Biosynthesis and Structure of the Burkholderia cenocepacia K56-2 Lipopolysaccharide Core Oligosaccharide
2009; Elsevier BV; Volume: 284; Issue: 32 Linguagem: Inglês
10.1074/jbc.m109.008532
ISSN1083-351X
AutoresXimena Ortega, Alba Silipo, M. Soledad Saldías, Christa C. Bates, Antonio Molinaro, Miguel A. Valvano,
Tópico(s)Bacterial biofilms and quorum sensing
ResumoBurkholderia cenocepacia is an opportunistic pathogen that displays a remarkably high resistance to antimicrobial peptides. We hypothesize that high resistance to antimicrobial peptides in these bacteria is because of the barrier properties of the outer membrane. Here we report the identification of genes for the biosynthesis of the core oligosaccharide (OS) moiety of the B. cenocepacia lipopolysaccharide. We constructed a panel of isogenic mutants with truncated core OS that facilitated functional gene assignments and the elucidation of the core OS structure in the prototypic strain K56-2. The core OS structure consists of three heptoses in the inner core region, 3-deoxy-d-manno-octulosonic acid, d-glycero-d-talo-octulosonic acid, and 4-amino-4-deoxy-l-arabinose linked to d-glycero-d-talo-octulosonic acid. Also, glucose is linked to heptose I, whereas heptose II carries a second glucose and a terminal heptose, which is the site of attachment of the O antigen. We established that the level of core truncation in the mutants was proportional to their increased in vitro sensitivity to polymyxin B (PmB). Binding assays using fluorescent 5-dimethylaminonaphthalene-1-sulfonyl-labeled PmB demonstrated a correlation between sensitivity and increased binding of PmB to intact cells. Also, the mutant producing a heptoseless core OS did not survive in macrophages as compared with the parental K56-2 strain. Together, our results demonstrate that a complete core OS is required for full PmB resistance in B. cenocepacia and that resistance is due, at least in part, to the ability of B. cenocepacia to prevent binding of the peptide to the bacterial cell envelope. Burkholderia cenocepacia is an opportunistic pathogen that displays a remarkably high resistance to antimicrobial peptides. We hypothesize that high resistance to antimicrobial peptides in these bacteria is because of the barrier properties of the outer membrane. Here we report the identification of genes for the biosynthesis of the core oligosaccharide (OS) moiety of the B. cenocepacia lipopolysaccharide. We constructed a panel of isogenic mutants with truncated core OS that facilitated functional gene assignments and the elucidation of the core OS structure in the prototypic strain K56-2. The core OS structure consists of three heptoses in the inner core region, 3-deoxy-d-manno-octulosonic acid, d-glycero-d-talo-octulosonic acid, and 4-amino-4-deoxy-l-arabinose linked to d-glycero-d-talo-octulosonic acid. Also, glucose is linked to heptose I, whereas heptose II carries a second glucose and a terminal heptose, which is the site of attachment of the O antigen. We established that the level of core truncation in the mutants was proportional to their increased in vitro sensitivity to polymyxin B (PmB). Binding assays using fluorescent 5-dimethylaminonaphthalene-1-sulfonyl-labeled PmB demonstrated a correlation between sensitivity and increased binding of PmB to intact cells. Also, the mutant producing a heptoseless core OS did not survive in macrophages as compared with the parental K56-2 strain. Together, our results demonstrate that a complete core OS is required for full PmB resistance in B. cenocepacia and that resistance is due, at least in part, to the ability of B. cenocepacia to prevent binding of the peptide to the bacterial cell envelope. Burkholderia cenocepacia is a Gram-negative opportunistic pathogen ubiquitously found in the environment (1Mahenthiralingam E. Urban T.A. Goldberg J.B. Nat. Rev. Microbiol. 