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Pseudomonas aeruginosa galU is required for a complete lipopolysaccharide core and repairs a secondary mutation in a PA103 (serogroup O11) wbpM mutant

Charles R. Dean, Joanna B. Goldberg
DOI: http://dx.doi.org/10.1111/j.1574-6968.2002.tb11193.x 277-283 First published online: 1 May 2002


Insertional inactivation of wbpM in Pseudomonas aeruginosa serogroup O11 strain PA103 resulted in mutants exhibiting three distinct lipopolysaccharide (LPS) phenotypes. One mutant, PA103 wbpM-C, had a truncated LPS core and lacked O antigen. These defects were not complemented by the cloned wbpM gene, suggesting a secondary mutation was present. When the wild-type galU gene was introduced into strain PA103 wbpM-C containing the cloned wbpM gene, both LPS defects were corrected. Construction of galU mutants in P. aeruginosa serogroups O11, O5, O6 and O17 strains led to truncation of the LPS core, indicating the involvement of GalU in P. aeruginosa LPS core synthesis.

Key words
  • Lipopolysaccharide
  • O antigen
  • Core oligosaccharide
  • Pseudomonas

1 Introduction

As with other Gram-negative bacteria, lipopolysaccharide (LPS) is a major virulence factor for the opportunistic pathogen Pseudomonas aeruginosa. It is composed of O antigen, the LPS core oligosaccharide, and lipid A. In P. aeruginosa there are 20 different serogroups that differ in their monosaccharide composition and/or linkages between sugar residues in the O antigen[1]. While P. aeruginosa O-antigen loci vary significantly, reflecting the diversity of polysaccharide structures present in different serogroup O antigens, one gene, wbpM, is conserved in all 20 P. aeruginosa serogroup strains [2,3]. Construction of wbpM mutants in serogroups O5, O3, O10 abolished production of O antigen, confirming the role of WbpM in O-antigen synthesis [2,4].

Recently, using purified WbpM, Creuzenet and Lam[5] determined that wbpM encodes a UDP–N-acetyl-d-glucosamine C6 dehydratase/C4 reductase that converts UDP–N-acetyl-d-glucosamine to UDP–N-acetyl-d-quinovosamine. This group originally proposed that WbpM was an epimerase[2] or a dehydratase/reductase[6] responsible for the synthesis of UDP–N-acetyl-d-fucosamine or UDP–N-acetyl-d-quinovosamine from UDP–N-acetyl-d-glucosamine. The conservation of wbpM was suggested to reflect the presence of fucosamine or quinovosamine in the majority of P. aeruginosa O antigens[2]. Mutational analysis of wbpM in serogroups O15 and O17, which have O antigens lacking these sugars, supported this idea; wbpM mutants of these serogroups make O antigen[4]. In our analysis of serogroup O17 wbpM mutants, we observed strains with two different LPS phenotypes: some contained O antigen showing a phenotype similar to that described by Burrows et al.[4] whereas others were O-antigen-deficient[7]. This result calls into question the exact role for wbpM in the synthesis of serogroup O17 O antigen. Moreover, it suggests that, in some cases, mutation of wbpM can lead to secondary mutations.

To continue the analysis of the role of WbpM in the synthesis of P. aeruginosa LPS, we inactivated the chromosomal wbpM gene in the serogroup O11 strain PA103. The serogroup O11 O antigen is one of the simplest and has the repeating unit structure [–2)-β-d-glucose-(1–3)-α-l-N-acetylfucosamine-(1–3)-β-d-N-acetylfucosamine-(1–][8]. In this background, strains with varying LPS phenotypes were again observed. In this case, defects were noted in the LPS core. This report identifies the galU gene as compensating for one of these secondary mutations.

2 Materials and methods

2.1 Bacterial strains, plasmids, and growth media

The bacterial strains and plasmids used in this study are listed in Table 1. Luria broth or agar was used for routine growth of bacteria. Media contained ampicillin (50–100 μg ml−1), carbenicillin (250 μg ml−1), gentamicin (15 μg ml−1 for Escherichia coli; 250 μg ml−1 for P. aeruginosa), tetracycline (10 μg ml−1 for E. coli; 50–100 μg ml−1 for P. aeruginosa), and sucrose (5% w/v) as needed. Bacteria were routinely grown at 37°C.

