Glycobiology Advance Access originally published online on March 12, 2008
Glycobiology 2008 18(6):447-455; doi:10.1093/glycob/cwn021
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A unique glycosyltransferase involved in the initial assembly of Moraxella catarrhalis lipooligosaccharides
2 Department of Microbiology and Immunology, State University of New York at Buffalo, Buffalo, NY, 14214, USA
3 Witebsky Center for Microbial Pathogenesis and Immunology, State University of New York at Buffalo, Buffalo, NY, 14214, USA
4 Department of Medicine, State University of New York at Buffalo, Buffalo, NY, 14214, USA
5 New York State Center of Excellence in Bioinformatics & Life Sciences, State University of New York at Buffalo, Buffalo, NY, 14214, USA
6 Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada
1 To whom correspondence should be addressed: Tel: +1-716-829-2673; Fax: +1-716-829-3889; e-mail: aac{at}buffalo.edu
Received on October 9, 2007; revised on February 29, 2008; accepted on March 2, 2008
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Abstract |
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Moraxella catarrhalis express three predominant forms of lipooligosaccharide (LOS) molecules on the bacterial surface. These major glycolipids contain specific carbohydrate epitopes that distinguish each glycoform into serotype A, B, or C LOS. All three serotypes, however, share a common glucose containing inner-core structure, consisting of an
-glucose attached to 2-keto-3-deoxyoctulosonic acid (KDO), which is unique among Gram-negative bacteria. Many of the LOS glycosyltransferase genes (lgt) responsible for assembly of the extended M. catarrhalis LOS structure have been identified. In this report, we now describe the identification and characterization of Lgt6, a unique glycosyltransferase that is responsible for the addition of the first glucose to the inner core thus initiating the assembly of full length LOS. Isogenic mutants defective in the expression of lgt6 were constructed in all three M. catarrhalis LOS serotypes and the resulting LOS glycoforms consisted of KDO2-lipid A-OH as analyzed by urea sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and mass spectrometry. In addition, the expression of lgt6 in trans in a heptose-deficient Neisseria meningitidis NMB gmhX mutant resulted in the addition of a hexose to the LOS of this strain. These studies demonstrate that Lgt6 functions as an -(1-5)-glucosyltransferase in M. catarrhalis adding the primary glucose to the KDO2-lipid A-OH in LOS biosynthesis. The function of Lgt6 is required for the completion of both the major and minor oligosaccharide chains in M. catarrhalis. Key words: glycosyltransferase / lipooligosaccharide (LOS) / Moraxella catarrhalis
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsFundingConflict of interest statementReferences |
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Moraxella catarrhalis is a frequent colonizer of the upper respiratory tract, leading to otitis media and sinusitis in children and exacerbations in patients with chronic obstructive pulmonary disease (COPD). This strict human pathogen is currently among the top three most prominent causes of middle ear disease among Gram-negative bacteria (Enright and McKenzie 1997
; Holme et al. 1999; Murphy et al. 2005). The primary reasons that M. catarrhalis continues to cause disease can be attributed to greater than 90% of the clinical isolates expressing beta-lactamase, the high frequency of recurrent disease observed in children that have recovered from infection and the lack of a vaccine (Murphy 1996; Enright and McKenzie 1997; Sethi et al. 2002; Verduin et al. 2002). Thus, while the identification of potential drug targets and vaccine antigens are clearly a priority, such research efforts require a more thorough knowledge of the bacterial components involved in the establishment of infection in the host.
