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Transcriptional Regulation of the 4-Amino-4-deoxy-L-arabinose Biosynthetic Genes in Yersinia pestis

2005; Elsevier BV; Volume: 280; Issue: 15 Linguagem: Inglês

10.1074/jbc.m413900200

ISSN

1083-351X

Autores

Mollie D. Winfield, Tammy Latifi, Eduardo A. Groisman,

Tópico(s)

Vibrio bacteria research studies

Resumo

Inducible membrane remodeling is an adaptive mechanism that enables Gram-negative bacteria to resist killing by cationic antimicrobial peptides and to avoid eliciting an immune response. Addition of 4-amino-4-deoxy-l -arabinose (4-aminoarabinose) moieties to the phosphate residues of the lipid A portion of the lipopolysaccharide decreases the net negative charge of the bacterial membrane resulting in protection from the cationic antimicrobial peptide polymyxin B. In Salmonella enterica serovar Typhimurium, the PmrA/PmrB two-component regulatory system governs resistance to polymyxin B by controlling transcription of the 4-aminoarabinose biosynthetic genes. Transcription of PmrA-activated genes is induced by Fe3+, which is sensed by PmrA cognate sensor PmrB, and by low Mg2+, in a mechanism that requires not only the PmrA and PmrB proteins but also the Mg2+-responding PhoP/PhoQ system and the PhoP-activated PmrD protein, a post-translational activator of the PmrA protein. Surprisingly, Yersinia pestis can promote PhoP-dependent modification of its lipid A with 4-aminoarabinose despite lacking a PmrD protein. Here we report that Yersinia uses different promoters to transcribe the 4-aminoarabinose biosynthetic genes pbgP and ugd depending on the inducing signal. This is accomplished by the presence of distinct binding sites for the PmrA and PhoP proteins in the promoters of the pbgP and ugd genes. Our results demonstrate that closely related bacterial species may use disparate regulatory pathways to control genes encoding conserved proteins. Inducible membrane remodeling is an adaptive mechanism that enables Gram-negative bacteria to resist killing by cationic antimicrobial peptides and to avoid eliciting an immune response. Addition of 4-amino-4-deoxy-l -arabinose (4-aminoarabinose) moieties to the phosphate residues of the lipid A portion of the lipopolysaccharide decreases the net negative charge of the bacterial membrane resulting in protection from the cationic antimicrobial peptide polymyxin B. In Salmonella enterica serovar Typhimurium, the PmrA/PmrB two-component regulatory system governs resistance to polymyxin B by controlling transcription of the 4-aminoarabinose biosynthetic genes. Transcription of PmrA-activated genes is induced by Fe3+, which is sensed by PmrA cognate sensor PmrB, and by low Mg2+, in a mechanism that requires not only the PmrA and PmrB proteins but also the Mg2+-responding PhoP/PhoQ system and the PhoP-activated PmrD protein, a post-translational activator of the PmrA protein. Surprisingly, Yersinia pestis can promote PhoP-dependent modification of its lipid A with 4-aminoarabinose despite lacking a PmrD protein. Here we report that Yersinia uses different promoters to transcribe the 4-aminoarabinose biosynthetic genes pbgP and ugd depending on the inducing signal. This is accomplished by the presence of distinct binding sites for the PmrA and PhoP proteins in the promoters of the pbgP and ugd genes. Our results demonstrate that closely related bacterial species may use disparate regulatory pathways to control genes encoding conserved proteins. The lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; ORF, open reading frame; 4-aminoarabinose, 4-amino-4-deoxy-l -arabinose. is a major component of the outer membrane of Gram-negative bacteria (1Raetz C.R. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (238) Google Scholar, 2Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc, New York1999: 31-38Google Scholar). 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Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar) and the ugd gene (19Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (516) Google Scholar, 23Zhou Z. Ribeiro A.A. Lin S. Cotter R.J. Miller S.I. Raetz C.R. J. Biol. Chem. 2001; 276: 43111-43121Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 24Breazeale S.D. Ribeiro A.A. Raetz C.R. J. Biol. Chem. 2003; 278: 24731-24739Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In Salmonella enterica serovar Typhimurium, expression of these genes is controlled by the PmrA/PmrB two-component system (25Soncini F.C. Groisman E.A. J. Bacteriol. 1996; 178: 6796-6801Crossref PubMed Scopus (181) Google Scholar). Transcription of PmrA-activated genes is induced by Fe3+, which is the signal sensed by the PmrA cognate sensor PmrB (26Wosten M.M. Kox L.F. Chamnongpol S. Soncini F.C. Groisman E.A. Cell. 2000; 103: 113-125Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), and by low Mg2+, in a mechanism that requires not only the PmrA and PmrB proteins but also the Mg2+-responsive PhoP/PhoQ system and the PhoP-activated PmrD protein, a post-translational activator of the PmrA protein (27Kox L.F. Wosten M.M. Groisman E.A. EMBO J. 2000; 19: 1861-1872Crossref PubMed Scopus (203) Google Scholar, 28Kato A. Groisman E.A. Genes Dev. 2004; 18: 2302-2313Crossref PubMed Scopus (162) Google Scholar) (Fig. 1A). The PmrA protein binds to the pbgP and ugd promoters at regions that include the hexanucleotide repeat (C/T)TTAAT separated by 5 bp, which has been termed the PmrA box (29Wosten M.M. Groisman E.A. J. Biol. Chem. 1999; 274: 27185-27190Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 30Aguirre A. Lejona S. Garcia Vescovi E. Soncini F.C. J. Bacteriol. 2000; 182: 3874-3876Crossref PubMed Scopus (40) Google Scholar, 31Kato A. Latifi T. Groisman E.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4706-4711Crossref PubMed Scopus (94) Google Scholar, 32Marchal K. De Keersmaecker S. Monsieurs P. van Boxel N. Lemmens K. Thijs G. Vanderleyden J. De Moor B. Genome Biology. 2004; 5: R9-R9.20Crossref PubMed Google Scholar). The PmrA protein promotes transcription of the pbgP and ugd genes using the same start sites whether the inducing condition is Fe3+, which is a PhoP/PhoQ- and PmrD-independent process, or low Mg2+, which is dependent on both PhoP/PhoQ and PmrD (28Kato A. Groisman E.A. Genes Dev. 2004; 18: 2302-2313Crossref PubMed Scopus (162) Google Scholar). Thus, wild-type Salmonella expresses PmrA-regulated genes and is resistant to polymyxin B following growth in the presence of Fe3+ (26Wosten M.M. Kox L.F. Chamnongpol S. Soncini F.C. Groisman E.A. Cell. 2000; 103: 113-125Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar) and/or low Mg2+ (27Kox L.F. Wosten M.M. Groisman E.A. EMBO J. 2000; 19: 1861-1872Crossref PubMed Scopus (203) Google Scholar), whereas pmrD and phoP mutants are resistant to polymyxin B if grown in the presence of Fe3+ but sensitive if the inducing condition is low Mg2+ (26Wosten M.M. Kox L.F. Chamnongpol S. Soncini F.C. Groisman E.A. Cell. 2000; 103: 113-125Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 27Kox L.F. Wosten M.M. Groisman E.A. EMBO J. 2000; 19: 1861-1872Crossref PubMed Scopus (203) Google Scholar). Salmonella modifies its lipid A with 4-aminoarabinose during infection of murine macrophages (33Gibbons H.S. Kalb S.R. Cotter R.J. Raetz C.R. Mol. Microbiol. 