View this table:
Table 1

Bacterial strains and plasmids used

Strain or plasmidGenotype or relevant characteristicsaReference or
P. aeruginosa
PA103IATS serogroup O11[18]
PAO1IATS serogroup O5[19]
PAKIATS serogroup O6[20]
IATSO17IATS serogroup O17T.L. Pitt (Central Public Health Laboratory, London, UK)
PA103 wbpM-AwbpM::aacC1; O-antigen-deficient, complementable by wbpM in transThis study
PA103 wbpM-BwbpM::aacC1; O-antigen-deficient, altered LPS core, O-antigen defect complementable by wbpM in transThis study
PA103 wbpM-CwbpM::aacC1; O-antigen-deficient, truncated LPS core, neither defect complementable by wbpM in transThis study
PA103 galUgalU::aacC1This study
PA103 ugdugd::aacC1This study
PAO1 galUgalU::aacC1This study
PAO1 ugdugd::aacC1This study
PAK galUgalU::aacC1This study
PAK ugdugd::aacC1This study
IATSO17 galUgalU::aacC1This study
IATSO17 ugdugd::aacC1This study
E. coli
SM10thi-1 thr leu tonA lacY supE recA RP4-2-Tc::Mu, Kmr[21]
HB101Triparental mating[22]
pEX100TMobilizable, counterselectable gene replacement vector; Apr/Cbr[23]
pUCGMSource of Gmr cassette, aacC1; Apr, Gmr[24]
pUCP18E. coli/P. aeruginosa shuttle vector; Apr/Cbr[25]
pCD203pUCP18 harboring a 3.5-kb Eco RI–Mlu I fragment encompassing wbpM from PA103[7]
pHP45Ω-TcSource of Ω-Tc interposon[26]
pUCP18Ω-TcpUCP18 with an approximately 2.1-kb Sma I Ω-Tc interposon fragment derived from pHP45-Ω-Tc inserted into the Sca I site within the bla gene; TcrThis study
pCD204pUCP18Ω-Tc harboring a 1036-bp PCR fragment encompassing galU from PA103 in the same orientation as the lac promoterThis study
pCD205pUCP18Ω-Tc harboring a 1590-bp PCR fragment encompassing ugd from PA103 in the same orientation as the lac promoterThis study
pRK2013Helper plasmid used in triparental mating; Kmr[27]
pLAFR1Broad host range cosmid; Tcr[28]
pLPS2pLAFR1 containing an approximately 26-kb DNA fragment from PA103 encompassing the serogroup O11 O-antigen locus[13]
pCD206pLAFR1 containing an approximately 33-kb PA103 chromosomal DNA insert encompassing ugd and galU; restores wild-type LPS production in PA103 wbpM-C (pCD203)This study
  • aIATS, International Antigenic Typing Scheme[29]; Apr, ampicillin resistant; Cbr, carbenicillin resistant; Gmr, gentamicin resistant; Tcr, tetracycline resistant; Kmr, kanamycin resistant.

2.2 DNA manipulations

Plasmid and genomic DNA isolation, restriction endonuclease digestions and modifications, agarose gel electrophoresis, DNA fragment isolation, Southern blot analysis, and polymerase chain reaction (PCR) were performed as previously described[3]. Oligonucleotide primers used in PCR and nucleotide sequence determinations were purchased from the Great American Gene Company or Ransom Hill (Ramona, CA, USA). Nucleotide sequencing was performed by the University of Virginia Biomolecular Research Facility. Sequence data were analyzed using Sequencher (Gene Codes Corp., Ann Arbor, MI, USA) and were compared to the sequence in the Pseudomonas Genome Project (http://www.pseudomonas.com) and using the BLAST functions of the interactive P. aeruginosa genome database (http://www.pseudomonas.bit.uq.edu.au).

Plasmids were introduced into E. coli and P. aeruginosa as described[3]. Cosmids were introduced into P. aeruginosa by triparental mating using the helper plasmid pRK2013 as described[9].

2.3 Cloning, in vitro mutagenesis, and gene replacement

The PA103 wbpM gene was inactivated on the chromosome by insertion of a gentamicin resistance cartridge (aacC1), using a previously described gene replacement strategy[7]. Gene replacement was confirmed by PCR amplification from chromosomal DNA or colonies using primers flanking the site of aacC1 insertion (Fig. 1). Southern blot analysis of wbpM::aacC1 mutants was also carried out; a 704-bp PCR fragment encompassing the 5′-half of wbpM (Fig. 1) was used to probe Eco RI/Sal I-digested genomic DNA from PA103 wild-type and wbpM::aacC1 mutated strains.

