggested by the DNase I footprinting results (Fig. 3B). These features of the ugtL promoter are atypical for PhoP-regulated promoters and could be the reason that ugtL transcription also requires the SlyA protein (Fig. 1A).
The SlyA protein bound to a region located downstream from the ugtL transcription start site (Fig. 2B), which is unusual for a regulatory protein that is promoting gene transcription (Fig. 1A). However, this is not unprecedented because both the response regulator PhoP from Bacillus subtilis (which is not related to the Salmonella PhoP) (33) and the regulatory protein Rns from E. coli (34) have been shown to have binding sites downstream of the ϩ1 that are necessary for gene transcription.
Feed-forward Control of ugtL Transcription-One of the most prevalent regulatory circuit configurations in E. coli is the coherent feed-forward loop in which a regulatory protein controls transcription of a second regulatory protein, and both regulatory proteins together regulate a third target gene (35). It has been proposed that a coherent feed-forward loop can prevent activation in response to transient signals and respond only to persistent signals (35). The PhoP and SlyA protein appear to be part of a feed-forward loop that controls transcription of the ugtL gene and possibly additional PhoPregulated genes.
The PhoP protein was required for slyA transcription (Ref. 20 and Fig. 4A) and footprinted two regions of the slyA promoter (Fig. 4B) that are part of a DNA fragment shown to be sufficient to mediate PhoP-mediated transcription of a plasmidborne slyA promoter (20). The upstream binding site exhibits higher affinity for the PhoP protein than the second site and consists of sequences partially resembling half of a PhoP box and located in the opposite strand (Fig. 4C), as found in the ugtL promoter. The second PhoP binding site overlaps the putative Ϫ10 sequence of the slyA promoter (Fig. 4C), also like the ugtL promoter (Fig. 2B). Although others have proposed that the PhoP protein regulates slyA transcription indirectly (i.e. by modulating the expression of another regulatory protein), our results argue that PhoP controls slyA transcription directly (i.e. by binding to the slyA promoter.) We ruled out a classical transcriptional cascade model involving the PhoP protein controlling transcription of the slyA gene, which, in turn, would be solely responsible for ugtL transcription because expression of the slyA gene from a heterologous promoter failed to restore ugtL transcription in a phoP mutant although it fully complemented a slyA mutant (Fig. 1B). Moreover, similar levels of a SlyA-FLAG protein produced from a heterologous promoter were detected in wildtype and phoP strains (Fig. 1C), suggesting that PhoP does not affect SlyA stability. Taken together with the identification of binding sites for both the SlyA and PhoP proteins in the ugtL promoter (Fig. 2C), these results support the notion of a feedforward design in the regulation of ugtL transcription.
One of the best characterized feed-forward loops is the one governing expression of the araBAD genes mediating the utilization of L-arabinose where two signals that are detected by two different proteins must be present for araBAD expression to occur. The CRP protein directs the synthesis of the AraC protein in response to high levels of cAMP (i.e. the primary signal), and then CRP together with AraC, which is active only in the presence of L-arabinose (i.e. the secondary signal), bind to the araBAD promoter and stimulate transcription (36). The primary signal in the PhoP-SlyA loop is low Mg 2ϩ , which controls the phosphorylated state of the PhoP protein (11). The identity and even the existence of a secondary signal controlling SlyA are presently unknown. Although binding of the SlyA protein to a small ligand has not been reported, the SlyA protein could respond to a signal present in the low Mg 2ϩ medium used in our experiments and/or generated as a result of activating the PhoP/PhoQ system.
Distinct Transcriptional Control of Different Lipid A-modifying Determinants by the PhoP Protein-PhoP-controlled resistance to antimicrobial peptides is mediated by a variety of gene products that typically produce distinct modifications in the lipid A moiety of the lipopolysaccharide. For example, the inner membrane UgtL protein is necessary for the formation of heptaacylmono-phosphorylated lipid A (21) whereas the PhoPactivated outer membrane protein PagP mediates the palmitoylation of lipid A (37). In addition, the PhoP-regulated PmrD protein activates the PmrA/PmrB two-component system (17), which promotes expression of proteins mediating the incorporation 4-aminoarabinose into lipid A (38). Although full resistance to polymyxin B requires ugtL and pmrA, these genes appear to be differentially regulated by PhoP in that transcription of the former (Fig. 1A), but not the latter, 1 is dependent on the SlyA protein. This indicates that, despite their role in polymyxin B resistance, these determinants are activated independently because the cell surface modifications they promote may help the bacterial cell adapt to the Mg 2ϩ -limiting environments that activate the PhoP/PhoQ system.
The polymyxin B susceptibility of the slyA mutant was the same as that exhibited by the ugtL mutant (Fig. 5A) and could be complemented to wild-type levels by a plasmid that expressed the ugtL gene from a heterologous promoter (Fig. 5B), suggesting that ugtL is the only SlyA-regulated gene mediating polymyxin B resistance. In contrast, the slyA mutant was more sensitive to magainin 2 than the ugtL mutant (Fig. 5C), and the ugtL-expressing plasmid could not restore wild-type levels of magainin 2 resistance to the slyA mutant (Fig. 5D), which implies that SlyA controls additional genes involved in magainin 2 resistance. Candidate genes include the PhoP-activated pagP and yqjA genes because they have recently been shown to exhibit increase susceptibility to magainin 2 (21).
Finally, it appears that some of the cell surface remodeling activities controlled by the PhoP/PhoQ system can be expressed in response to other signals. For example, the PmrA/PmrB system promotes expression of the 4-aminoarabinose biosynthesis products that modify lipid A in response to Fe 3ϩ (31,38).
Likewise, the SlyA protein may promote ugtL expression independently of the PhoP protein because the phoP mutant exhibited a residual level of ugtL transcription (Fig. 1A), and slyA promoters have been identified which do not require the PhoP protein for expression (20). This suggests that cell surface modifications originally identified as PhoP-regulated may help bacterial survival in environments in addition to those of low Mg 2ϩ .