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• W1990180328 abstract "Lipopolysaccharide (LPS) is one of the main constituents of the Gram-negative bacterial outer membrane. It usually consists of a highly variable O-antigen, a less variable core oligosaccharide, and a highly conserved lipid moiety, designated lipid A. Several bacteria are capable of modifying their lipid A architecture in response to external stimuli. The outer membrane-localized lipid A 3-O-deacylase, encoded by the pagL gene of Salmonella enterica serovar Typhimurium, removes the fatty acyl chain from the 3 position of lipid A. Although a similar activity was reported in some other Gram-negative bacteria, the corresponding genes could not be identified. Here, we describe the presence of pagL homologs in a variety of Gram-negative bacteria. Although the overall sequence similarity is rather low, a conserved domain could be distinguished in the C-terminal region. The activity of the Pseudomonas aeruginosa and Bordetella bronchiseptica pagL homologs was confirmed upon expression in Escherichia coli, which resulted in the removal of an R-3-hydroxymyristoyl group from lipid A. Upon deacylation by PagL, E. coli lipid A underwent another modification, which was the result of the activity of the endogenous palmitoyl transferase PagP. Furthermore, we identified a conserved histidine-serine couple as active site residues, suggesting a catalytic mechanism similar to serine hydrolases. The biological function of PagL remains unclear. However, because PagL homologs were found in both pathogenic and nonpathogenic species, PagL-mediated deacylation of lipid A probably does not have a dedicated role in pathogenicity. Lipopolysaccharide (LPS) is one of the main constituents of the Gram-negative bacterial outer membrane. It usually consists of a highly variable O-antigen, a less variable core oligosaccharide, and a highly conserved lipid moiety, designated lipid A. Several bacteria are capable of modifying their lipid A architecture in response to external stimuli. The outer membrane-localized lipid A 3-O-deacylase, encoded by the pagL gene of Salmonella enterica serovar Typhimurium, removes the fatty acyl chain from the 3 position of lipid A. Although a similar activity was reported in some other Gram-negative bacteria, the corresponding genes could not be identified. Here, we describe the presence of pagL homologs in a variety of Gram-negative bacteria. Although the overall sequence similarity is rather low, a conserved domain could be distinguished in the C-terminal region. The activity of the Pseudomonas aeruginosa and Bordetella bronchiseptica pagL homologs was confirmed upon expression in Escherichia coli, which resulted in the removal of an R-3-hydroxymyristoyl group from lipid A. Upon deacylation by PagL, E. coli lipid A underwent another modification, which was the result of the activity of the endogenous palmitoyl transferase PagP. Furthermore, we identified a conserved histidine-serine couple as active site residues, suggesting a catalytic mechanism similar to serine hydrolases. The biological function of PagL remains unclear. However, because PagL homologs were found in both pathogenic and nonpathogenic species, PagL-mediated deacylation of lipid A probably does not have a dedicated role in pathogenicity. Lipopolysaccharide (LPS), 1The abbreviations used are: LPS, lipopolysaccharide; EDDHA, ethylenediamine-di(o-hydroxyphenyl)acetic acid; ESI/MS, electrospray ionization-mass spectrometry; GC/MS, gas chromatographymass spectrometry; IPTG, isopropyl-1-thio-β-d-galactopyranoside; l-Ara4N, 4-amino-4-deoxy-l-arabinose; LB, Luria-Bertani