2005; 3: 144-156Crossref PubMed Scopus (661) Google Scholar, 2Balandreau J. Viallard V. Cournoyer B. Coenye T. Laevens S. Vandamme P. Appl. Environ. Microbiol. 2001; 67: 982-985Crossref PubMed Scopus (129) Google Scholar). Although generally harmless to healthy individuals, B. cenocepacia affects immunocompromised patients (1Mahenthiralingam E. Urban T.A. Goldberg J.B. Nat. Rev. Microbiol. 2005; 3: 144-156Crossref PubMed Scopus (661) Google Scholar) such as those with cystic fibrosis and chronic granulomatous disease. Infected cystic fibrosis patients commonly develop chronic lung infections that are very difficult to treat because these bacteria are intrinsically resistant to virtually all clinically useful antibiotics as well as antimicrobial peptides (APs) 5The abbreviations used are: APantimicrobial peptideDQF-COSYdouble quantum filtered correlation spectroscopyd-QuiNd-quinovosamineHMBCheteronuclear multiple bond correlationHSQCheteronuclear single quantum coherenceKdo3-deoxy-d-manno-octulosonic acidKod-glycero-d-talo-octulosonic acidl-Ara4N4-amino-4-deoxy-l-arabinoseLPSlipopolysaccharidel-Rhal-rhamnoseMALDImatrix-assisted laser desorption ionizationMSmass spectrometryNOEnuclear Overhauser effectOSoligosaccharidePmBpolymyxin BTOCSYtotal correlation spectroscopyTOFtime-of-flightT-ROESYtransverse rotating-frame Overhauser enhancement spectroscopyDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumdansyl5-dimethylaminonaphthalene-1-sulfonylTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineBcCVB. cenocepacia-containing vacuole. (1Mahenthiralingam E. Urban T.A. Goldberg J.B. Nat. Rev. Microbiol. 2005; 3: 144-156Crossref PubMed Scopus (661) Google Scholar, 3Aaron S.D. Ferris W. Henry D.A. Speert D.P. Macdonald N.E. Am. J. Respir. Crit. Care Med. 2000; 161: 1206-1212Crossref PubMed Scopus (213) Google Scholar). antimicrobial peptide double quantum filtered correlation spectroscopy d-quinovosamine heteronuclear multiple bond correlation heteronuclear single quantum coherence 3-deoxy-d-manno-octulosonic acid d-glycero-d-talo-octulosonic acid 4-amino-4-deoxy-l-arabinose lipopolysaccharide l-rhamnose matrix-assisted laser desorption ionization mass spectrometry nuclear Overhauser effect oligosaccharide polymyxin B total correlation spectroscopy time-of-flight transverse rotating-frame Overhauser enhancement spectroscopy Dulbecco's modified Eagle's medium fetal bovine serum 5-dimethylaminonaphthalene-1-sulfonyl N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine B. cenocepacia-containing vacuole. Lipopolysaccharide (LPS) is the major surface component of Gram-negative bacteria and consists of lipid A, core oligosaccharide (OS), and in some bacteria O-specific polysaccharide or O antigen (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3530) Google Scholar, 5Caroff M. Karibian D. Carbohydr. Res. 2003; 338: 2431-2447Crossref PubMed Scopus (392) Google Scholar). The O antigen acts as a protective barrier against desiccation, phagocytosis, and serum complement-mediated killing, whereas the core OS and the lipid A contribute to maintain the integrity of the outer membrane (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3530) Google Scholar, 5Caroff M. Karibian D. Carbohydr. Res. 2003; 338: 2431-2447Crossref PubMed Scopus (392) Google Scholar). The lipid A also anchors the LPS molecule to the outer leaflet of the outer membrane and accounts for the endotoxic activity of LPS (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3530) Google Scholar, 6Raetz C.R. Reynolds C.M. Trent M.S. Bishop R.E. Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (976) Google Scholar). Lipid A is a bisphosphorylated Β-1,6-linked glucosamine disaccharide substituted with fatty acids ester-linked at positions 3 and 3′ and amide-linked at positions 2 and 2′ (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3530) Google Scholar). The core OS can be subdivided into the inner core and outer core. The inner core OS typically consists of one or two 3-deoxy-d-manno-octulosonic acid (Kdo) residues linked to the lipid A and three l-glycero-d-manno-heptose residues linked to the first Kdo (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3530) Google Scholar). The outer core OS in enteric bacteria typically consists of 8–12 branched sugars linked to heptose II of the inner core. As a result of phosphate groups on the lipid A and core OS, the bacterial surface has a net negative charge. This plays an important role in the interaction of the bacterial surface with positively charged compounds such as cationic APs, which are cationic amphipathic molecules that kill bacteria by membrane permeabilization. In response to a series of environmental conditions such as low magnesium or high iron, bacteria can express modified LPS molecules that result in a less negative surface. This reduces the binding of APs and promotes resistance to these compounds. Previous studies have shown that Burkholderia LPS molecules possess unique properties. For example, Kdo cannot be detected by classic colorimetric methods in LPS from Burkholderia pseudomallei and Burkholderia cepacia, and the covalent linkage between Kdo and lipid A is more resistant to acid hydrolysis than in conventional LPS molecules (7Isshiki Y. Kawahara K. Zähringer U. Carbohydr. Res. 1998; 313: 21-27Crossref PubMed Scopus (56) Google Scholar). In B. cepacia, 4-amino-4-deoxy-l-arabinose (l-Ara4N) is bound to the lipid A by a phosphodiester linkage at position 4 of the nonreducing glucosamine (GlcN II) (8Silipo A. Molinaro A. Cescutti P. Bedini E. Rizzo R. Parrilli M. Lanzetta R. Glycobiology. 2005; 15: 561-570Crossref PubMed Scopus (50) Google Scholar) and is also present as a component of the core OS. Also, instead of two Kdo molecules, the B. cepacia core OS has only one Kdo and the unusual Kdo analog, d-glycero-d-talo-octulosonic acid (Ko), which is nonstoichiometrically substituted with l-Ara4N forming a 1→8 linkage with α-Ko (7Isshiki Y. Kawahara K. Zähringer U. Carbohydr. Res. 1998; 313: 21-27Crossref PubMed Scopus (56) Google Scholar, 9Isshiki Y. Zähringer U. Kawahara K. Carbohydr. Res. 2003; 338: 2659-2666Crossref PubMed Scopus (29) Google Scholar). Although this is also the case for the inner core OS of B. cenocepacia J2315 (10Silipo A. Molinaro A. Ieranó T. De Soyza A. Sturiale L. Garozzo D. Aldridge C. Corris P.A. Khan C.M. Lanzetta R. Parrilli M. Chemistry. 2007; 13: 3501-3511Crossref PubMed Scopus (58) Google Scholar), it is not a common feature for the core OS in all Burkholderia. For example, the inner core of Burkholderia caryophylli consists of two Kdo residues and does not possess l-Ara4N (11Molinaro A. De Castro C. Lanzetta R. Evidente A. Parrilli M. Holst O. J. Biol. Chem. 2002; 277: 10058-10063Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Burkholderia species, including B. cenocepacia, are intrinsically resistant to human and non-human APs such as these produced by airway epithelial cells (12Baird R.M. Brown H. Smith A.W. Watson M.L. Immunopharmacology. 1999; 44: 267-272Crossref PubMed Scopus (29) Google Scholar, 13Devine D.A. Mol. Immunol. 2003; 40: 431-443Crossref PubMed Scopus (76) Google Scholar), human Β-defensin 3 (14Sahly H. Schubert S. Harder J. Rautenberg P. Ullmann U. Schröder J. Podschun R. Antimicrob. Agents Chemother. 