The CpxR/CpxA Two-component System Up-regulates Two Tat-dependent Peptidoglycan Amidases to Confer Bacterial Resistance to Antimicrobial Peptide

Abstract

We demonstrate that the twin arginine translocation (Tat) system contributes to bacterial resistance to cationic antimicrobial peptides (CAMPs). Our results show that a deletion at the tatC gene, which encodes a subunit of the Tat complex, caused Salmonella and Escherichia coli to become susceptible to protamine. We screened chromosomal loci that encode known and predicted Tat-dependent proteins and found that two N-acetylmuramoyl-l-alanine amidases, encoded by amiA and amiC, elevated bacterial resistance to protamine and α-helical peptides magainin 2 and melittin but not to β-sheet defensin HNP-1 and lipopeptide polymyxin B. Genetic analysis suggests that transcription of both amiA and amiC loci in Salmonella is up-regulated by the CpxR/CpxA two-component system when nlpE is overexpressed. A footprinting analysis reveals that CpxR protein can interact with amiA and amiC promoters at the CpxR box, which is localized between the predicted −10 and −35 regions but present on different strands in these two genes. In addition, our results show that activation of the CpxR/CpxA system can facilitate protamine resistance because nlpE overexpression elevates this resistance in the wild-type strain but not the cpxR deletion mutant. Thus, we uncover a new transcriptional regulation pathway in which the Cpx envelope stress response system modulates the integrity of the cell envelope in part by controlling peptidoglycan amidase activity, which confers bacterial resistance to protamine and α-helical CAMPs. Our studies have important implications for understanding transcriptional regulation of peptidoglycan metabolism and also provide new insights into the role of the bacterial envelope in CAMP resistance. We demonstrate that the twin arginine translocation (Tat) system contributes to bacterial resistance to cationic antimicrobial peptides (CAMPs). Our results show that a deletion at the tatC gene, which encodes a subunit of the Tat complex, caused Salmonella and Escherichia coli to become susceptible to protamine. We screened chromosomal loci that encode known and predicted Tat-dependent proteins and found that two N-acetylmuramoyl-l-alanine amidases, encoded by amiA and amiC, elevated bacterial resistance to protamine and α-helical peptides magainin 2 and melittin but not to β-sheet defensin HNP-1 and lipopeptide polymyxin B. Genetic analysis suggests that transcription of both amiA and amiC loci in Salmonella is up-regulated by the CpxR/CpxA two-component system when nlpE is overexpressed. A footprinting analysis reveals that CpxR protein can interact with amiA and amiC promoters at the CpxR box, which is localized between the predicted −10 and −35 regions but present on different strands in these two genes. In addition, our results show that activation of the CpxR/CpxA system can facilitate protamine resistance because nlpE overexpression elevates this resistance in the wild-type strain but not the cpxR deletion mutant. Thus, we uncover a new transcriptional regulation pathway in which the Cpx envelope stress response system modulates the integrity of the cell envelope in part by controlling peptidoglycan amidase activity, which confers bacterial resistance to protamine and α-helical CAMPs. Our studies have important implications for understanding transcriptional regulation of peptidoglycan metabolism and also provide new insights into the role of the bacterial envelope in CAMP resistance. IntroductionCationic antimicrobial peptides (CAMPs) 3The abbreviations used are: CAMPcationic antimicrobial peptideIPTGisopropyl β-d-1-thiogalactopyranosideTattwin arginine translocation. are short peptides (12–50 amino acids) that carry positive charges in physiological conditions and generally contain abundant hydrophobic residues to allow for interaction with the bacterial membrane as well as other components of the bacterial envelope (1Brogden K.A. Nat. Rev. Microbiol. 2005; 3: 238-250Crossref PubMed Scopus (4272) Google Scholar, 2Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6655) Google Scholar). The specific interaction allows CAMPs to weaken the integrity of the inner and outer membranes and subsequently kill bacterial cells. On the other hand, bacteria have developed a number of mechanisms against CAMPs. For instance, a two-component system in Salmonella, PmrA/PmrB, mediates modification of lipopolysaccharide that reduces the negative charges in lipid A moiety, resulting in a decrease of electrostatic interactions between the outer membrane and CAMPs, thus conferring resistance to these peptides (for a recent review, see Ref. 3Gunn J.S. Trends Microbiol. 