broth; OMPLA, outer membrane phospholipase A; ORF, open reading frame; S. Typhimurium, Salmonella enterica serovar Typhimurium; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.1The abbreviations used are: LPS, lipopolysaccharide; EDDHA, ethylenediamine-di(o-hydroxyphenyl)acetic acid; ESI/MS, electrospray ionization-mass spectrometry; GC/MS, gas chromatographymass spectrometry; IPTG, isopropyl-1-thio-β-d-galactopyranoside; l-Ara4N, 4-amino-4-deoxy-l-arabinose; LB, Luria-Bertani broth; OMPLA, outer membrane phospholipase A; ORF, open reading frame; S. Typhimurium, Salmonella enterica serovar Typhimurium; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. a major component of the Gram-negative bacterial outer membrane, is known to be important for the functioning of this membrane as a permeability barrier and for the resistance against complement-mediated cell lysis (for review, see Ref. 1Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3281) Google Scholar). It consists of three covalently linked domains: lipid A, the core, and the O-antigen. Lipid A forms the hydrophobic membrane anchor and is responsible for the endotoxic activity of LPS. In Escherichia coli, it consists of a 1,4′-bisphosphorylated β-1,6-linked glucosamine disaccharide, which is replaced by R-3-hydroxymyristic acid residues at positions 2, 3, 2′, and 3′ via ester or amide linkage. Secondary lauroyl and myristoyl groups replace the hydroxyl group of R-3-hydroxymyristoyl at the 2′ and 3′ positions, respectively (Fig. 1A). Previous studies have shown that the phosphate groups, the glucosamine disaccharide, and the correct number and length of the acyl chains are important for the biological activity of lipid A (1Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3281) Google Scholar, 2Loppnow H. Brade H. Durrbaum I. Dinarello C.A. Kusumoto S. Rietschel E.T. Flad H.D. J. Immunol. 1989; 142: 3229-3238PubMed Google Scholar, 3Steeghs L. Berns M. ten Hove J. de Jong A. Roholl P. van Alphen L. Tommassen J. van der Ley P. Cell. Microbiol. 2002; 4: 599-611Crossref PubMed Scopus (27) Google Scholar).The basic structure of lipid A is reasonably well conserved among Gram-negative bacteria, although slight variations in the pattern of the substitutions of the two phosphates and the acyl chain number and length are observed (4Nikaido H. Vaara M. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1987: 7-22Google Scholar, 5Caroff M. Karibian D. Cavaillon J.-M. Haeffner-Cavaillon N. Microbes Infect. 2002; 4: 915-926Crossref PubMed Scopus (157) Google Scholar). Additional modifications of lipid A (Fig. 1B) are regulated in Salmonella enterica serovar Typhimurium (S. Typhimurium) by the two-component regulatory system PhoP/PhoQ (6Guo L. Lim K.B. Gunn J.S. Bainbridge B. Darveau R.P. Hackett M. Miller S.I. Science. 1997; 276: 250-253Crossref PubMed Scopus (472) Google Scholar, 7Guo L. Lim K.B. Poduje C.M. Daniel M. Gunn J.S. Hackett M. Miller S.I. Cell. 1998; 95: 189-198Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). In response to low Mg2+ levels, the sensor kinase PhoQ phosphorylates and thereby activates the transcriptional activator PhoP, which leads to the activation or repression of 40 different genes (6Guo L. Lim K.B. Gunn J.S. Bainbridge B. Darveau R.P. Hackett M. Miller S.I. Science. 1997; 276: 250-253Crossref PubMed Scopus (472) Google Scholar, 8Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (62) Google Scholar). A second regulatory system involved in lipid A modification is the PmrA/PmrB two-component system, which itself is PhoP/PhoQ-regulated (9Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (496) Google Scholar, 10Gunn J.S. Ryan S.S. Van Velkinburgh J.C. Ernst R.K. Miller S.I. Infect. Immun. 