2005; 55: 425-440Crossref PubMed Scopus (100) Google Scholar). This modification appears to be PhoP/PhoQ-dependent because it was detected only under PhoP-inducing conditions when bacteria were grown in defined media (33Gibbons H.S. Kalb S.R. Cotter R.J. Raetz C.R. Mol. Microbiol. 2005; 55: 425-440Crossref PubMed Scopus (100) Google Scholar), and because expression of the ugd gene inside macrophages required a functional PhoP/PhoQ system even though ugd transcription can be promoted by other two-component systems independently of PhoP/PhoQ (34Mouslim C. Groisman E.A. Mol. Microbiol. 2003; 47: 335-344Crossref PubMed Scopus (96) Google Scholar). Escherichia coli cannot modify its lipid A with 4-aminoarabinose in response to the low Mg2+ signal that induces the PhoP/PhoQ system (33Gibbons H.S. Kalb S.R. Cotter R.J. Raetz C.R. Mol. Microbiol. 2005; 55: 425-440Crossref PubMed Scopus (100) Google Scholar, 35Zhou Z. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) because its highly divergent PmrD protein fails to activate the PmrA protein, which prevents E. coli from expressing PmrA-dependent genes in low Mg2+ (36Winfield M.D. Groisman E.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17162-17167Crossref PubMed Scopus (120) Google Scholar). The plague agent Yersinia pestis can promote PhoP-dependent modification of its LPS with 4-aminoarabinose (14Rebeil R. Ernst R.K. Gowen B.B. Miller S.I. Hinnebusch B.J. Mol. Microbiol. 2004; 52: 1363-1373Crossref PubMed Scopus (217) Google Scholar). This is surprising because Yersinia lacks a PmrD protein. Moreover, it indicates that Salmonella and Yersinia must use different strategies to promote expression of lipid A modifying genes under PhoP-inducing conditions. Here we report the mechanism by which Y. pestis regulates expression of the 4-aminoarabinose biosynthetic genes mediating resistance to polymyxin B. Our results demonstrate that closely related bacterial species adopt distinct regulatory strategies for expression of conserved genes encoding structural proteins, and highlight the difficulty of deducing bacterial behavior solely on the basis of gene content. Bacterial Strains and Growth Conditions—Bacterial strains used in this study are listed in Table I. Y. pestis strains were derived from KIM6 (37Deng W. Burland V. Plunkett G.I. Boutin A. Mayhew G.F. Liss P. Perna N.T. Rose D.J. Mau B. Zhou S. Schwartz D.C. Fetherston J.D. Lindler L.E. Brubaker R.R. Plano G.V. Straley S.C. McDonough K.A. Nilles M.L. Matson J.S. Blattner F.R. Perry R.D. J. Bacteriol. 2002; 184: 4601-4611Crossref PubMed Scopus (449) Google Scholar, 38Staggs T.M. Perry R.D. J. Bacteriol. 1991; 173: 417-425Crossref PubMed Google Scholar), and grown at the optimal growth temperature of 28 °C (39Holt J.G. Krieg N.R. Sneath P.H.A. Staley J.T. Williams S.T. Bergey's Manual of Determinative Bacteriology. 9th Ed. Williams & Wilkins, Baltimore, MD1994: 189Google Scholar) in a modified defined medium (40Perry R.D. Brubaker R.R. J. Bacteriol. 1979; 137: 1290-1298Crossref PubMed Google Scholar), pH 7.0, supplemented with 0.1% casamino acids, 10 mm (d)-glucosamine, and 10 μm MgS04, 10 mm MgS04, or 10 μm MgS04 + 100 μm FeS04 as indicated. Ampicillin was used at 50 μg/ml, chloramphenicol at 25 μg/ml, and kanamycin at 50 μg/ml.Table IList of strains and plasmids used in this studyDescriptionRef.StrainKIM6Pgm- Pst+ Lcr- Fra+ (pmT1, pPCP1)38Staggs T.M. Perry R.D. J. Bacteriol. 