There are a number of potential virulence factors identified for M. catarrhalis; however their roles in pathogenesis are not completely understood. A predominant surface structure expressed on the outer membrane of this bacterium, the lipooligosaccharide (LOS), is one of the components that have been implicated in virulence (Fomsgaard et al. 1991). LOS is composed of lipid A and a carbohydrate core structure, while it is devoid of the variable repeating O-antigen subunits characteristic of lipopolysaccharide (LPS). M. catarrhalis LOS has been divided into three major serotypes, A, B, and C, as previously defined by polyclonal antisera and structural analyses (Edebrink et al. 1994, 1995, 1996; Masoud et al. 1994). Recently, we have developed a multiplex polymerase chain reaction (PCR) method that can rapidly and specifically predict the type of LOS a M. catarrhalis strain produces and confirmed that serotypes A and B are the predominant glycoforms observed in most clinical isolates analyzed to date (Edwards, Schwingel et al. 2005). More importantly, the LOS of M. catarrhalis contains specific carbohydrate epitopes that have been linked to virulence in other Gram-negative human pathogens including Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenzae (Campagnari et al. 1990; Klein et al. 1996; Nassif 2000; Song et al. 2000; Swords et al. 2000, 2002; Gorter et al. 2003; Pridmore et al. 2003). In addition, more recent animal studies suggest that M. catarrhalis LOS has potential as a vaccine antigen (Hu et al. 2000; Jiao et al. 2002; Yu and Gu 2005). Despite these important observations, the steps involved in the biosynthesis and assembly of each M. catarrhalis LOS type remain poorly understood.
In contrast to the LOS structures assembled by many Gram-negative human pathogens, M. catarrhalis LOS does not contain heptose residues (Caroff and Karibian 2003), and the unique inner core is exclusively composed of glucose residues. Previous reports have identified a cluster of glycosyltransferase genes involved in the biosynthesis of the LOS expressed by M. catarrhalis (Edwards, Allen et al. 2005; Wilson et al. 2006; Peak et al. 2007); however, the transferase involved in the first step of LOS assembly remained elusive. In this study, we have identified and characterized Lgt6, a unique glycosyltransferase that is essential for the initiation of the oligosaccharide chain assembly in all three major M. catarrhalis LOS serotypes.
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Results and discussion |
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Identification of glycosyltransferase gene, lgt6
A search of the NCBI database identified a possible lipooligosaccharide glycosyltransferase (lgt) gene located directly downstream of a putative lipid A acyltransferase gene in the patented sequence of M. catarrhalis strain 43617 (Figure 1). In contrast to the previously identified glycosyltransferases, this putative lgt was not present within these known clusters (Edwards, Allen et al. 2005
; Edwards, Schwingel et al. 2005; Wilson et al. 2006; Peak et al. 2007), but was located in a distinct region of the chromosome. Although BLAST searches of the current NCBI database resulted in some weak similarities with putative glycosyltransferases in other bacterial genomes, there were no significant homologies to any glycosyltransferase genes or proteins with a specifically defined function. Further analyses confirmed that this lgt did not exhibit any significant homologies with M. catarrhalis genes involved in carbohydrate utilization or modification, nor with the previously described lgt1–5 (Edwards, Allen et al. 2005; Wilson et al. 2006; Peak et al. 2007); thus we designated this gene lgt6. PCR primers were designed to this putative lgt and used to amplify the predicted size product in the chromosome of M. catarrhalis strains 25238 (LOS serotype A), 103P14B1 (LOS serotype A), 7169 (LOS serotype B), and 26291 (LOS serotype C). Sequence analysis of each amplicon confirmed an open-reading frame (ORF) of 747 nt encoding a predicted protein of 248 aa. Comparative analysis of this sequence data demonstrated that lgt6 exhibited a significant degree of homology among all three LOS serotypes at both the nucleotide (98–99% similarity) and amino acid levels (98–100% identity). In addition to the computer analysis performed above, reverse transcription-polymerase chain reaction (RT-PCR) confirmed that lgt6 was transcribed in each major LOS serotype background (data not shown).