2000; 68: 6139-6146Crossref PubMed Scopus (309) Google Scholar). Mutants with alterations in the PhoP/PhoQ system exhibit reduced virulence and an increased susceptibility to anti-microbial peptides (11Miller S.I. Kukral A.M. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5054-5058Crossref PubMed Scopus (686) Google Scholar, 12Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (332) Google Scholar). Homologs of the PhoP/PhoQ and PmrA/PmrB systems have been identified in other Gram-negative bacteria, including E. coli, Yersinia pestis, and Pseudomonas aeruginosa (13Ernst R.K. Guina T. Miller S.I. J. Infect. Dis. 1999; 179: 326-330Crossref PubMed Scopus (131) Google Scholar, 14Ernst R.K. Yi E.C. Guo L. Lim K.B. Burns J.L. Hackett M. Miller S.I. Science. 1999; 286: 1561-1565Crossref PubMed Scopus (405) Google Scholar).Until now, several lipid A-modifying enzymes have been identified (Fig. 1B). Substitution of the 1- and 4′-phosphate groups with one or two 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties in S. Typhimurium was found to be dependent on the enzyme ArnT (15Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Recently, the PmrC protein was identified to mediate the addition of phosphoethanolamine to lipid A in S. enterica (16Lee H. Hsu F.F. Turk J. Groisman E.A. J. Bacteriol. 2004; 186: 4124-4133Crossref PubMed Scopus (234) Google Scholar). Another enzyme, designated LpxO, catalyzes the O2-dependent hydroxylation of lipid A (17Gibbons H.S. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2000; 275: 32940-32949Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), and a lipid A 1-phosphatase was identified in Rhizobium leguminosarum (18Karbarz M.J. Kalb S.R. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2003; 278: 39269-39279Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). All these enzymes are thought to reside within the inner membrane or periplasmic space (15Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 16Lee H. Hsu F.F. Turk J. Groisman E.A. J. Bacteriol. 2004; 186: 4124-4133Crossref PubMed Scopus (234) Google Scholar, 17Gibbons H.S. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2000; 275: 32940-32949Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 18Karbarz M.J. Kalb S.R. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2003; 278: 39269-39279Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Recently, a new class of outer membrane-localized lipid A-modifying enzymes was discovered. One of them is the palmitoyl transferase PagP (19Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R.H. EMBO J. 2000; 19: 5071-5080Crossref PubMed Scopus (273) Google Scholar). Palmitoylation of lipid A leads to an increased resistance to cationic antimicrobial peptides (7Guo L. Lim K.B. Poduje C.M. Daniel M. Gunn J.S. Hackett M. Miller S.I. Cell. 1998; 95: 189-198Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). Furthermore, palmitoylated lipid A antagonizes LPS-induced activation of human cells (20Tanamoto K. Azumi S. J. Immunol. 2000; 164: 3149-3156Crossref PubMed Scopus (70) Google Scholar). Homologs of PagP are found, among others, in S. Typhimurium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella parapertussis, Legionella pneumophila, E. coli, and Y. pestis (19Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R.H. EMBO J. 2000; 19: 5071-5080Crossref PubMed Scopus (273) Google Scholar, 21Robey M. O'Connell W. Cianciotto N.P. Infect. Immun. 