1991; 173: 417-425Crossref PubMed Google ScholarEG14325KIM6 ΔpmrA::CmRThis workEG14737KIM6 ΔphoP::CmRThis workEG14738KIM6 ΔphoP::CmR, ΔpmrA::KnRThis workEG14736KIM6 ΔpbgP::KnRThis workPlasmidsPkD3repR6Kγ ApR FRT CmR FRT41Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11555) Google ScholarpKD4repR6Kγ ApR FRT KnR FRT41Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11555) Google ScholarpKD46rep(Ts)pSC101 AmpR paraBAD γ β exo+41Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11555) Google Scholar Open table in a new tab Construction of Strains with Defined Deletions—All mutant strains were constructed according to the one-step disruption protocol (41Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11555) Google Scholar) using the following primer pairs: 3387 (5′-GCCGCCTCTATAAAGATATTTATCAGAATAACTGAGAGCTCATATGAATATCCTCCTTAG-3′) and 3388 (5′-ATCAGCGCCAGCATTAACAGCAAACGACGGCGCATGCTGAGTGTAGGCTGGAGCTGCTTC-3′) for pmrA; 3385 (5′-TATCCTGTTATCCGGTTAACGTTTTATCAAGGATTGGTGTCATATGAATATCCTCCTTAG-3′) and 3386 (5′-CGAAGGGAGAAGGGTTTATTATTTTTTCTGAGCATAGTGTGTGTAGGCTGGAGCTGCTTC-3′) for phoP; 4340 (5′-ATAGTTAATAGTCCATGAAGGTGTCCTAAGGGATTTATTAGTGTAGGCTGGAGCTGCTTC-3′) and 4341 (5′-GTTATAGACAGGAATAACAATGGAGACCTTCTTAATTGGTCATATGAATATCCTCCTTAG-3′) for pbgP. The Y. pestis phoP pmrA double mutant was constructed using the one-step method to delete the pmrA gene from the phoP mutant strain EG14738. The chromosomal structure of the mutated loci was verified both by PCR as described (41Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11555) Google Scholar) and by Southern hybridization using probes specific to: (i) the antibiotic resistance genes used during the construction of the chromosomal deletions, and (ii) sequences flanking the inactivated loci (data not shown). S1 Nuclease Assay—To prepare RNA, overnight cultures grown in the defined medium described above containing 10 mm Mg2+, washed and diluted 1:50 into 50 ml of the defined medium containing either 10 μm MgSO4, 10 mm MgSO4, or 10 μm MgSO4 + 100 μm FeSO4. Because the ΔphoP mutant was unable to grow in defined medium containing 10 μm MgSO4, strains were not washed prior to subculture into defined medium containing 10 μm MgSO4, which allowed growth. Total RNA was extracted from early-logarithmic phase cultures (A600, 0.250) with the MasterPure RNA purification kit (Epicenter Technologies) according to the manufacturer's protocol. Primers 1429 (5′-CCACAAGAAGCGTATAACGC-3′) and 5298 (5′-GTAGCTTGAACAAGTCCCAC-3′) were labeled at the 5′-end by phosphorylation with [32P]ATP (Amersham Biosciences) using T4 polynucleotide kinase (Invitrogen). Double-stranded DNA probes to the pbgP and ugd promoter regions were generated by PCR using primer pairs 1428 (5′-CTTCACTACCTATTGCTGGC-3′), 1429 and 5297 (5′-GCTGATGCTTGCTGCTGAAG-3′), 5298, respectively. S1 nuclease reactions were performed as described (42Garcia Vescovi E. Soncini F.C. Groisman E.A. Cell. 1996; 84: 165-174Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar). In brief, total RNA (30 μg for pbgP and 100 μg for ugd) and the labeled DNA probe were combined with 50 μl of hybridization buffer (80% formamide, 20 mm HEPES pH 6.5, 0.4 m NaCl). The mixture was incubated at 95 °C for 5 min and then left to cool down in an incubator at 30 °C overnight. Hybridization reactions were treated with 10 units of S1 nuclease (Promega) for 30 min at 37 °C. The reaction was stopped by the addition of 200 μl of phenol-chloroform, and the aqueous phase was precipitated with ethanol. The precipitate was dissolved in sequence loading buffer and electrophoresed on a 6% acrylamide, 7.5 m urea gel together with a Maxam-Gilbert DNA ladder generated from the pbgP and ugd promoter DNA probes. Assays were performed in duplicate. DNase I Footprinting—DNase I footprinting was performed as reported (31Kato A. Latifi T. Groisman E.