2003; 47: 1739-1741Crossref PubMed Scopus (68) Google Scholar), human neutrophil peptides (15Loutet S.A. Flannagan R.S. Kooi C. Sokol P.A. Valvano M.A. J. Bacteriol. 2006; 188: 2073-2080Crossref PubMed Scopus (112) Google Scholar), and polymyxin B (PmB) (15Loutet S.A. Flannagan R.S. Kooi C. Sokol P.A. Valvano M.A. J. Bacteriol. 2006; 188: 2073-2080Crossref PubMed Scopus (112) Google Scholar, 16Burtnick M.N. Woods D.E. Antimicrob. Agents Chemother. 1999; 43: 2648-2656Crossref PubMed Google Scholar). The minimum inhibitory concentration determined for some of these peptides in several Burkholderia species is greater than 500 μg/ml, which could aid these microorganisms during colonization of the respiratory epithelia (13Devine D.A. Mol. Immunol. 2003; 40: 431-443Crossref PubMed Scopus (76) Google Scholar). It has been proposed that the resistance of B. cepacia to cationic APs stems from ineffective binding to the outer membrane, as a consequence of the low number of phosphate and carboxylate groups in the lipopolysaccharide (17Cox A.D. Wilkinson S.G. Mol. Microbiol. 1991; 5: 641-646Crossref PubMed Scopus (84) Google Scholar), but a systematic analysis of the molecular basis of AP resistance in B. cenocepacia and other Burkholderia is lacking. We have previously reported that a heptoseless B. cenocepacia mutant (SAL1) is significantly more sensitive than the parental clinical strain K56-2 to APs (15Loutet S.A. Flannagan R.S. Kooi C. Sokol P.A. Valvano M.A. J. Bacteriol. 2006; 188: 2073-2080Crossref PubMed Scopus (112) Google Scholar). This mutant has a truncated inner core and lacks the outer core, suggesting that a complete core OS is required for resistance of B. cenocepacia to APs. Apart from heptoses, the role of other sugar moieties of the B. cenocepacia core OS in AP resistance is not known. In this study, we report the structure of the core OS for B. cenocepacia strain K56-2 and its isogenic mutants XOA3, XOA7, and XOA8, which carry various core OS truncations. The structural analysis, combined with mutagenesis, allowed us to assign function to the majority of the genes involved in core OS biosynthesis and ligation of the O antigen and to establish that the degree of truncation of the core OS correlates with increased binding and bacterial sensitivity to PmB in vitro and reduced bacterial intracellular survival in macrophages. Strains and plasmids used in this study are listed in Table 1. B. cenocepacia strain K56-2 was grown at 37 °C in LB medium supplemented, as required, with 100 μg/ml trimethoprim and 50 μg/ml gentamicin. This strain is a clinical isolate that belongs to the same clonal group as the type strain J2315 (18Mahenthiralingam E. Coenye T. Chung J.W. Speert D.P. Govan J.R. Taylor P. Vandamme P. J. Clin. Microbiol. 2000; 38: 910-913Crossref PubMed Google Scholar). Escherichia coli strains were grown at 37 °C in LB medium supplemented with trimethoprim (50 μg/ml), kanamycin (40 μg/ml), or chloramphenicol (30 μg/ml), as required. Conditional mutants were grown at 37 °C in M9 medium supplemented with 5 mg/ml yeast extract and 0.5% (w/v) rhamnose (permissive conditions) or 0.5% (w/v) glucose (nonpermissive conditions) as described previously (19Ortega X.P. Cardona S.T. Brown A.R. Loutet S.A. Flannagan R.S. Campopiano D.J. Govan J.R. Valvano M.A. J. Bacteriol. 2007; 189: 3639-3644Crossref PubMed Scopus (86) Google Scholar).TABLE 1Bacterial strains and plasmids used in this studyStrain or plasmidGenotype and relevant characteristicsReference or sourceB. cenocepacia strains CCB1K56-2, waaC ::pGPΩTp, TpRThis study K56-2ET12 clone, cystic fibrosis clinical isolateCBCCRRRaCanadian B. cepacia complex Research and Referral Repository. XOA3K56-2, wbxE ::pGPΩTp, TpRThis study XOA6K56-2, wabP ::pGPΩTp, TpRThis study XOA7K56-2, waaL ::pGPΩTp, TpRThis study XOA8K56-2, wabO ::pGPΩTp, TpRThis study XOA9K56-2, wabQ ::pGPΩTp, TpRThis study XOA15K56-2, wabR ::pGPΩTp, TpRThis study XOA17K56-2, wabS ::pGPApTp, TpRThis study XOA19K56-2, waaF ::pSC200, TpRThis studyPlasmids pAP20oripBBR1, CmR, mob+, PdhfrS. Cardona pGPΩTPoriR6K, ΩTpR cassette, mob+20Flannagan R.S. Aubert D. Kooi C. Sokol P.A. Valvano M.A. Infect. Immun. 