2001; 69: 4276-4286Crossref PubMed Scopus (98) Google Scholar). Another outer membrane-localized lipid A-modifying enzyme is the 3-O-deacylase PagL (22Trent M.S. Pabich W. Raetz C.R.H. Miller S.I. J. Biol. Chem. 2001; 276: 9083-9092Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). This enzyme was discovered in S. Typhimurium and shown to hydrolyze the ester bond at the 3 position of lipid A, thereby releasing the primary 3-hydroxymyristoyl moiety (22Trent M.S. Pabich W. Raetz C.R.H. Miller S.I. J. Biol. Chem. 2001; 276: 9083-9092Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). At that time, no obvious homologs of this protein could be found in the nonredundant or unfinished microbial databases, except in the closely related strains S. enterica serovars Typhi and Paratyphi (22Trent M.S. Pabich W. Raetz C.R.H. Miller S.I. J. Biol. Chem. 2001; 276: 9083-9092Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Nevertheless, some other Gram-negative bacteria, including P. aeruginosa (14Ernst R.K. Yi E.C. Guo L. Lim K.B. Burns J.L. Hackett M. Miller S.I. Science. 1999; 286: 1561-1565Crossref PubMed Scopus (405) Google Scholar), R. leguminosarum (23Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar), Helicobacter pylori (24Moran A.P. Lindner B. Walsh E.J. J. Bacteriol. 1997; 179: 6453-6463Crossref PubMed Google Scholar), and Porhyromonas gingivalis (25Kumada H. Haishima Y. Umemoto T. Tanamoto K. J. Bacteriol. 1995; 177: 2098-2106Crossref PubMed Google Scholar) contain 3-O-deacylated lipid A species, suggesting that these organisms contain enzymes with an activity similar to that of PagL. We report now the identification of PagL homologs in a variety of Gram-negative bacteria. The limited sequence similarity among the various proteins was used to identify active site residues.MATERIALS AND METHODSBacterial Strains and Growth Conditions—All bacterial strains used in this study are described in Table I. Unless otherwise notified, the . coli and P. aeruginosa strains were grown at 37 °C and 30 °C, respectively, in a modified Luria-Bertani broth, designated LB (26Tommassen J. van Tol H. Lugtenberg B. EMBO J. 1983; 2: 1275-1279Crossref PubMed Scopus (116) Google Scholar), supplemented with 0.2% glucose, or in minimal medium (SV) (49Winkler K.C. de Haan P.G. Arch. Biochem. 1948; 18: 97-107PubMed Google Scholar) supplemented with 0.5% glucose, while shaking at 200 rpm. To induce expression of the pagL genes cloned behind the T7 promoter, the bacteria were grown in LB supplemented with glucose until an absorbance at 600 nm (A600) of 0.4–0.6 was reached. Expression of the pagL genes was then induced by adding 1 mm isopropyl-1-thio-β-d-galactopyranoside (IPTG), and incubation at 37 °C was continued. When appropriate, bacteria were grown in the presence of 100 μg/ml ampicillin, 50 μg/ml kanamycin, 10 μg/ml tetracycline, or 100 μg/ml streptomycin, for plasmid maintenance. S. Typhimurium SR11 was grown on LB agar plates at 37 °C. B. bronchiseptica and B. pertussis strains were grown at 35 °C on Borduet-Gengou agar (Difco) supplemented with 15% defibrinated sheep blood (Biotrading). Stain JG101, carrying a tetracycline-resistance transposon insertion in pagP, was obtained by P1 transduction by using E. coli BL21 Star™ (DE3) and E. coli SK2257 (Table I) as the acceptor and donor, respectively.Table IBacterial strains and plasmids used in this studyStrain or plasmidGenotype or descriptionSource or referenceStrainsB. bronchisepticaB505Wild-type strainN.V.I.aNetherlands Vaccine Institute, Bilthoven, The NetherlandsB. pertussisBP509Dutch vaccine strainN.V.I.aNetherlands Vaccine Institute, Bilthoven, The NetherlandsBP134Dutch vaccine strainN.V.I.aNetherlands Vaccine Institute, Bilthoven, The NetherlandsP. aeruginosaPAO1Wild-type strain45Jacobs M.A. Alwood A. Thaipisuttikul I. Spencer D. Haugen E. Ernst S. Will O. Kaul R. Raymond C. Levy R. Chun-Rong L. Guenthner D. Bovee D. Olson M.V. Manoil C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14339-14344Crossref PubMed Scopus (840) Google ScholarPAO25PAO1 leu arg46Haas D. Holloway B.W. Mol. Gen. Genet. 