Figure 1

Genetic organization of P. aeruginosa serogroup O11 strain PA103 O-antigen locus. The genes are shown as arrows below the restriction site map. The site of insertion of the gentamicin resistance (Gm) determinant in wbpM is shown. Region 1 (hatched) represents the region of the wzxPaO11 gene PCR-amplified to confirm the serogroup O11 locus. Region 2 represents the region PCR-amplified to confirm the wbpM insertion. Region 3 represents the region used as a probe for Southern blot hybridization. The insert DNA containing the wbpM gene in the recombinant plasmid pCD203 is shown below. Restriction enzyme sites shown are for E, Eco R1; EV, Eco RV; M, Mlu 1; S, Sal 1; and X, Xho 1.

The ugd and galU genes were PCR-amplified from the complementing cosmid, pCD206, and subcloned into the vector pUCP18Ω-Tc to give pCD205 and pCD204, respectively. This vector is a derivative of pUCP18 with the ampicillin resistance (bla) gene inactivated by insertion of a tetracycline resistance cartridge.

To construct a non-polar mutation of galU, a 1036-bp PCR fragment encompassing galU was generated from cosmid pCD206 using Vent DNA polymerase and ligated into Sma I-digested pEX100T. This plasmid was linearized at the unique Eco RV site within galU and ligated to aacC1, isolated as an 855-bp Sma I fragment from pUCGM. The resultant plasmid with the gentamicin resistance cartridge in the same orientation as galU was used to generate knockouts on the chromosomes of strains PA103 (serogroup O11), PAO1 (serogroup O5), PAK (serogroup O6) and IATSO17 (serogroup O17) as described[7]. A non-polar knockout of ugd was constructed by cloning ugd, as a 1590-bp PCR fragment from cosmid pCD206 using Vent DNA polymerase, into Sma I-digested pEX100T. This construct was digested with Asp 718I, liberating a 23-bp fragment which was replaced with the aacC1 cartridge, isolated as an 847-bp Asp 718I fragment from pUCGM. A construct having aacC1 in the same orientation as ugd was used to generate chromosomal ugd knockouts in strains PA103, PAO1, PAK and IATSO17, as described above. The insertions in galU and ugd were confirmed by PCR amplification from chromosomal DNA or colonies, using primers flanking the aacC1 insertion sites.

2.4 Isolation of LPS

P. aeruginosa LPS was isolated by the proteinase K digestion protocol[10], or by this protocol followed by hot phenol extraction with modifications, as previously described[3].

2.5 SDS–PAGE, Western immunoblotting, and colony immunoblot

LPS samples were separated on 10% NuPAGE bis-Tris–polyacrylamide gels (Novex, Los Angeles, CA, USA) and visualized by silver staining with the Bio-Rad silver-staining kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the directions provided. LPS separated by SDS–PAGE was transferred to NitroBind nitrocellulose membrane (Micron Separations Inc., Westboro, MA, USA) for Western immunoblotting, using the Trans-Blot Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions. For colony immunoblotting, colonies were picked and patched onto agar plates. After overnight growth, these colonies were transferred to nitrocellulose circles (Osmonics, Inc., Phoenix, AZ, USA) and baked at 80°C for 30 min. The filters were then extensively washed with PBS/0.05% Tween 20 to remove visible cellular material. Immunoblots were performed as described[11]. Monoclonal antibodies (mAb) specific to P. aeruginosa serogroup O11 (Rougier Bio-Tech Ltd, Montreal, QC, Canada) were used for colony blots. Polyclonal antisera specific to P. aeruginosa serogroups O11 and O5[12] were used for Western immunoblots. The blots were developed at room temperature with anti-[mouse IgM]–alkaline phosphatase (Boehringer Mannheim Corp., Indianapolis, IN, USA) for mAb or anti-[rabbit IgG]–alkaline phosphatase (Sigma Chemical Co., St. Louis, MO, USA) for polyclonal antisera, respectively. Sigma Fast 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets (Sigma Chemical Co., St. Louis, MO, USA) were used as the alkaline phosphatase substrate according to the instructions provided.