2007; 75: 1679-1689Crossref PubMed Scopus (68) Google Scholar pGPApTppGP704, TpR, ApR, mob+21Flannagan R.S. Valvano M.A. Microbiology. 2008; 154: 643-653Crossref PubMed Scopus (26) Google Scholar pJR1pMLBAD ((56Lefebre M.D. Valvano M.A. Appl. Environ. Microbiol. 2002; 68: 5956-5964Crossref PubMed Scopus (122) Google Scholar)) expressing the red fluorescent protein mRFP1 under the control of a constitutive Pdhfr promoter, TpR40Lamothe J. Huynh K.K. Grinstein S. Valvano M.A. Cell. Microbiol. 2007; 9: 40-53Crossref PubMed Scopus (84) Google Scholar pRK2013oricolE1, RK2 derivative, KanR, mob+, tra+23Figurski D.H. Helinski D.R. Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 1648-1652Crossref PubMed Scopus (2520) Google Scholar pSC200oriR6K, PRhaB rhamnose-inducible promoter, ΩTpR cassette, mob+19Ortega X.P. Cardona S.T. Brown A.R. Loutet S.A. Flannagan R.S. Campopiano D.J. Govan J.R. Valvano M.A. J. Bacteriol. 2007; 189: 3639-3644Crossref PubMed Scopus (86) Google Scholar pXO13pAP20, wabO under the control of PdhfrThis study pXO14pAP20, waaL under the control of PdhfrThis study pXO20pAP20, wabS under the control of PdhfrThis study pXO21pAP20, waaC under the control of PdhfrThis study pXO22pAP20, waaL-wabR under the control of PdhfrThis studya Canadian B. cepacia complex Research and Referral Repository. Open table in a new tab Murine macrophage (RAW 264.7; ATCC TIB-71) and human lung epithelial cell lines (A549; CCL-185) were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and were grown at 37 °C in a humidified atmosphere with 5% CO2. DMEM and FBS were purchased from Wisent Inc. (St. Bruno, Quebec, Canada). An internal 300-bp fragment near the 5′ end of the gene targeted for mutagenesis was PCR-amplified and cloned into the XbaI and EcoRI restriction sites of the suicide vector pGPΩTp to provide the homology region for recombination. Vector and recombinant plasmids were maintained in E. coli SY327 (araD Δlac-pro argE recA56 nalA λpir RifR). The integration vector has a replication origin that cannot function in B. cenocepacia and also contains transcriptional and translational stops causing polar effects on genes downstream from the insertion point (20Flannagan R.S. Aubert D. Kooi C. Sokol P.A. Valvano M.A. Infect. Immun. 2007; 75: 1679-1689Crossref PubMed Scopus (68) Google Scholar). Similarly, the suicide plasmid pGPApTp (21Flannagan R.S. Valvano M.A. Microbiology. 2008; 154: 643-653Crossref PubMed Scopus (26) Google Scholar) was used to construct nonpolar insertional mutants, and the plasmid pSC200 was used to construct conditional mutants as described (19Ortega X.P. Cardona S.T. Brown A.R. Loutet S.A. Flannagan R.S. Campopiano D.J. Govan J.R. Valvano M.A. J. Bacteriol. 2007; 189: 3639-3644Crossref PubMed Scopus (86) Google Scholar). These plasmids were introduced into B. cenocepacia strain K56-2 by triparental mating (22Craig F.F. Coote J.G. Parton R. Freer J.H. Gilmour N.J. J. Gen. Microbiol. 1989; 135: 2885-2890PubMed Google Scholar) using E. coli MM294 (endA hsdR pro) containing the helper plasmid pRK2013 (23Figurski D.H. Helinski D.R. Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 1648-1652Crossref PubMed Scopus (2520) Google Scholar). Exconjugants were selected on LB agar plates supplemented with 50 μg/ml gentamicin (to kill the E. coli donor and helper bacteria) and 100 μg/ml trimethoprim for selection of K56-2 exconjugants. Exconjugants were screened for plasmid integration into the chromosome by colony PCR using a primer annealing to a region of the mutagenesis vector and another primer annealing to chromosomal sequences upstream of the insertion site. Integration was confirmed by Southern blot hybridization using a probe corresponding to a DNA fragment spanning the homology region used for recombination. The sequences of the DNA primers used for mutagenesis are listed in supplemental Table S1. To construct the complementing plasmids pXO13, pXO14, pXO20, pXO21, and pXO22, the corresponding genes were PCR-amplified using ProofStart polymerase (Qiagen Inc., Valencia, CA). The PCR products were digested with XbaI and EcoRI and ligated into pAP20, which was also cut with the same restriction enzymes. Ligation mixtures were introduced into E. coli DH5α (F− ϕ80lacZ M15 endA recA hsdR[rK−mK−] supE thi gyrA relA Δ[lacZYA-argF]U169) competent cells by the calcium chloride method (24Cohen S.N. Chang A.C. Hsu L. Proc. Natl. Acad. Sci. U.S.A. 1972; 69: 2110-2114Crossref PubMed Scopus (1878) Google Scholar), and transformants were plated on LB agar plates supplemented with 30 μg/ml chloramphenicol. The correct DNA inserts were verified by colony PCR using primers 1630 (5′-CGCAGCAGGGTAGTCGCCCT-3′) and 1631 (5′-ACTCTCGCATGGGGAGACCC-3′), which anneal to sequences flanking the cloning sites of pAP20. Plasmids from PCR-positive colonies were isolated, initially confirmed by restriction digestion and ultimately by DNA sequencing of the DNA insert. These plasmids were mobilized by conjugation into the corresponding B. cenocepacia mutant strains as described above. For electrophoresis analysis, LPS samples were extracted as described previously (25Marolda C.L. Welsh J. Dafoe L. Valvano M.A. J. Bacteriol. 1990; 172: 3590-3599Crossref PubMed Google Scholar). LPS was resolved by electrophoresis in 16.4% polyacrylamide gels using a Tricine-SDS system and visualized by silver staining. In the case of conditional mutants, the LPS was extracted from bacteria grown in liquid cultures at nonpermissive conditions. Large scale LPS preparations for structural analysis were obtained by the method of Westphal and Jann (26Westphal O. Jann K. Whistler R.L. BeMiller J.N. Wolfrom M.L. Methods in Carbohydrate Chemistry. Academic Press, New York1965: 83-91Google Scholar) from 1-liter cultures of B. cenocepacia strains XOA3, XOA7, and XOA8. The quality of the purified LPS samples was confirmed by Tricine-SDS-PAGE as described above. Fractions containing the core OS were obtained by mild acid hydrolysis in sodium acetate buffer, pH 4.4, for 3 h at 100 °C as described previously (10Silipo A. Molinaro A. Ieranó T. De Soyza A. Sturiale L. Garozzo D. Aldridge C. Corris P.A. Khan C.M. Lanzetta R. Parrilli M. Chemistry. 2007; 13: 3501-3511Crossref PubMed Scopus (58) Google Scholar). The determination of sugar residues, their absolute configuration, and linkage analysis, as well as the characterization of total fatty acids content and their absolute configuration were all carried out as described previously (10Silipo A. Molinaro A. Ieranó T. De Soyza A. Sturiale L. Garozzo D. Aldridge C. Corris P.A. Khan C.M. Lanzetta R. Parrilli M. Chemistry. 