1976; 144: 243-251Crossref PubMed Scopus (170) Google Scholar32751PA4661 (pagL) mutant-derivative of PAO145Jacobs M.A. Alwood A. Thaipisuttikul I. Spencer D. Haugen E. Ernst S. Will O. Kaul R. Raymond C. Levy R. Chun-Rong L. Guenthner D. Bovee D. Olson M.V. Manoil C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14339-14344Crossref PubMed Scopus (840) Google ScholarS. enterica TyphimuriumSR11Wild-type strain47Pace J. Hayman M.J. Galan J.E. Cell. 1993; 72: 505-514Abstract Full Text PDF PubMed Scopus (221) Google ScholarE. coliTOP10F′F′ {lacIq Tn10 (TetR)} mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR recA1 araD139 (ara-leu)7697 galU galK rpsL endA1 nupGInvitrogenDH5αF- Δ(lacZYA-algF)U169 thi-1 hsdR17 gyrA96 recA1 endA1 supE44 relA1 phoA φ80 dlacZΔM1548Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8124) Google ScholarBL21 Star™ (DE3)F- ompT hsdS B (rB- mB-) gal dcm rne131 (DE3)InvitrogenSK2257F- crcA280::Tn10bE. coli genetic stock center, Yale University, New Haven, CTthyA6 rpsL120(StrR) deoC1CGSCcpagP is also known as crcAJG101BL21 Star (DE3) crcA280::Tn10bE. coli genetic stock center, Yale University, New Haven, CTThis studyPlasmidspCRII-TOPOE. coli cloning vector AmpR KanRInvitrogenpET-11aE. coli high-copy expression vector, AmpR, T7 promotorNovagenpPagL(Pa)pET-11a derivative harboring P. aeruginosa pagLThis studypPagL(Bb)pET-11a derivative harboring B. bronchiseptica pagLThis studypPagL(St)pET-11a derivative harboring S. typhimurium pagLThis studypPagL(Pa)(-)pET-11a derivative encoding P. aeruginosa PagL without signal sequenceThis studypPagL(Pa) (H81A)pPagL(Pa) encoding PagL(Pa) with H81A substitutionThis studypPagL(Pa) (H81N)pPagL(Pa) encoding PagL(Pa) with H81N substitutionThis studypPagL(Pa) (S84A)pPagL(Pa) encoding PagL(Pa) with S84A substitutionThis studypPagL(Pa) (S84C)pPagL(Pa) encoding PagL(Pa) with S84C substitutionThis studypPagL(Pa) (H149A)pPagL(Pa) encoding PagL(Pa) with H149A substitutionThis studypPagL(Pa) (H149N)pPagL(Pa) encoding PagL(Pa) with H149N substitutionThis studypPagL(Pa) (S151A)pPagL(Pa) encoding PagL(Pa) with S151A substitutionThis studypPagL(Pa) (S151C)pPagL(Pa) encoding PagL(Pa) with S151C substitutionThis studya Netherlands Vaccine Institute, Bilthoven, The Netherlandsb E. coli genetic stock center, Yale University, New Haven, CTc pagP is also known as crcA Open table in a new tab Recombinant DNA Techniques—The plasmids used are described in Table I. Plasmid DNA was isolated using the Promega Wizard® Plus SV minipreparations system. Calf intestine alkaline phosphatase and restriction endonucleases were used according to the instructions of the manufacturer (Fermentas). DNA fragments were isolated from agarose gels using the Qiagen quick gel extraction kit. Ligations were performed by using the rapid DNA ligation kit (Roche Applied Science).The pagL genes from S. Typhimurium SR11 (pagL(St)), B. bronchiseptica B505 (pagL(Bb)), and P. aeruginosa PAO25 (pagL(Pa)) were cloned into pET-11a (Novagen) behind the T7 promoter. The genes were amplified by PCR using chromosomal DNA as template. Template DNA was prepared by resuspending ∼109 bacteria in 50 μl of distilled water, after which the suspension was heated for 15 min at 95 °C. The suspension was then centrifuged for 1 