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Spectrometric analysis of Lgt 6
To characterize the function of this putative glycosyltransferase gene (lgt6), isogenic mutants were constructed in M. catarrhalis strains 25238, 103P14B1, 7169, and 26291, representing the three major LOS serotypes, including a COPD and a middle ear clinical isolate. Sequence analysis of chromosomal DNA and RT-PCR analysis of the lgt6 mutant strains confirmed that a nonpolar gene disruption had occurred (data not shown). Proteinase K lysates of wild-type LOS glycoforms were compared to the LOS assembled by each lgt6 mutant by urea sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The silver-stained gel in Figure 2 shows that the LOS isolated from each of the wild-type strains is consistent with the expected size glycoform whereas the LOS expressed by each respective lgt6 mutant exhibits a severely truncated glycoform. There were no other contaminating bands detected which was consistent with the purity of these samples as confirmed by the analyses below.
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Electrospray mass spectrometry (ES-MS) analysis confirmed that the LOS of the lgt6 mutants consisted only of KDO2-lipid A-OH (Table I), confirming that disruption of this gene prevented the addition of the first glucose of the LOS structure. For example, the serotype A parent strain, 25238, gave ions consistent with a composition of PEA, HexNAc, 7Hex, 2KDO, and lipid A-OH when the ES-MS spectrum was examined, whereas the corresponding lgt6 mutant gave ions consistent with a severely truncated LOS-OH molecule of PEA, 2KDO, and lipid A-OH. Similar observations were obtained from strains of the other serotypes (Table I).
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Restoration of the wild-type phenotype
To confirm that lgt6 was responsible for the loss of the
-(1-5)-glucose addition to the KDO2-lipid A core, the wild-type lgt6 was reintroduced into each respective mutant in cis by natural transformation. Our group has previously used this approach (Furano and Campagnari 2004; Edwards, Allen et al. 2005) because currently there are no reliable shuttle vectors that work in all strains of M. catarrhalis and we have experienced some instability problems in using the recently reported pWW115 vector (Wang and Hansen 2006). Figure 2 shows that the LOS isolated from each complemented mutant expressed a glycoform that was consistent with that of each parental LOS. While these data support the conclusion that disruption of lgt6 alone is responsible for the resulting truncated LOS phenotype expressed by the mutants, LOS was isolated from each complemented mutant and analyzed by MS (Table I) to confirm that these glycoforms were identical to those expressed by the wild-type strains. Thus, the lgt6 mutant of serotype A strain, 25238, reverted from the severely truncated LOS-OH molecule of PEA, 2KDO, and lipid A-OH to a spectrum consistent with a wild-type composition, when a functional lgt6 gene was supplied in cis. Similar observations were obtained from the complemented strains of the other mutants (Table I).
Demonstration that Lgt6 is a glycosyltransferase
While the preceding data suggested that Lgt6 functions as a glucosyltransferase, these data provide indirect support for this conclusion. To confirm that Lgt6 functioned as a glycosyltransferase, we obtained a previously described N. meningitidis NMB mutant defective in expression of gmhX. This meningococcal mutant is deficient in the heptose biosynthetic pathway, resulting in the expression of an LOS containing KDO2-lipid A-OH (Shih et al. 2001). This structure represents the hypothesized acceptor molecule for M. catarrhalis Lgt6. We expressed M. catarrhalis lgt6 on a plasmid in the N. meningitidis NMB gmhX mutant and analyzed the resulting LOS glycoforms by SDS–PAGE. Figure 3 shows that the glycoform assembled by the N. meningitidis gmhX mutant contains multiple bands, consistent with the previously published report, where the lowest glycoform (arrow A) represents KDO2-lipid A (Shih et al. 2001). More importantly, the gmhX mutant expressing lgt6 from M. catarrhalis shows a slight but definite shift to a larger glycoform (arrow B) than the LOS assembled by the N. meningitidis gmhX mutant itself. This same shift in size was not observed in a N. meningitidis gmhX mutant harboring a control plasmid that did not contain lgt6 (data not shown). These data support the conclusion that the expression of lgt6 in the N. meningitidis gmhX mutant resulted in an addition to the KDO2-lipid A-OH glycoform.