2007; 13: 3501-3511Crossref PubMed Scopus (58) Google Scholar). For structural assignments of OS fractions, one-dimensional and two-dimensional 1H NMR spectra were recorded on Bruker 600 DRX equipped with a cryoprobe on a solution of 300 μl of D2O using Shigemi tubes. T-ROESY experiments were recorded using data sets (t1 × t2) of 4096 × 256 points with mixing times between 100 and 400 ms. The spin lock field was attenuated (∼ 4000 Hz) with respect to that employed for the hard pulses. No correction for Hartmann-Hahn effects was applied, because T-ROESY effectively removed most of these effects. Double quantum-filtered phase-sensitive COSY experiments were performed using data sets of 4096 × 512 points. TOCSY were performed with spin lock times from 20 to 100 ms, using data sets (t1 × t2) of 4096 × 256 points. In all homonuclear experiments the data matrix was zero-filled in both dimensions to give a matrix of 4000 × 2000 points and was resolution-enhanced in both dimensions by a cosine-bell function before Fourier transformation. Coupling constants were determined on a first order basis from high resolution one-dimensional spectra or by two-dimensional phase-sensitive DQF-COSY. HSQC and HMBC experiments were measured in the 1H-detected mode via single quantum coherence with proton decoupling in the 13C domain, using data sets of 2048 × 256 points. Experiments were carried out in the phase-sensitive mode. A 60-ms delay was used for the evolution of long range connectivity in the HMBC experiment. In all heteronuclear experiments the data matrix was extended to 2048 × 1024 points using forward linear prediction extrapolation. MALDI-TOF mass spectra were recorded in the negative and positive polarity in linear mode on a Voyager STR from PerSeptive Biosystems (Framingham, MA) equipped with delayed extraction technology. Ions formed by a pulsed UV laser beam (nitrogen laser, λ = 337 nm) were accelerated at 24 kV. The mass spectra reported are the result of 256 laser shots. Resolution was about 1500. Bacteria were grown overnight in LB medium or LB medium supplemented with the appropriate antibiotics as required. The next day cultures were diluted to an A600 of 0.01, and 50 μl of this suspension were added to LB medium or LB medium supplemented with 100 μg/ml trimethoprim to make a final volume of 5 ml. 500 μl of this bacterial suspension were aliquoted into Eppendorf tubes, and 10 μl of buffer or PmB (5 mg/ml stock) were added to each tube to reach a final concentration of 100 μg/ml. Cells were incubated with PmB at 37 °C for 22 h with constant rotation using a Barnstead Thermolyne LABQUAKE (Barnstead International, Dubuque, IA), and the A600 was recorded. To determine the MIC50 (concentration of PmB causing 50% reduction in bacterial growth), cells were incubated as described above with PmB at final concentrations of 0, 25, 50, 100, and 200 μg/ml. Dansyl/PmB was prepared from PmB and dansyl/chloride by the method of Schindler and Teuber (27Schindler P.R. Teuber M. Antimicrob. Agents Chemother. 1975; 8: 95-104Crossref PubMed Scopus (114) Google Scholar) and quantified by the dinitrophenylation assay (28Bader J. Teuber M. Z. Naturforsch. 1973; 28: 422-430Crossref PubMed Scopus (5) Google Scholar). For the binding assay bacteria were grown overnight in LB medium or LB medium supplemented with 100 μg/ml trimethoprim, as required. The overnight cultures were diluted in LB to an A600 of 0.1 and grown at 37 °C for an additional 4 h. Bacteria were collected by centrifugation, suspended in 1 ml of 5 mm Hepes, pH 7.4, 10 mm sodium azide buffer, and diluted to an A600 of 0.5. Aliquots (80 μl) of this suspension were added to 96-well plates (Microfluor 2 white, flat bottom 96-well microtiter plates, ThermoLabsystems, Franklin, MA) and mixed with 20-μl aliquots of Hepes/sodium azide buffer (negative control) or the corresponding solutions of dansyl-PmB prepared by dilution of a 1.7-μg/μl stock solution in Hepes/sodium azide buffer. Fluorescence was read in a Varian Cary Eclipse fluorescence spectrofluorometer using an excitation wavelength of 340 nm and an emission wavelength of 485 nm as described (29Moore R.A. Bates N.C. Hancock R.E. Antimicrob. Agents Chemother. 