min at 16,100 × g, after which the supernatant was used as template DNA. The sequences of the forward primers, which contained an NdeI site (underlined), including an ATG start codon, were 5′-AACATATGAAGAGAATATTTATATATC-3′ (pagL(St)), 5′-AACATATGAAGAAACTACTTCCGCTGG-3′ (pag-L(Pa)), and 5′-AACATATGCAATTTCTCAAGAAAAACA-3′ (pagL(Bb)). The sequences of the reverse primers, which contained a BamHI site (underlined) and included a stop codon, were 5′-AAGGATCCTCAGAAATTATAACTAATT-3′ (pagL(St)), 5′-AAGGATCCCTAGATCGGGATCTTGTAG-3′ (pagL(Pa)), and 5′-AAGGATCCTCAGAACTGGTACGTATA-G-3′ (pagL(Bb)). The PCRs were done under the following conditions: 50-μl total reaction volume, 25 pmol of each primer, 0.2 mm dNTPs, 3 μl of template DNA solution, 1.5% dimethyl sulfoxide, 1.75 units of Expand High Fidelity enzyme mix with buffer supplied by the manufacturer (Roche Applied Science). The temperature program was as follows: 95 °C for 3 min, a cycle of 1 min at 95 °C, 1 min at 60 °C, and 1 min 30 s at 72 °C repeated 30 times, followed by 10 min at 72 °C and subsequent cooling to 4 °C. The PCR products were purified from agarose gel and subsequently cloned into pCRII-TOPO. Plasmid DNA from correct clones was digested with NdeI and BamHI, and the PagL-encoding fragments were ligated into NdeI/BamHI-digested pET-11a. The ligation mixture was used to transform E. coli DH5α using the CaCl2 method (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 1.82-1.84Google Scholar). Plasmid DNA from transformants was checked for presence of the correct PagL-encoding insert by digestion with NdeI and BamHI. Plasmids that gave a correct digestion profile were designated pPagL(Pa), pPagL(Bb), and pPagL(St) (Table I). The correct coding sequences of the cloned pagL genes were confirmed by nucleotide sequencing in both directions. Mutations were introduced in pagL by using the QuikChange site-directed mutagenesis kit (Stratagene) and the primers listed in Table II. Plasmid pPagL(Pa) was used as the template in which the mutations were created. The presence of the correct mutations was confirmed by nucleotide sequencing in both directions.Table IIPrimers used for site-directed mutagenesisNameaThe primer name gives the amino acid substitution, e.g. H81A_FW indicates that the oligonucleotide shown was used as the forward primer in a site-directed mutagenesis procedure to replace the histidine at position 81 of the precursor PagL(Pa) with an alanineSequence (5′-3′)bIntroduced mutations are underlinedH81A_FWGAAGGCGCCGGCAAGGCGTCGCTGTCGTTCGCTH81A_REVAGCGAACGACAGCGACGCCTTGCCGGCGCCTTCH81N_FWGAAGGCGCCGGCAAGAACTCGCTGTCGTTCGCTH81N_REVAGCGAACGACAGCGAGTTCTTGCCGGCGCCTTCS84A_FWGGCAAGCATTCGCTGGCGTTCGCTCCGGTATTCS84A_REVGAATACCGGAGCGAACGCCAGCGAATGCTTGCCS84C_FWGGCAAGCATTCGCTGTGCTTCGCTCCGGTATTCS84C_REVGAATACCGGAGCGAAGCACAGCGAATGCTTGCCH149A_FWGGCGTTCGGGCGATCGCGTATTCCAACGCCGGCH149A_REVGCCGGCGTTGGAATACGCGATCGCCCGAACGCCH149N_FWGGCGTTCGGGCGATCAACTATTCCAACGCCGGCH149N_REVGCCGGCGTTGGAATAGTTGATCGCCCGAACGCCS151A_FWCGGGCGATCCACTATGCGAACGCCGGCCTGAAAS151A_REVTTTCAGGCCGGCGTTCGCATAGTGGATCGCCCGS151C_FWCGGGCGATCCACTATTGCAACGCCGGCCTGAAAS151C_REVTTTCAGGCCGGCGTTGCAATAGTGGATCGCCCGa The primer name gives the amino acid substitution, e.g. H81A_FW indicates that the oligonucleotide shown was used as the forward primer in a site-directed mutagenesis procedure to replace the histidine at position 81 of the precursor PagL(Pa) with an alanineb Introduced mutations are underlined Open table in a new tab Isolation of Cell Envelopes—Cells were harvested by centrifugation for 10 min at 1,500 × g and washed once in 50 ml of cold 0.9% sodium chloride solution. The cell pellets were frozen