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4706-4711Crossref PubMed Scopus (94) Google Scholar). Briefly, the pbgP promoter region was amplified as described above with labeling of primer 1429 for the coding strand and primer 1428 for the non-coding strand. The ugd promoter region was amplified using two different primer pairs, 3383 (5′-CACCTTGATGGACAGTTTCC-3′), 3384 (5′-TTCATACCAGACTTACTCCC-3′) for foot-printing with the PhoP protein and primers 5297, 5298 for footprinting with the PmrA protein. Primers 3383 and 5297 were labeled for the coding strand and primers 3384 and 5298 were labeled for the non-coding strand. The Salmonella PhoP and PmrA proteins were purified as described (29Wosten M.M. Groisman E.A. J. Biol. Chem. 1999; 274: 27185-27190Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 43Chamnongpol S. Groisman E.A. J. Mol. Biol. 2000; 300: 291-305Crossref PubMed Scopus (56) Google Scholar). Binding reactions with the PhoP and PmrA proteins were carried out as follows. Proteins were incubated with 25 fmol of DNA probe in 100 μl of 2mm Hepes (pH 7.9), 10 mm KCl, 20 μm EDTA, 500 μg of bovine serum albumin, 20 μg/ml poly(dI-dC), and 2% glycerol for 20 min at room temperature. DNase I (Invitrogen) (0.01 units), 100 μm CaCl2, and 100 μm MgCl2 were added and incubated for 3 min at room temperature. The reactions were stopped by the addition of phenol-chloroform, and the aqueous phase was precipitated. Samples were analyzed by electrophoresis on a 6% polyacrylamide, 7.5 m urea gel and compared with a Maxam-Gilbert A+G DNA ladder generated from the same DNA probe. Polymyxin B Susceptibility Assay—Strains were grown to logarithmic phase in the defined medium described above containing either 10 μm MgSO4, 10 mm MgSO4, or 10 μm MgSO4 + 100 μm FeSO4, washed and incubated in the presence of 5.0 μg/ml polymyxin B at 28 °C for 1 h. Samples were serially diluted in phosphate-buffered saline, plated on BHI and incubated for 36–48 h at 28 °C for viability counts. Survival values were calculated by dividing the number of bacteria following treatment with polymyxin B relative to those incubated in the presence of PBS and then multiplied by 100. Assays were performed in triplicate. Identification of Putative Transcription Factor Binding Sites—The promoter regions of the pbgP and ugd genes were examined manually, as well as using the GPS program (soar-tools.wustl.edu/) to identify putative binding sites for the PhoP (44Kato A. Tanabe H. Utsumi R. J. Bacteriol. 1999; 181: 5516-5520Crossref PubMed Google Scholar, 45Yamamoto K. Ogasawara H. Fujita N. Utsumi R. Ishihama A. Mol. Microbiol. 2002; 45: 423-438Crossref PubMed Scopus (87) Google Scholar, 46Lejona S. Aguirre A. Cabeza M.L. Garcia Vescovi E. Soncini F.C. J. Bacteriol. 2003; 185: 6287-6294Crossref PubMed Scopus (102) Google Scholar) and PmrA (29Wosten M.M. Groisman E.A. J. Biol. Chem. 1999; 274: 27185-27190Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 30Aguirre A. Lejona S. Garcia Vescovi E. Soncini F.C. J. Bacteriol. 2000; 182: 3874-3876Crossref PubMed Scopus (40) Google Scholar, 31Kato A. Latifi T. Groisman E.