2008; 16: 284-290Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Protamine, an arginine-rich 33-residue CAMP isolated from salmon sperm nuclei (4Ando T. Yamasaki M. Suzuki K. Mol. Biol. Biochem. Biophys. 1973; 12: 1-114Crossref PubMed Google Scholar), as well as magainin 2 and melittin, both α-helical peptides from frog skin and honeybee (5Zasloff M. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 5449-5453Crossref PubMed Scopus (1999) Google Scholar, 6Habermann E. Science. 1972; 177: 314-322Crossref PubMed Scopus (1314) Google Scholar), have been used as model CAMPs to select chromosomal loci required for bacterial resistance to antimicrobial peptides. It is suggested that protamine may exert its bactericidal effect by disrupting cytoplasmic membrane energization (7Aspedon A. Groisman E.A. Microbiology. 1996; 142: 3389-3397Crossref PubMed Scopus (63) Google Scholar), whereas magainin 2 and melittin induce leakage of cytoplasmic components by forming pores in the bacterial membrane (8Yang L. Harroun T.A. Heller W.T. Weiss T.M. Huang H.W. Biophys. J. 1998; 75: 641-645Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 9Yang L. Harroun T.A. Weiss T.M. Ding L. Huang H.W. Biophys. J. 2001; 81: 1475-1485Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar, 10Lee M.T. Chen F.Y. Huang H.W. Biochemistry. 2004; 43: 3590-3599Crossref PubMed Scopus (242) Google Scholar).Several independent pathways have been implicated in resistance to these peptides in the Gram-negative species Salmonella typhimurium based the following observations. (i) The PhoP/PhoQ system plays a critical role in Salmonella resistance to varied CAMPs, including protamine, magainin 2, and melittin (11Fields P.I. Groisman E.A. Heffron F. Science. 1989; 243: 1059-1062Crossref PubMed Scopus (403) Google Scholar, 12Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (332) Google Scholar), mediated in part by the yqjA gene encoding an inner membrane protein of unknown function (13Shi Y. Cromie M.J. Hsu F.F. Turk J. Groisman E.A. Mol. Microbiol. 2004; 53: 229-241Crossref PubMed Scopus (117) Google Scholar). Transcription of the yqjA gene is regulated in a PhoP- and CpxR-dependent manner (13Shi Y. Cromie M.J. Hsu F.F. Turk J. Groisman E.A. Mol. Microbiol. 2004; 53: 229-241Crossref PubMed Scopus (117) Google Scholar, 14Yamamoto K. Ishihama A. Biosci. Biotechnol. Biochem. 2006; 70: 1688-1695Crossref PubMed Scopus (113) Google Scholar). The yqjA mutant is susceptible to protamine and α-helical CAMPs, including magainin 2 and melittin (13Shi Y. Cromie M.J. Hsu F.F. Turk J. Groisman E.A. Mol. Microbiol. 2004; 53: 229-241Crossref PubMed Scopus (117) Google Scholar). In addition, it has been shown that the PhoP-dependent pgtE gene contributes to the resistance to several α-helical CAMPs (15Guina T. Yi E.C. Wang H. Hackett M. Miller S.I. J. Bacteriol. 2000; 182: 4077-4086Crossref PubMed Scopus (267) Google Scholar). The Escherichia coli outer membrane protease, OmpT, which shares significant similarity with PgtE, has been demonstrated to confer bacterial resistance to protamine by mediating cleavage of this peptide (16Stumpe S. Schmid R. Stephens D.L. Georgiou G. Bakker E.P. J. Bacteriol. 1998; 180: 4002-4006Crossref PubMed Google Scholar). (ii) A set of sap loci, encoding factors required for potassium and oligopeptide transport, is essential for Salmonella resistance to protamine (17Parra-Lopez C. Lin R. Aspedon A. Groisman E.A. EMBO J. 1994; 13: 3964-3972Crossref PubMed Scopus (101) Google Scholar). The sapABCDF operon encodes a periplasmic oligopeptide-binding protein and its inner membrane transporter, which is proposed to transport protamine into the cytoplasm to yield resistance (18Parra-Lopez C. Baer M.T. Groisman E.A. EMBO J. 1993; 12: 4053-4062Crossref PubMed Scopus (161) Google Scholar). (iii) The yejABEF operon, which encodes a putative ATP-binding cassette transporter, is suggested to be responsible for resistance to antimicrobial peptides with different structures, including protamine, α-helical melittin, lipopeptide polymyxin B, and β-sheet human defensins (19Eswarappa S.M. Panguluri K.K. Hensel M. Chakravortty D. Microbiology. 