3 Results and discussion

3.1 Phenotype of PA103 wbpM mutants

To investigate the role of wbpM in synthesis of the serogroup O11 O antigen, a gentamicin resistance cassette (aacC1) was used to disrupt wbpM on the chromosome of strain PA103 (Fig. 1). Mutant strains exhibiting three distinct colony morphologies were isolated. Compared to the wild-type colonies, PA103 wbpM-A was similar in size and appearance, PA103 wbpM-B appeared smaller and more compact, and PA103 wbpM-C appeared drier with rough edges. Since these mutant strains had different morphologies, PCR was carried out using primers corresponding to the serogroup O11-specific gene wzxPaO11[3], to confirm that each mutant was indeed a PA103 derivative. In each case an intense PCR band of the correct predicted size was obtained (data not shown). To verify insertion of aacC1 into wbpM in each mutant, PCR using primers flanking the aacC1 insertion site in wbpM and Southern blot analysis were performed. All three mutant strains gave the anticipated results for both procedures (data not shown). For PCR, a band of 322 bp was amplified from the chromosome of PA103, and was increased in the mutant strains by an amount consistent with the size of aacC1. By Southern blot hybridization, the 2333-bp probe-reactive Eco RI–Sal I fragment derived from PA103 was similarly lengthened in the mutants by an amount consistent with the size of aacC1.

LPS isolated from all three mutants lacked the characteristic O-antigen ladder observed for the parent strain (Fig. 2), consistent with the proposed role of WbpM in O-antigen synthesis. In addition, LPS from PA103 wbpM-B and PA103 wbpM-C displayed alterations of the LPS core. The LPS core of PA103 wbpM-B (Fig. 2, lane 4) appears on silver-stained PAGE as a doublet with two bands of slightly smaller size than the single core band visualized from the parent strain, indicating that the LPS core is likely truncated. Mutant PA103 wbpM-C (Fig. 2, lane 6) exhibited an LPS core band migrating faster than that of the wild-type strain, suggesting that significant truncation of the LPS core structure had occurred.

Figure 2

LPS phenotype of PA103 wbpM mutants. LPS was separated by SDS–PAGE and visualized by silver staining. Migration of the O antigen and the LPS core are shown. Lane 1, PA103; lane 2, PA103 wbpM-A; lane 3, PA103 wbpM-A (pCD203); lane 4, PA103 wbpM-B; lane 5, PA103 wbpM-B (pCD203); lane 6, PA103 wbpM-C; and lane 7, PA103 wbpM-C (pLPS2).

3.2 Complementation patterns of PA103 wbpM mutants

Plasmid pCD203 (Fig. 1), containing wbpM, complemented the O-antigen defect of PA103 wbpM-A and PA103 wbpM-B (Fig. 2, lanes 3 and 5). The LPS core defect of PA103 wbpM-B was not complemented by the cloned wbpM gene (Fig. 2, lane 5). Finding that plasmid-borne wbpM complements the O-antigen defect in PA103 wbpM-B despite an inability to complete the LPS core implies that the remaining genes of the serogroup O11 O-antigen gene cluster are likely functional and suggests that the secondary mutation affecting LPS core synthesis probably lies outside the O-antigen locus. Work is underway in our laboratory to determine the nature of the secondary mutation in PA103 wbpM-B.

In the case of PA103 wbpM-C, plasmid pCD203 did not restore either a normal-sized LPS core or O antigen (data not shown), indicating the presence of a secondary mutation outside wbpM. In fact, the LPS defect in this strain was not complemented by plasmid pLPS2 (Fig. 2, lane 7), which contains the entire serogroup O11 O-antigen biosynthetic locus[13], including the wbpM gene[3]. The inability of plasmid pLPS2 to complement either the O-antigen or LPS core defect in PA103 wbpM-C confirmed that the secondary mutation in PA103 wbpM-C resided outside the O-antigen locus.

3.3 Identification of the secondary mutation in PA103 wbpM-C

The inability of strain PA103 wbpM-C containing plasmid-borne wbpM to express O antigen was exploited to screen a PA103 cosmid gene bank[13] for chromosomal DNA fragments that, together with pCD203, restored O-antigen expression. After mobilization of the PA103 gene bank into PA103 wbpM-C (pCD203), eight transconjugants were identified expressing O antigen as detected by colony immunoblot using serogroup O11 O-antigen-specific mAb. Silver-stained PAGE analysis of LPS revealed a banding pattern consistent with the expression of normal O antigen and a complete LPS core in these strains (Fig. 3).