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4706-4711Crossref PubMed Scopus (94) Google Scholar, 32Marchal K. De Keersmaecker S. Monsieurs P. van Boxel N. Lemmens K. Thijs G. Vanderleyden J. De Moor B. Genome Biology. 2004; 5: R9-R9.20Crossref PubMed Google Scholar) proteins. Transcription of the Yersinia pbgP Gene Is Mediated by Distinct PhoP- and PmrA-dependent Promoters—We searched the genomes of the three sequenced Y. pestis strains (37Deng W. Burland V. Plunkett G.I. Boutin A. Mayhew G.F. Liss P. Perna N.T. Rose D.J. Mau B. Zhou S. Schwartz D.C. Fetherston J.D. Lindler L.E. Brubaker R.R. Plano G.V. Straley S.C. McDonough K.A. Nilles M.L. Matson J.S. Blattner F.R. Perry R.D. J. Bacteriol. 2002; 184: 4601-4611Crossref PubMed Scopus (449) Google Scholar, 47Parkhill J. Wren B.W. Thomson N.R. Titball R.W. Holden M.T.G. Prentice M.B. Sebaihia M. James K.D. Churcher C. Mungall K.L. Baker S. Basham D. Bentley S.D. Brooks K. Cerdeno-Tarraga A.M. Chillingworth T. Cronin A. Davies R.M. Davis P. Dougan G. Feltwell T. Hamlin N. Holroyd S. Jagels K. Leather S. Karlyshev A.V. Moule S. Oyston P.C.F. Quail M. Rutherford K. Simmonds M. Skelton J. Stevens K. Whitehead S. Barrell B.G. Nature. 2001; 413: 523-527Crossref PubMed Scopus (1007) Google Scholar) (www.ncbi.nlm.nih.gov) but found no open reading frame with sequence similarity to the Salmonella PmrD protein. Because Y. pestis harbors a Mg2+-responsive PhoP/PhoQ system (48Oyston P.C. Dorrell N. Williams K. Li S.R. Green M. Titball R.W. Wren B.W. Infect. Immun. 2000; 68: 3419-3425Crossref PubMed Scopus (190) Google Scholar), can modify its lipid A with 4-aminoarabinose in a PhoP-dependent manner (14Rebeil R. Ernst R.K. Gowen B.B. Miller S.I. Hinnebusch B.J. Mol. Microbiol. 2004; 52: 1363-1373Crossref PubMed Scopus (217) Google Scholar), and encodes a conserved PmrA/PmrB system (37Deng W. Burland V. Plunkett G.I. Boutin A. Mayhew G.F. Liss P. Perna N.T. Rose D.J. Mau B. Zhou S. Schwartz D.C. Fetherston J.D. Lindler L.E. Brubaker R.R. Plano G.V. Straley S.C. McDonough K.A. Nilles M.L. Matson J.S. Blattner F.R. Perry R.D. J. Bacteriol. 2002; 184: 4601-4611Crossref PubMed Scopus (449) Google Scholar, 47Parkhill J. Wren B.W. Thomson N.R. Titball R.W. Holden M.T.G. Prentice M.B. Sebaihia M. James K.D. Churcher C. Mungall K.L. Baker S. Basham D. Bentley S.D. Brooks K. Cerdeno-Tarraga A.M. Chillingworth T. Cronin A. Davies R.M. Davis P. Dougan G. Feltwell T. Hamlin N. Holroyd S. Jagels K. Leather S. Karlyshev A.V. Moule S. Oyston P.C.F. Quail M. Rutherford K. Simmonds M. Skelton J. Stevens K. Whitehead S. Barrell B.G. Nature. 2001; 413: 523-527Crossref PubMed Scopus (1007) Google Scholar) (www.ncbi.nlm.nih.gov), we reasoned that the low Mg2+ induction of the 4-aminoarabinose biosynthetic genes taking place in Yersinia must involve a mechanism different from the PmrD-dependent pathway described in Salmonella (27Kox L.F. Wosten M.M. Groisman E.A. EMBO J. 2000; 19: 1861-1872Crossref PubMed Scopus (203) Google Scholar, 28Kato A. Groisman E.A. Genes Dev. 2004; 18: 2302-2313Crossref PubMed Scopus (162) Google Scholar) (Fig. 1A). To determine the transcription start site of the pbgP operon in Y. pestis, we conducted S1 mapping experiments using RNA harvested from organisms grown under conditions known to modulate pbgP transcription in Salmonella (25Soncini F.C. Groisman E.A. J. Bacteriol. 1996; 178: 6796-6801Crossref PubMed Scopus (181) Google Scholar, 26Wosten M.M. Kox L.F. Chamnongpol S. Soncini F.C. Groisman E.A. 