2008; 154: 666-678Crossref PubMed Scopus (79) Google Scholar).The twin arginine translocation (Tat) complex is a transporter responsible for the export of folded proteins across the cytoplasmic membrane (20Berks B.C. Sargent F. Palmer T. Mol. Microbiol. 2000; 35: 260-274Crossref PubMed Scopus (462) Google Scholar). This Sec-independent system consists of three major subunits (TatA, TatB, and TatC), and is known for transporting substrates involved for various cellular activities, such as acquisition of metal ions, energy metabolism, cell wall biogenesis, and virulence (for a review, see Ref. 21Palmer T. Sargent F. Berks B.C. Trends Microbiol. 2005; 13: 175-180Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). In this study, we used a mutant library containing single deletions at Salmonella loci and demonstrated that a Tat-dependent pathway is responsible for resistance to protamine and α-helical CAMPs in Salmonella. With genetic analysis of the known and predicted Tat-dependent loci, we can begin to understand a novel regulatory circuit required for CAMP resistance through modulation of the peptidoglycan metabolism in response to the envelope stress.DISCUSSIONAccording to the results shown in this study, we present a transcriptional regulation pathway that contributes to resistance of Gram-negative bacteria Salmonella and E. coli to specific CAMPs. We demonstrate that (i) the Tat system is required for the resistance to protamine and α-helical CAMPs magainin 2 and melittin; (ii) the Tat-dependent resistance is in part mediated by two of its amidase substrates, AmiA and AmiC; (iii) the CpxR/CpxA two-component system up-regulates amiA and amiC transcription; (iv) the CpxR protein binds to divergent CpxR boxes in the amiA and amiC promoters; and (v) the CpxR/CpxA system facilitates resistance to protamine.The Tat-dependent Cell Wall Amidases Contribute to Resistance to Protamine and α-Helical Antimicrobial Peptides in Enteric BacteriaEnzymes that can cleave peptidoglycan and damage the integrity of bacterial cell wall play a role in cell division, growth, and cell wall remodeling. The Tat-dependent N-acetylmuramoyl-l-alanine amidases, AmiA and AmiC, catalyze cleavage of the cross-link formed by the peptide strand and polysaccharide strand of peptidoglycan (21Palmer T. Sargent F. Berks B.C. Trends Microbiol. 2005; 13: 175-180Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 33Tomioka S. Nikaido T. Miyakawa T. Matsuhashi M. J. Bacteriol. 1983; 156: 463-465Crossref PubMed Google Scholar, 34Heidrich C. Templin M.F. Ursinus A. Merdanovic M. Berger J. Schwarz H. de Pedro M.A. Höltje J.V. Mol. Microbiol. 2001; 41: 167-178Crossref PubMed Scopus (252) Google Scholar). We have shown that tatC, amiA, and amiC mutants exhibit increased susceptibility to protamine and α-helical CAMPs magainin 2 and melittin (Fig. 2A) (data not shown). This phenotype is probably caused by an altered outer membrane that allows penetration of more peptides to the sites of action (e.g. cytoplasmic membrane) that are located within this barrier. This hypothesis is based on the observation that interference of the AmiA and AmiC function by either disruption of the Tat activity or mutation of the amiA and amiC genes renders the integrity of the E. coli cell envelope highly defective, resulting in increased permeability of the outer membrane to hydrophobic antibiotics and detergents (27Ize B. Stanley N.R. Buchanan G. Palmer T. Mol. Microbiol. 2003; 48: 1183-1193Crossref PubMed Scopus (164) Google Scholar). Consistent with this notion, both tatC and amiA amiC mutants of Salmonella displayed increased susceptibility to the antibiotic vancomycin (Fig. 2B), which targets the peptidoglycan of Gram-positive bacteria. However, this antibiotic is prohibited, probably due to its high molecular weight, by the outer membrane of Gram-negative bacteria. Furthermore, Gram-negative bacteria could be sensitized to many antibiotics when pretreated with polymyxin B nonapeptide, a polymyxin B analog that could enhance the permeability of bacterial outer membrane but not kill bacteria by itself (42Vaara M. Vaara T. Antimicrob. Agents Chemother. 1983; 24: 107-113Crossref PubMed Scopus (161) Google Scholar).Notably, antimicrobial peptide nisin exhibits bactericidal activity against Gram-positive bacteria by specifically binding to lipid II, a membrane-bound component involved in peptidoglycan synthesis (43Bauer R. Dicks L.M. Int. J. Food Microbiol. 