for at least 15 min at -80 °C and then suspended in 20 ml of 3 mm EDTA, 10 mm Tris-HCl (pH 8.0) containing Complete protease inhibitor mixture (Roche Applied Science). The cells were disrupted by sonication, after which unbroken cells were removed by centrifugation for 10 min at 1,500 × g. The cell envelopes were pelleted from the supernatant by centrifugation for 1.5 h at 150,000 × g and resuspended in 2 mm Tris-HCl (pH 7.4). The cell envelopes were stored at -80 °C in aliquots.SDS-PAGE and Immunoblotting—Proteins were analyzed by SDS-PAGE (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar) with 0.2% SDS in the running gel using the Bio-Rad Mini-PROTEAN®3 apparatus. Samples were applied to a 13% polyacrylamide gel with a 4% stacking gel and subjected to electrophoresis at 150 V. Proteins were stained with Coomassie Brilliant Blue. Prestained or unstained Precision Plus Protein™ Standard from Bio-Rad was used to determine the relative molecular mass. For Western blotting, proteins were transferred from SDS-polyacrylamide gels onto nitrocellulose membranes. The membranes were blocked overnight in phosphate-buffered saline (pH 7.6), 0.5% nonfat dried milk, 0.1% Tween 20 and incubated with guinea pig antibodies directed against PagL(Pa) in blocking buffer followed by an incubation with horseradish peroxidase-conjugated rabbit anti-guinea pig IgG antibodies (Sigma) in blocking buffer. Blots were developed using SuperSignal® WestPico Chemiluminescent Substrate (Pierce).Polyclonal Antibodies—For antibody production, the pagL gene from P. aeruginosa PAO25 without the signal sequence-encoding part was PCR amplified by using the forward primer (5′-AACATATGGCGGACGTCTCGGCCGCCG-3′), which contained an NdeI site (underlined), including an ATG start codon, and the reverse primer (5′-AAGGATCCCTAGATCGGGATCTTGTAG-3′), which contained an BamHI site (underlined) and included a stop codon. The PCR product was cloned into pET-11a, and the resulting plasmid, pPagL(Pa)(-), was used to transform E. coli BL21 Star™ (DE3) to allow for expression of the truncated pagL gene. The PagL(Pa) protein, accumulating in inclusion bodies, was isolated (29Dekker N. Merck K. Tommassen J. Verheij H.M. Eur. J. Biochem. 1995; 232: 214-219Crossref PubMed Scopus (86) Google Scholar), purified from a preparative SDS-polyacrylamide gel, and used for immunization of guinea pigs at Eurogentec.Microsequencing—Proteins were transferred from SDS-polyacrylamide gels to an Immobilon-P polyvinylidene difluoride membrane (Millipore Corp.) in 192 mm glycine, 25 mm Tris (pH 8.3), 10% methanol (v/v) at 100 V for 1 h using the Bio-Rad Mini-PROTEAN®2 blotting apparatus. After transfer, the membrane was washed three times for 15 min with distilled water. Transferred proteins were stained with Coomassie Brilliant Blue. The membrane was dried in the air, and the putative PagL bands were excised and subjected to microsequencing at the Sequencing Center Facility, Utrecht University, The Netherlands.LPS Analysis by Tricine-SDS-PAGE—Approximately 109 bacteria were suspended in 50 μl of sample buffer (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar), and 0.5 mg/ml proteinase K (end concentration) was added. The samples were incubated for 60 min at 55 °C followed by 10 min at 95 °C to inactivate proteinase K. The samples were then diluted 10-fold by adding sample buffer, after which 2 μl of each sample was applied to a Tricine-SDS-polyacrylamide gel (30Lesse A.J. Campagnari A.A. Bittner W.E. Apicella M.A. J. Immunol. Methods. 