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There was weak expression from both promoters in wild-type cells following growth in high Mg2+ (Fig. 2A), a condition that represses pbgP transcription in Salmonella (27Kox L.F. Wosten M.M. Groisman E.A. EMBO J. 2000; 19: 1861-1872Crossref PubMed Scopus (203) Google Scholar, 50Soncini F.C. Garcia Vescovi E. Solomon F. Groisman E.A. J. Bacteriol. 1996; 178: 5092-5099Crossref PubMed Scopus (271) Google Scholar). Deletion of the pmrA gene eliminated transcription from the distal but not from the proximal promoter (Fig. 2A). In contrast, inactivation of the phoP gene abolished transcription from the proximal but not from the distal promoter (Fig. 2A). There was no transcription from either promoter in a phoP pmrA double mutant, regardless of the growth condition (Fig. 2A). These results demonstrate that Y. pestis harbors a Fe3+-responding PmrA/PmrB system, which is consistent with the presence of the Fe3+-binding motif in the putative periplasmic region of the PmrB protein (26Wosten M.M. Kox L.F. Chamnongpol S. Soncini F.C. Groisman E.A. Cell. 2000; 103: 113-125Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Moreover, they indicate that, unlike what happens in Salmonella, the PmrA and PhoP proteins use different promoters to transcribe the pbgP gene (Fig. 2B). Interestingly, pbgP transcription from the pmrA-dependent promoter was still present in a phoP mutant experiencing low Mg2+ (Fig. 2A), indicative that the PmrA protein is activated in this media, albeit at lower levels. The PhoP and PmrA Proteins Bind to the pbgP Promoter— Analysis of the Yersinia pbgP promoter region revealed the presence of sequences resembling a PhoP box: the hexanucleotide repeat (G/T)TTTA(A/T) separated by 5 bp (44Kato A. Tanabe H. Utsumi R. J. Bacteriol. 1999; 181: 5516-5520Crossref PubMed Google Scholar, 45Yamamoto K. Ogasawara H. Fujita N. Utsumi R. Ishihama A. Mol. Microbiol. 2002; 45: 423-438Crossref PubMed Scopus (87) Google Scholar, 46Lejona S. Aguirre A. Cabeza M.L. Garcia Vescovi E. Soncini F.C. J. Bacteriol. 2003; 185: 6287-6294Crossref PubMed Scopus (102) Google Scholar), and a PmrA box: the hexanucleotide repeat (C/T)TTAAT separated by 5 bp (29Wosten M.M. Groisman E.A. J. Biol. Chem. 1999; 274: 27185-27190Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 30Aguirre A. Lejona S. Garcia Vescovi E. Soncini F.C. J. Bacteriol. 2000; 182: 3874-3876Crossref PubMed Scopus (40) Google Scholar, 32Marchal K. De Keersmaecker S. Monsieurs P. van Boxel N. Lemmens K. Thijs G. Vanderleyden J. De Moor B. Genome Biology. 2004; 5: R9-R9.20Crossref PubMed Google Scholar) (Fig. 2B), suggesting that the PhoP and PmrA proteins regulate pbgP transcription by binding to its promoter region. To test this hypothesis, we conducted DNase I footprinting analysis of the pbgP promoter using purified PhoP and PmrA proteins from Salmonella, which are 78.9 and 56.1% identical to the Yersinia PhoP and PmrA proteins, respectively. We established the following. (i) The PhoP protein protected nucleotides –17 to –46 and –28 to –53 from the PhoP-dependent start site on the coding and non-coding strands, respectively (Fig. 2C), which include the predicted PhoP box (Fig. 2B). (ii) The PmrA protein protected nucleotides –10 to –47 and –28 to –54 from the PmrA-dependent start site on the coding and non-coding strands, respectively (Fig. 2D), which include the predicted PmrA box (Fig. 2B). The regions protected by the PhoP and PmrA proteins are followed by distinct –10 regions (Fig. 2B), consistent with separate transcriptional control of the pbgP operon mediated by these two proteins. Transcr

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