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To further confirm the nature of this addition by M. catarrhalis lgt6 to the heptose-deficient N. meningitidis mutant, ES-MS analyses were used. As detailed in Table II and illustrated in Figure 4(A), the N. meningitidis gmhX mutant gave the expected glycoform profile of 2KDO and lipid A-OH as indicated by the doubly and triply charged ions at m/z 695.82– and 463.63–, respectively. Other ions consistent with the loss of water and a hydrazide, formed during O-deacylation, were also observed. Examination of the lgt6 complemented gmhX mutant mass spectrum (Table II and Figure 4(B)) revealed ions at m/z 776.82– and 517.63– consistent with the incorporation of an additional hexose moiety, by virtue of a mass increase of 162 amu over the gmhX mutant, to give a glycoform profile of Hex, 2KDO, and lipid A-OH. Again, similar ions consistent with loss of water and hydrazide formation were observed, as in the gmhX mutant. These MS data clearly corroborate and extend the PAGE analyses (Figure 3) and the previous data in the Moraxella background to illustrate that Lgt6 is responsible for the addition of a hexose moiety to the KDO2-lipid A region.
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A thorough search of the current databases emphasizes the truly unique nature of Lgt6 and in particular the function of this glycosyltransferase in M. catarrhalis LOS assembly (Figure 5). Despite the extensive amount of literature describing numerous glycosyltransferases involved in LPS and LOS biosynthesis, we were unable to identify a single functional glucosyltransferase that exhibited significant homology with Lgt6. A recent review of bacterial lipopolysaccharide structures confirms that there are no other descriptions of enteric or nonenteric LPS core structures that express an inner core composed of glucose and KDO (Caroff and Karibian 2003
). Most of the enteric bacterial species that share a similar ecological niche as M. catarrhalis, such as nontypeable H. influenzae and N. meningitidis, express an LOS inner core containing heptose added directly to KDO. In addition, while there are numerous examples of nonenteric bacteria that express unusual LPS inner cores devoid of heptose, none of these species assemble an inner core similar to M. catarrhalis LOS (Caroff and Karibian 2003).
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Based on the data presented here we conclude that Lgt6 is an
-(1-5)-glucosyltransferase, which functions by transferring the -glucose to the KDO2-lipid A in all three major M. catarrhalis LOS serotypes (Figure 5). This represents the primary step in the assembly of full-length M. catarrhalis LOS irrespective of the specific glycoform that is expressed. In addition, the resulting Glc-KDO2-lipid A core is the acceptor substrate that is necessary for the function of the subsequent glycosyltransferase enzymes involved in the elongation of both the major and minor oligosaccharide chains. Thus, the data in this report describe the characterization of a novel glucosyltransferase involved in the assembly of the LOS core. Perhaps more importantly, the demonstration that Lgt6 functions in the essential first step in oligosaccharide chain assembly fills a previous gap in the knowledge and understanding of M. catarrhalis LOS biosynthesis. |
Materials and methods |
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TopAbstractIntroductionResults and discussionMaterials and methodsFundingConflict of interest statementReferences |
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Bacterial strains and culture conditions
The bacterial strains used in this study are described in Table III. M. catarrhalis strains were cultured on brain heart infusion (BHI) agar plates at 35.5°C in 5% CO2 or shaking aerobically in BHI broth at 37°C. Escherichia coli XL1-Blue, cultured in the Luria-Bertani (LB) medium at 35.5°C in 5% CO2, was used as the host strain for plasmid DNA manipulations. N. meningitidis strains were cultured on GC agar plates at 35.5°C in 5% CO2 or shaking aerobically in BHI broth at 37°C. Antibiotics were supplemented as necessary with 50 µg/mL kanamycin (Kan) and 3 µg/mL erythromycin (Erm) for M. catarrhalis and N. meningitidis. E. coli was selected on 100 µg/mL ampicillin (Amp), 50 µg/mL kanamycin, and 300 µg/mL erythromycin.