1986; 29: 496-500Crossref PubMed Scopus (189) Google Scholar). Macrophage infections were performed as described previously (30Saldías M.S. Lamothe J. Wu R. Valvano M.A. Infect. Immun. 2008; 76: 1059-1067Crossref PubMed Scopus (57) Google Scholar). Briefly, bacterial suspensions were added to RAW 264.7 cells grown on glass coverslips at a multiplicity of infection of 50 and incubated at 37 °C in 5% CO2 for 4 h. When needed, 0.5 mm LysoTracker Red DND-99 (Invitrogen) was added for 1 min prior to visualization. Fluorescence and phase contrast images were acquired using a Qimaging (Burnaby, British Columbia, Canada) cooled, charged-coupled device camera on an Axioscope 2 (Carl Zeiss) microscope. Images were digitally processed using the Northern Eclipse version 6.0 imaging analysis software (Empix Imaging, Mississauga, Ontario, Canada). Each experiment was independently repeated at least three times. Monolayers for adhesion assays were prepared by seeding 7 × 104 human lung epithelial A549 cells in DMEM, 10% FBS into a 48-multiwell plate and incubating at 37 °C for 20 h in a humidified atmosphere containing 5% CO2. Overnight bacterial cultures were washed and resuspended in DMEM, 10% FBS and added to the cells at multiplicity of infection of 50, centrifuged for 2 min at 300 × g, and incubated for 30 min at 4 °C. Nonadherent bacteria were removed by rinsing five times with ice-cold phosphate-buffered saline. Cells were lysed with 100 ml of 0.5% sodium deoxycholate. Serial dilutions were performed in LB and plated in duplicated. The percentage of adhesion was calculated as follows: 100 × (number of cell-associated bacteria/initial number of bacteria added). Data were calculated from at least three independent experiments performed in triplicate and are expressed as means ± S.E. The statistical significance of differences in the data were determined using the one-way analysis of variance test and the Tukey post-test, provided in the Prism GraphPad software version 4.0. We examined the genome of strain J2315 (31Holden M.T. Seth-Smith H.M. Crossman L.C. Sebaihia M. Bentley S.D. Cerdeño-Tárraga A.M. Thomson N.R. Bason N. Quail M.A. Sharp S. Cherevach I. Churcher C. Goodhead I. Hauser H. Holroyd N. Mungall K. Scott P. Walker D. White B. Rose H. Iversen P. Mil-Homens D. Rocha E.P. Fialho A.M. Baldwin A. Dowson C. Barrell B.G. Govan J.R. Vandamme P. Hart C.A. Mahenthiralingam E. Parkhill J. J. Bacteriol. 2009; 191: 261-277Crossref PubMed Scopus (273) Google Scholar) for genes predicted to encode enzymes for the synthesis of the core OS. Unlike enteric bacteria, the B. cenocepacia core OS genes are not found within a single cluster but rather dispersed into three different locations in chromosome 1 (Fig. 1). One of these regions is located between nucleotides 2,656,960 and 2,669,740 and contains eight genes named BCAL2402 to BCAL2409. The last two genes, BCAL2409 and BCAL2408, have also been annotated as dnaE (encoding DNA polymerase C or PolC) and msbA (encoding the ATPase transported for lipid A-core oligosaccharide (6Raetz C.R. Reynolds C.M. Trent M.S. Bishop R.E. Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (976) Google Scholar)), respectively (31Holden M.T. Seth-Smith H.M. Crossman L.C. Sebaihia M. Bentley S.D. Cerdeño-Tárraga A.M. Thomson N.R. Bason N. Quail M.A. Sharp S. Cherevach I. Churcher C. Goodhead I. Hauser H. Holroyd N. Mungall K. Scott P. Walker D. White B. Rose H. Iversen P. Mil-Homens D. Rocha E.P. Fialho A.M. Baldwin A. Dowson C. Barrell B.G. Govan J.R. Vandamme P. Hart C.A. Mahenthiralingam E. Parkhill J. J. Bacteriol. 2009; 191: 261-277Crossref PubMed Scopus (273) Google Scholar). According to the established norms for nomenclature of bacterial polysaccha
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