2005; 101: 201-216Crossref PubMed Scopus (128) Google Scholar). In Gram-negative bacteria, the pentapeptide moiety of the lipid II intermediates, l-alanyl-γ-d-glutamyl-diaminopimelyl-d-alanyl-d-alanine, should carry negative charges in the physiological condition due to the carboxyl group of the glutamate residue. We hypothesize that the defective outer membrane in the tatC and amidase mutants may allow more CAMP molecules to pass through and interact with the pentapeptide domain via electrostatic force. Thus, an alternative mechanism for protamine and α-helical CAMPs to implement their bactericidal activities might be to bind to the glycan chain during peptidoglycan biosynthesis in the periplasm. It is possible that a pentapeptide in the lipid II intermediate, to protamine, into the peptidoglycan via the On the other hand, the periplasmic amidases, AmiA and AmiC, could probably cleave the to the complex thus conferring resistance to this It to be AmiA and AmiC are not required for Salmonella resistance to defensins and polymyxin B are also a Salmonella mutant deletion at the displayed wild-type resistance to protamine (Fig. It is possible that a periplasmic amidase de Mol. Microbiol. 2003; 48: PubMed Scopus Google Scholar), has a different to the formed between the and which is located in a of the peptidoglycan AmiA and AmiC both to resistance to CAMPs, the AmiC amidase to on the peptidoglycan at the and is located at the during cell division, whereas the AmiA amidase can be the de Mol. Microbiol. 2003; 48: PubMed Scopus Google Scholar). N-acetylmuramoyl-l-alanine are bacterial the mechanism in this may a mechanism for bacterial resistance to α-helical CAMPs and probably other peptides with CpxR/CpxA to Resistance to Protamine and α-Helical Antimicrobial Peptides in Enteric CpxR/CpxA two-component system in the cell envelope and up-regulates the of genes encoding factors that the damage (for a review, see Ref. Microbiol. PubMed Scopus Google Scholar). In with its we demonstrate that the CpxR/CpxA system contributes to Salmonella resistance to protamine and α-helical CAMPs in this Our results show that the amiA and amiC genes are required for resistance to these peptides (Fig. 2A) (data not and are up-regulated by the CpxR/CpxA system (Fig. the action of these Tat-dependent is to cell wall should play a role in the of bacterial envelope stress because of the amiA and amiC loci or the tatC caused a of integrity of the bacterial cell envelope (27Ize B. Stanley N.R. Buchanan G. Palmer T. Mol. Microbiol. 2003; 48: 1183-1193Crossref PubMed Scopus (164) Google Scholar). Thus, it is that the CpxR/CpxA system confers the resistance to CAMPs by damage of the cell envelope caused by these peptides or by the outer membrane to CAMPs from of the CpxR/CpxA system by nlpE is to resistance of the tatC mutant to protamine, that other CpxR-dependent loci to the peptide resistance in a of these loci is the yqjA gene, transcription is up-regulated by both the PhoP/PhoQ and CpxR/CpxA (13Shi Y. Cromie M.J. Hsu F.F. Turk J. Groisman E.A. Mol. Microbiol. 2004; 53: 229-241Crossref PubMed Scopus (117) Google Scholar, 14Yamamoto K. Ishihama A. Biosci. Biotechnol. Biochem. 2006; 70: 1688-1695Crossref PubMed Scopus (113) Google Scholar). The yqjA gene encodes a protein with unknown function but to be a Tat and is required for magainin 2 and protamine resistance (13Shi Y. Cromie M.J. Hsu F.F. Turk J. Groisman E.A. Mol. Microbiol. 2004; 53: 229-241Crossref PubMed Scopus (117) Google Scholar). In to the CpxR/CpxA other regulatory should also be responsible for activation of the amiA and amiC genes. amiA and amiC are to in and the cpxR mutant when in nlpE overexpression (Fig. and the cpxR mutant not increased susceptibility to protamine when with the wild-type (Fig. is shown that CAMPs are to induce regulatory activity of two-component A number of antimicrobial including polymyxin B and Chen Wang J. J. 2000; PubMed Scopus Google Scholar), the thus resistance to these peptides in S. Mol. Microbiol. 2003; PubMed Scopus Google Scholar). In addition, as well as other CAMPs, shown to induce the PhoP/PhoQ resulting in of the gene, in Salmonella S. H. Miller S.I. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). However, CAMPs are to the CpxR/CpxA system because activity to that of the and in with or of protamine and magainin 2 (data not CpxR to the amiA and amiC via the CpxR CpxR-dependent regulation of the amiA and amiC genes is by the sites in their the CpxR protein used not in the in these genes similarity to the CpxR box, as the from demonstrated or predicted genes Lin E.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The CpxR in both amiA and amiC promoters is localized within the −35 and −10 A that of the CpxR CpxR-dependent activation of amiA transcription (data not shown). However, we could not the that the amiA due to these located the −35 and −10 A that the box, the for the binding S. A. E. J. Bacteriol. 2003; PubMed Scopus Google Scholar), from a that with the −35 in many genes S. A. E. J. Bacteriol. 2003; PubMed Scopus Google Scholar). Thus, it is that binding of these two-component to the regions located −35 and −10 could but not interaction of the to specific regions On the other hand, the CpxR box, to the amiA is also located between the −35 and −10 regions of the gene in E. which mediates of its transcription Lin E.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is to CpxR can in transcription of amiA and the CpxR is located in the strand at the amiC (Fig. its transcription is up-regulated in a CpxR-dependent manner (Fig. A found in the of the operon, in which a CpxR in the with the −35 for it responsible for negative regulation of transcription Lin E.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). to these it is that CpxR with different thus binding or binding when it is located on the strand of a Furthermore, it has been shown that the CpxR is located of the −35 in three loci, and J. D. Lin E.C. J. PubMed Scopus Google Scholar). these results divergent for the CpxR regulatory which provide for of this stress response is that bacteria such as Salmonella to confer resistance to antimicrobial peptides. A that might this is that these can different cellular components in of bacterial various thus different targets from by CAMPs. In we demonstrate that the CpxR/CpxA system confers Salmonella resistance to protamine and α-helical CAMPs on the Tat pathway via amidases, AmiA and in our provide new insights into the regulatory that to bacterial resistance to antimicrobial peptides and our understanding of the new mechanism for envelope stress response by the CpxR/CpxA IntroductionCationic antimicrobial peptides (CAMPs) 3The abbreviations used are: CAMPcationic antimicrobial peptideIPTGisopropyl β-d-1-thiogalactopyranosideTattwin arginine translocation. are short peptides (12–50 amino acids) that carry positive charges in physiological conditions and generally contain abundant hydrophobic residues to allow for interaction with the bacterial membrane as well as other components of the bacterial envelope (1Brogden K.A. Nat. Rev. Microbiol. 2005; 3: 238-250Crossref PubMed Scopus (4272) Google Scholar, 2Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6655) Google Scholar). The specific interaction allows CAMPs to weaken the integrity of the inner and outer membranes and subsequently kill bacterial cells. On the other hand, bacteria have developed a number of mechanisms against CAMPs. For instance, a two-component system in Salmonella, PmrA/PmrB, mediates modification of lipopolysaccharide that reduces the negative charges in lipid A moiety, resulting in a decrease of electrostatic interactions between the outer membrane and CAMPs, thus conferring resistance to these peptides (for a recent review, see Ref. 3Gunn J.S. Trends Microbiol. 2008; 16: 284-290Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Protamine, an arginine-rich 33-residue CAMP isolated from salmon sperm nuclei (4Ando T. Yamasaki M. Suzuki K. Mol. Biol. Biochem. Biophys. 1973; 12: 1-114Crossref PubMed Google Scholar), as well as magainin 2 and melittin, both α-helical peptides from frog skin and honeybee (5Zasloff M. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 5449-5453Crossref PubMed Scopus (1999) Google Scholar, 6Habermann E. Science. 1972; 177: 314-322Crossref PubMed Scopus (1314) Google Scholar), have been used as model CAMPs to select chromosomal loci required for bacterial resistance to antimicrobial peptides. It is suggested that protamine may exert its bactericidal effect by disrupting cytoplasmic membrane energization (7Aspedon A. Groisman E.A. Microbiology. 