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Nucleic acid techniques
Standard molecular biological techniques were used as previously described (Sambrook et al. 1989
). Plasmids were isolated using QIAprep Spin Miniprep kits (QIAGEN, Chatsworth, CA). Chromosomal DNA was obtained using standard methods and DNA was purified using QIAGEN purification kits. PCR was performed using Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA). Fast-Link DNA Ligase (EPICENTRE, Madison, WI) and all enzymes obtained from New England Biolabs (Beverly, MA) were used according to company protocols. PCR amplicons were ligated into the TA cloning vector pGEM-T Easy (Promega, Madison, WI) for further modifications. DNA nucleotide sequences were obtained for all cloning and mutant constructs via automated DNA sequencing at the RPCI Biopolymer Facility (Roswell Park Cancer Institute, Buffalo, NY), and all sequences were analyzed using Sequencher, version 4.5 (Gene Codes Corporation, Ann Arbor, MI) and MacVector, version 7.2 (Accelrys, San Diego, CA), software. RNA was isolated from cells at the mid-log phase of growth using a RNeasy Mini Kit (QIAGEN) followed by RQ1 RNase-Free DNase treatment (Promega).
Construction of isogenic mutants
The PCR primers used to clone and sequence the region surrounding lgt6 are listed in Table IV. The primers were designed based on the sequence homologies between the M. catarrhalis genome patent number WO0078968 (patent located in the NCBI database under Incyte Genomics, Inc. accession number AX067448
[GenBank]
) and preliminary genomic sequence from M. catarrhalis strain 7169. An inverse PCR strategy was used to construct isogenic mutants as previously described (Ling and Robinson 1997; Furano and Campagnari 2003; Luke et al. 2003; Edwards, Allen et al. 2005). This resulted in an internal deletion of the cloned lgt6 gene with engineered restriction sites in each individual gene that allowed for the directed insertion of a nonpolar kanamycin resistance cassette (see Table III) (Menard et al. 1993). The resulting insertionally inactivated lgt6-Kan mutant constructs were amplified by PCR and purified linear amplicons were introduced into M. catarrhalis by natural transformation (Furano and Campagnari 2003; Luke et al. 2003). Kanamycin-resistant transformants were selected. The inactivated gene and the surrounding region were amplified from chromosomal DNA and sequenced to ensure proper integration of the mutant construct. RT-PCR analysis was completed using a OneStep RT-PCR Kit (QIAGEN) with primers shown in Table IV, to ensure that surrounding genes are still transcribed in the disrupted mutants.
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Restoration of the wild-type LOS phenotype
A 2.2-Kb PCR fragment (Table IV; primers 1522 and 1525) containing the full-length wild-type lgt6 was used to complement the lgt6 mutants in cis by natural transformation (Furano and Campagnari 2003
; Luke et al. 2003). Transformants that lost resistance to kanamycin were selected for further analysis.
Expression of M. catarrhalis lgt6 in N. meningitidis NMB gmhX
A complementation method, similar to that previously described, was used to express lgt6 in N. meningitidis (Tzeng et al. 2002; Gudlavalleti et al. 2004). Briefly, primers (Table IV) were used to PCR amplify the coding sequence of lgt6 from M. catarrhalis 7169. The amplicon was digested with XhoI and BglII was ligated into a similarly digested pFLAG-CTC (Sigma) and the resulting plasmid was electroporated into E. coli XL1-Blue (Stratagene). DNA sequencing was used to confirm that the lgt6 ORF was not disrupted. The pFLAG-CTC plasmid containing the proper gene insertion was then digested with DraIII, blunted with the Klenow fragment, and cloned into the EcoRV site of pYT250. The resulting plasmids were transformed into N. meningitidis NMB gmhX (Janik et al. 1976; Shih et al. 2001). Transformants were selected with kanamycin and erythromycin and screened by PCR. Transformants were grown in the presence of 500 µM isopropyl-β-D-thiogalactopyranoside to induce transcription. Plasmids were isolated from the N. meningitidis transformants and then confirmed by restriction digest and PCR.