1990; 126: 109-117Crossref PubMed Scopus (287) Google Scholar). The bromphenol blue was allowed to run into the separating gel at 35 V, after which the voltage was increased to 105 V. After the front reached the bottom of the gel, the samples were left running for another 45 min. The gels were fixed overnight in water/ethanol/acetic acid 11:8:1 (v/v/v) and subsequently stained with silver as described previously (31Tsai C.M. Frasch C.E. Anal. Biochem. 1982; 119: 115-119Crossref PubMed Scopus (2294) Google Scholar).Gas Chromatography-Mass Spectrometry (GC/MS) and Electrospray Ionization-Mass Spectrometry (ESI/MS)—LPS was isolated using the hot phenol/water extraction method (32Westphal O. Jann J.K. Methods Carbohydr. Chem. 1965; 5: 83-91Google Scholar). For fatty acid analysis by GC/MS, a 5-fold volume excess of acetone was added to an aliquot of the isolated LPS (0.2 mg/ml), after which the solution was dried at 60 °C under a nitrogen flow. Subsequently, 10 μg of C12:0(2OH) (1 mg/ml in ethanol) was added as an internal standard, as well as 100 μl of acetylchloride/ethanol 1:9 (v/v), after which the samples were derivatized for 1 h at 90 °C. After cooling, the reaction was stopped by adding 200 μl of 1 m K2HPO4 (pH 8.0), followed by extraction of the acylethyl esters with 200 μl of ethyl acetate. A 1-μl volume of the upper phase was used for analysis by GC/MS on a Finnigan MAT SSQ in the electron-impact mode. For ESI/MS, an aliquot of isolated LPS was freeze-dried and taken up in 1.8 ml of 12.5 mm sodium acetate (pH 4.5) containing 1% SDS. The mixture was boiled for 30 min to hydrolyze the LPS and release the lipid A moiety, after which the mixture was cooled to room temperature and converted into a two-phase Bligh and Dyer mixture by adding 2 ml of methanol and 2 ml of chloroform. Phases were separated by centrifugation, after which the lower phase was collected and washed twice with the upper phase of a fresh two-phase Bligh and Dyer mixture, consisting of chloroform/methanol/water (2:2: 1.8, v/v). Structural analysis of purified lipid A was performed by nanoelectrospray tandem MS on a Finnigan LCQ in the positive ion mode (33Wilm M. Mann M. Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1691) Google Scholar).RESULTSIdentification of PagL Homologs in various Gram-negative Bacteria—The 187-amino acid sequence of the S. Typhimurium PagL precursor protein (GenBank accession no. AAL21147) was used as a lead to identify putative PagL homologs in other Gram-negative bacteria, by searching all completed and unfinished genomes of Gram-negative bacteria present in the NCBI data base (www.ncbi.nlm.nih.gov/sutils/genom_table.cgi). BLAST search (34Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69088) Google Scholar) revealed the presence of putative homologs in the Bordetella spp. B. pertussis, B. bronchiseptica, and B. parapertussis (Fig. 2). The PagL homologs of B. bronchiseptica and B. parapertussis are two mutually identical 178-amino acid polypeptides (Fig. 2) with, as predicted by the SignalP server (35Nielsen H. Brunak S. von Heijne G. Protein Eng. 1999; 12: 3-9Crossref PubMed Scopus (534) Google Scholar), a 25-amino acid N-terminal signal peptide. A gene for a PagL homolog was also found in the genome of the B. pertussis Tohama I strain (36Parkhill J. Sebaihia M. Preston A. Murphy L.D. Thomson N. Harris D.E. Holden M.T. Churcher C.M. Bentley S.D. Mungall K.L. Cerdeno-Tarraga A.M. Temple L. James K. Harris B. Quail M.A. Achtman M. Atkin R. Baker S. Basham D. Bason N. Cherevach I. Chilling" @default.
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• W1990180328 title "Dissemination of Lipid A Deacylases (PagL) among Gram-negative Bacteria" @default.
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