1996; 142: 3389-3397Crossref PubMed Scopus (63) Google Scholar), whereas magainin 2 and melittin induce leakage of cytoplasmic components by forming pores in the bacterial membrane (8Yang L. Harroun T.A. Heller W.T. Weiss T.M. Huang H.W. Biophys. J. 1998; 75: 641-645Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 9Yang L. Harroun T.A. Weiss T.M. Ding L. Huang H.W. Biophys. J. 2001; 81: 1475-1485Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar, 10Lee M.T. Chen F.Y. Huang H.W. Biochemistry. 2004; 43: 3590-3599Crossref PubMed Scopus (242) Google Scholar).Several independent pathways have been implicated in resistance to these peptides in the Gram-negative species Salmonella typhimurium based the following observations. (i) The PhoP/PhoQ system plays a critical role in Salmonella resistance to varied CAMPs, including protamine, magainin 2, and melittin (11Fields P.I. Groisman E.A. Heffron F. Science. 1989; 243: 1059-1062Crossref PubMed Scopus (403) Google Scholar, 12Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (332) Google Scholar), mediated in part by the yqjA gene encoding an inner membrane protein of unknown function (13Shi Y. Cromie M.J. Hsu F.F. Turk J. Groisman E.A. Mol. Microbiol. 2004; 53: 229-241Crossref PubMed Scopus (117) Google Scholar). Transcription of the yqjA gene is regulated in a PhoP- and CpxR-dependent manner (13Shi Y. Cromie M.J. Hsu F.F. Turk J. Groisman E.A. Mol. Microbiol. 2004; 53: 229-241Crossref PubMed Scopus (117) Google Scholar, 14Yamamoto K. Ishihama A. Biosci. Biotechnol. Biochem. 2006; 70: 1688-1695Crossref PubMed Scopus (113) Google Scholar). The yqjA mutant is susceptible to protamine and α-helical CAMPs, including magainin 2 and melittin (13Shi Y. Cromie M.J. Hsu F.F. Turk J. Groisman E.A. Mol. Microbiol. 2004; 53: 229-241Crossref PubMed Scopus (117) Google Scholar). In addition, it has been shown that the PhoP-dependent pgtE gene contributes to the resistance to several α-helical CAMPs (15Guina T. Yi E.C. Wang H. Hackett M. Miller S.I. J. Bacteriol. 2000; 182: 4077-4086Crossref PubMed Scopus (267) Google Scholar). The Escherichia coli outer membrane protease, OmpT, which shares significant similarity with PgtE, has been demonstrated to confer bacterial resistance to protamine by mediating cleavage of this peptide (16Stumpe S. Schmid R. Stephens D.L. Georgiou G. Bakker E.P. J. Bacteriol. 1998; 180: 4002-4006Crossref PubMed Google Scholar). (ii) A set of sap loci, encoding factors required for potassium and oligopeptide transport, is essential for Salmonella resistance to protamine (17Parra-Lopez C. Lin R. Aspedon A. Groisman E.A. EMBO J. 1994; 13: 3964-3972Crossref PubMed Scopus (101) Google Scholar). The sapABCDF operon encodes a periplasmic oligopeptide-binding protein and its inner membrane transporter, which is proposed to transport protamine into the cytoplasm to yield resistance (18Parra-Lopez C. Baer M.T. Groisman E.A. EMBO J. 1993; 12: 4053-4062Crossref PubMed Scopus (161) Google Scholar). (iii) The yejABEF operon, which encodes a putative ATP-binding cassette transporter, is suggested to be responsible for resistance to antimicrobial peptides with different structures, including protamine, α-helical melittin, lipopeptide polymyxin B, and β-sheet human defensins (19Eswarappa S.M. Panguluri K.K. Hensel M. Chakravortty D. Microbiology. 2008; 154: 666-678Crossref PubMed Scopus (79) Google Scholar).The twin arginine translocation (Tat) complex is a transporter responsible for the export of folded proteins across the cytoplasmic membrane (20Berks B.C. Sargent F. Palmer T. Mol. Microbiol. 2000; 35: 260-274Crossref PubMed Scopus (462) Google Scholar). This Sec-independent system consists of three major subunits (TatA, TatB, and TatC), and is known for transporting substrates involved for various cellular activities, such as acquisition of metal ions, energy metabolism, cell wall biogenesis, and virulence (for a review, see Ref. 21Palmer T. Sargent F. Berks B.C. Trends Microbiol. 2005; 13: 175-180Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). In this study, we used a mutant library containing single deletions at Salmonella loci and demonstrated that a Tat-dependent pathway is responsible for resistance to protamine and α-helical CAMPs in Salmonella. With genetic analysis of the known and predicted Tat-dependent loci, we can begin to understand a novel regulatory circuit required for CAMP resistance through modulation of the peptidoglycan metabolism in response to the envelope