Electrophoretic analysis of LOS
LOS was extracted from M. catarrhalis and N. meningitidis strains to access mutant phenotypes by a whole cell proteinase K procedure. Briefly, plate grown organisms were washed with sterile PBS and boiled in a lysing buffer (2% SDS, 10% glycerol, and 4% β-mercaptoethanol in 1 M Tris, pH 6.8) for 10 min, followed by an at least 2 h 60°C incubation with proteinase K at a final concentration of 0.4 mg/mL. The LOS was resolved by urea SDS–PAGE for M. catarrhalis (18%) and N. meningitidis (15%) strains and visualized by silver staining as previously described (Tsai and Frasch 1982; Inzana and Apicella 1999).
Preparation of LOS
LOS was extracted using a modified phenol–water method (Apicella et al. 1994). For mass spectrometric analyses, plate grown bacterial cells were treated with proteinase K followed by successive treatment DNase and RNase to release the LOS, which was O-deacylated in situ with anhydrous hydrazine (Li et al. 1998). This microanalytical procedure proved particularly convenient for rapid profiling of LOS glycoforms since a single plate growth provided sufficient material for subsequent analysis by ES-MS.
Glycoform profiling
Lyophylized O-deacylated LOS samples were dissolved in the ammonium acetate solution (1 M) and analyzed directly on a Crystal Model 310 CE instrument (ATI Unicam, Boston, MA, USA) coupled to a 4000 QTRAP mass spectrometer (Applied Biosystems/MDS Sciex, Canada). Separations were obtained on 90 cm length bare fused silica using 30 mM aqueous morpholine acetate, pH 9, containing 5% methanol. A voltage of –5 kV was applied at the injection end of the capillary and spectra were obtained in the negative ion mode. The calculation of molecular mass based on proposed compositions was done by using average mass units as follows: Hexose (Hex), 162.14; Heptose (Hep), 192.17; N-acetylhexosamine (HexNAc), 203.19; 2-keto-3-deoxyoctulosonic acid (KDO), 220.20; phosphoethanolamine (PEA), 123.05; and lipid A-OH, 1020 (Mc) and 952 (Nm).
Nucleotide sequence accession numbers
The sequence of the cluster of genes reported in this paper were deposited with GenBank and assigned the accession numbers EU108003
[GenBank]
, EU108004
[GenBank]
, EU108005
[GenBank]
, and EU108006.
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Funding |
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TopAbstractIntroductionResults and discussionMaterials and methodsFundingConflict of interest statementReferences |
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Public Health Service Research (AI46422 and DC005837 [GenBank] to A.A.C.).
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Conflict of interest statement |
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TopAbstractIntroductionResults and discussionMaterials and methodsFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We thank Perry Fleming for bacterial growth and Jacek Stupak for recording ESI-MS data. We would like to thank David S. Stephens, MD, for the N. meningitidis strains and corresponding plasmid. In addition, we thank Pascale Plamondon for helpful suggestions, Alan Lesse, MD, for assistance with database searches, and Michael Apicella, MD, for LOS preparation.
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Abbreviations |
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Amp, ampicillin; BHI, brain heart infusion; COPD, chronic obstructive pulmonary disease; Erm, erythromycin; ES-MS, electrospray mass spectrometry; Gal, galactose; Glc, Glucose; GlcNAc, N-acetylglucosamine; Hep, Heptose; Hex, hexose; HexNAc, N-acetylhexosamine; Kan, kanamycin; KDO, 2-keto-3-deoxyoctulosonic acid; LB, Luria-Bertani; lgt, lipooligosaccharide glycosyltransferase genes; LOS, lipooligosaccharide; LPS, lipopolysaccharide; ORFs, open-reading frames; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PEA, phosphoethanolamine; RT-PCR, reverse-transcriptase-polymerase chain reaction; SDS, sodium dodecyl sulfate
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References |
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