Figure 3

Complementation of the LPS defect of PA103 wbpM-C(pCD203) by eight cosmids. LPS was extracted and visualized by silver staining after SDS–PAGE. Lanes 1 and 10, PA103 wbpM-C(pCD203); lanes 2–9, PA103 wbpM-C (pCD203) containing cosmids pCD206–pCD213.

Comparison of nucleotide sequences from the ends of the insert DNA from eight complementing cosmids to the recently determined P. aeruginosa PAO1 genome sequence (http://www.pseudomonas.com) revealed that they all overlapped the same region of the genome. The ends of cosmid pCD206 corresponded to the open reading frames (ORFs) PA2012 and PA2041 (Fig. 4), which are approximately 31 kb apart on the PAO1 chromosome, consistent with the estimated size of the insert DNA. Thus, the PA103 DNA in this cosmid likely spanned this same region. Two adjacent ORFs within this insert DNA were candidates for involvement in LPS biosynthesis due to their presumed role in sugar metabolism: the genes ugd (PA2022; encoding UDP–glucose 6-dehydrogenase) and galU (PA2023; encoding UDP–glucose pyrophosphorylase). While ugd and galU are transcribed in the same direction, the genes adjacent to them are transcribed in the opposite direction indicating that these genes are not part of a larger operon.

Figure 4

Genetic map of the insert DNA from cosmid pCD206. Ends of the insert corresponded to PA2012 and PA2041 on the PAO1 chromosome (http://www.pseudomonas.com). Within this region were PA2022 and PA2023, corresponding to ugd and galU, respectively. Recombinant plasmids containing each of these genes referred to as pCD205 and pCD204 are shown below. Each of these genes was insertionally inactivated by the gentamicin resistance (Gm) cartridge as described in Section 2. Restriction sites are abbreviated as follows: A, Asp 718I; and EV, Eco RV.

Transformation of PA103 wbpM-C with wbpM (on pCD203) with ugd or galU (on pCD205 or pCD204, respectively) was performed. Co-transformants were then assessed for expression of serogroup O11 O antigen. Neither the cloning vector nor the ugd gene restored serogroup O11 expression. The galU-containing clone on the plasmid pCD204 did complement the LPS defect, although O antigen was made at reduced levels compared to the wild-type strain. When the galU gene was transferred to PA103 wbpM-C in the absence of wbpM on plasmid pCD203, O antigen was not made, but the LPS core was increased in size, although some truncated LPS core still remained (data not shown). In both cases, we noted that not all transformants were complemented by the cloned DNA, suggesting problems associated with expressing galU on a high copy number plasmid or spontaneous mutation of the cloned galU gene. This effect is currently under investigation.

Complementation of the LPS defects of PA103 wbpM-C, with and without the cloned wbpM gene, with the galU gene supports the notion that galU is the site of the secondary mutation. Determination of the nucleotide sequence of the galU gene from PA103 wbpM-C revealed a six-nucleotide deletion, resulting in a missense mutation at position 52 (glycine to valine) and the deletion of amino acids isoleucine and valine at positions 53–54. The valine at position 54 is conserved among all related GalU proteins[14].

3.4 Inactivation of galU results in LPS core truncation

We next confirmed that loss of galU alone in PA103 would confer the same truncation of LPS core as observed in PA103 wbpM-C. Indeed, non-polar inactivation of galU in PA103 resulted in production of LPS having a truncated core of identical size to that of PA103 wbpM-C (Fig. 5). The size of the LPS cores from PA103 galU and PA103 wbpM-C were the same as that isolated from a PAO1 algC::tet mutant (data not shown). AlgC has phosphoglucomutase activity that interconverts glucose 6-phosphate and glucose 1-phosphate[11]. Glucose 1-phosphate and UTP are the substrates for UDP–glucose pyrophosphorylase (encoded by galU) in the formation UDP–glucose. Therefore, AlgC and GalU are critical for the production of UDP–glucose, which is one of the precursors required for LPS core oligosaccharide biosynthesis.

Figure 5

LPS phenotype of galU mutants. A: Western immunoblot using polyclonal antisera to O11 (lanes 1–4) and O5 (lanes 5–7). B: Silver-stained SDS–PAGE. Lane 1, PA103; lane 2, PA103 galU; lane 3, PA103 wbpM-C; lane 4, PA103 galU (pCD204); lane 5, PAO1; lane 6, PAO1 galU; and lane 7, PAO1 galU (pCD204).

Construction of a galU mutation in PAO1 (serogroup O5) (Fig. 5), PAK (serogroup O6) and IATSO17 (serogroup O17) (data not shown) resulted in LPS core truncation, indicating that the galU function in P. aeruginosa LPS core synthesis is likely universal. Complementation with plasmid-borne galU gene on pCD204 did not completely eliminate the truncation of the LPS core, but did restore both O-antigen expression and completion of a portion of the LPS core, confirming the non-polarity of the galU knockout (Fig. 5).

3.5 Ugd is not involved in LPS synthesis

Although the ugd gene did not complement the secondary mutation in PA103 wbpM-C, it was unknown whether it played a role in LPS biosynthesis in P. aeruginosa. A non-polar insertion in the ugd gene had no discernible effect on LPS synthesis in strains PA103, PAO1, PAK or IATSO17, as detected by silver-stained SDS–PAGE (data not shown).

3.6 Inactivation of wbpM in other strains

In PAO1, construction of the wbpM mutant led to the isolation of colonies exhibiting two different morphologies, small and large. The LPS isolated from both colony types were devoid of O antigen and had seemingly normal-sized LPS cores and both could be complemented with wbpM from strain PA103 (data not shown). These results are consistent with the finding of Burrows et al.[2] and suggest that the pressure giving rise to secondary mutations affecting LPS core synthesis observed in PA103 may not be present in PAO1. In PAK, construction of wbpM mutants also led to isolation of small and large colonies. LPS isolated from both strains had apparently normal-sized LPS cores but only the LPS core from the small PAK-derived colonies reacted with LPS core-specific polyclonal antisera, implying that some alteration of the LPS core structure had occurred in the larger colonies. The O-antigen defect of the small colony could be complemented with wbpM isolated from PA103, while attempts to complement the LPS defect in the larger colonies with wbpM have been unsuccessful. Thus, the development of secondary mutations in a wbpM background resulting in an LPS core defect can be extended to at least one other serogroup. Whether a similar effect on the LPS core was observed in the serogroup O6 wbpM mutant constructed by Belanger et al.[15] was not reported. Also, how these secondary mutations affecting the LPS core relate to the situation in serogroup O17 is not known. In this latter case, secondary mutations can apparently compensate for the wbpM defect and lead to re-expression of the serogroup O17 O antigen[7].

The mechanism resulting in the emergence of secondary mutations occurring in the wbpM background is not yet known. We speculate that the product of WbpM (UDP–N-acetyl-d-fucosamine or UDP–N-acetyl-d-quinovosamine) acts a precursor for a complete and functional LPS core. Perhaps in PA103, and possibly strains of other serogroups, LPS lacking this structure is detrimental to the bacterial cell. Alternatively, the presence of the terminal glucose in the LPS core could be deleterious. In some cases, a secondary mutation develops and stabilizes the LPS core at its native size. We believe that PA103 wbpM-A represents such an isolate. In other cases, stabilization results from truncation of the LPS core. The LPS core of PA103 wbpM-B is truncated, but can still act as an acceptor for the O antigen; when PA103 wbpM-B contained the cloned wbpM gene, O antigen was restored although the LPS core was not completed. We believe that mutations in other genes can result in truncation to a form that can not act as an acceptor for O antigen, and that PA103 wbpM-C represents such a case. The LPS core is truncated in PA103 wbpM-C (by a mutation in galU), and this does not permit O antigen to be attached even when the complete O-antigen gene cluster is present on pLPS2, which is capable of directing the synthesis of serogroup O11 O antigen in E. coli, Salmonella sp. and numerous P. aeruginosa strains [13,16,17]. The chemical composition and structural analysis of the LPS these wbpM mutants is currently under investigation and should confirm the effects of the secondary mutations and suggest how they emerge.


This research was supported by research grants from the NIH (R01 AI37632) and Cystic Fibrosis Foundation (GOLDBE00G0) to J.B.G. We thank Michael J. Noto and Amy Staab for excellent technical assistance and Robert J. Kadner for helpful discussions.


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