Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide

Myriam Bélanger1, Lori L. Burrowsa,1 and Joseph S. Lam1

Department of Microbiology, University of Guelph, Guelph, Ontario , Canada N1G 2W11

Author for correspondence: Joseph S. Lam. Tel: +1 519 824 4120 Ext. 3823. Fax: +1 519 837 1802. e-mail: jlam{at}uoguelph.ca


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This study reports the organization of the wbp gene cluster and characterization of a number of genes that are essential for B-band O antigen biosynthesis in the clinically prevalent Pseudomonas aeruginosa serotype O6. Twelve genes were identified that share homology with other LPS and polysaccharide biosynthetic genes. This cluster contains homologues of wzx (encoding the O antigen flippase/translocase) and wzz (which modulates O antigen chain length distribution) genes, typical of a wzy- dependent pathway. However, a complete wzy gene (encoding the O-polymerase) was not found within the cluster. Four biosynthetic genes, wbpO, wbpP, wbpV and wbpM, and four putative glycosyltransferase genes, wbpR, wbpT , wbpU and wbpL, were identified in the cluster. To characterize their roles in LPS biosynthesis, null mutants of wbpO, wbpP, wbpV, wbpL and wbpM were generated using a gene-replacement strategy. Mutations in each of these genes caused deficiency in B-band synthesis. The wbpL mutant was deficient in both A-band and B-band LPS. WbpLO6 is a bi-functional enzyme which could initiate B-band synthesis through the addition of QuiNAc to undecaprenol phosphate, and A-band synthesis by transferring either a GalNAc or a GlcNAc residue. Another approach used to assign function to the wbpO6 genes was by complementation analysis. Two genes from Salmonella typhi, wcdA and wcdB, responsible for the synthesis of a homopolymer of GalNAcA called Vi antigen were used in complementation experiments to verify the functions of wbpO and wbpP. wcdA and wcdB restored B-band synthesis in wbpO and wbpP mutants respectively, implying that wbpO and wbpP are involved in UDP-GalNAcA synthesis. Although wbpV has homology to wbpK of the serotype O5 B-band LPS synthesis cluster, complementation analysis using the respective null mutants showed that the genes are not interchangeable. A knockout mutation of wbpN (located downstream of wbpM) did not abrogate LPS synthesis in either O5 or O6; therefore, it has been renamed orf48.5. These results establish the organization of genes involved in P. aeruginosa B-band O antigen synthesis and provide the evidence to assign functions to a number of LPS biosynthetic genes.

Keywords: Pseudomonas aeruginosa, lipopolysaccharide, rfb, wbp, O antigen

Abbreviations: GlcNAc, N-acetylglucosamine; GalNAc, N- acetylgalactosamine; GalNAcA, 2-deoxy-2-N- acetylgalactosaminuronic acid; GalNAcAN, 2-acetamido-2-deoxy-D- galacturonic acid (N-acetyl-D-galactosaminuronic acid); GalNFmA, 2-deoxy-2-formamido-D-galacturonic acid (N- formylgalactosaminuronic acid); QuiNAc, 2-acetamido-2,6- dideoxy-D-glucose (N-acetylquinovosamine); Rha, rhamnose; Fuc2NAc, 2-acetamido-2,6-dideoxy-D-galactose (6- deoxy-N-acetylgalactosamine)

The GenBank accession number for the sequence reported in this paper is AF035937.

a Present address: Center for Infection and Biomaterials Research, Toronto General Hospital, 200 Elizabeth St, Toronto, ON, Canada M5G 2C4.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pseudomonas aeruginosa, an opportunistic pathogen, is a major cause of morbidity and mortality in patients with compromised respiratory function, including those with cystic fibrosis. The lungs of cystic fibrosis patients are highly susceptible to colonization by P. aeruginosa, and the ensuing cycles of infection and inflammation damage lung tissue. Several virulence factors have been identified in the pathogenesis of P. aeruginosa infections, including exopolysaccharides such as alginate and LPS (Cryz et al. , 1984 ; May et al., 1991 ). Significantly, O6 is the most prevalent serotype among all clinical P. aeruginosa isolates (Bert & Lambert-Zechovsky, 1996 ; Pitt, 1988 ; Vachee et al., 1997 ). Smooth LPS from the International Antigenic Typing System (IATS) serotype O6 has been shown to be the predominant protective component in the commercially available Polyvalent Extract Vaccine (PEV, Wellcome Biotechnology) (MacIntyre et al., 1986 ). However, despite its importance, little is known regarding the biosynthesis and underlying genetic organization of P. aeruginosa O6 LPS.

P. aeruginosa can coexpress two distinct forms of LPS: A- band and B-band. A-band LPS, the common antigen, consists primarily of a trisaccharide repeating unit of D-Rha (Arsenault et al., 1991 ). To date, 20 immunologically distinct serotypes of P. aeruginosa (Liu et al., 1983 ; Liu & Wang, 1990 ) have been identified. The serotype-specific antigen is B-band LPS, the O antigen of which is a heteropolymer composed of di-to-pentasaccharide repeats (Knirel & Kochetkov, 1994 ). Structural studies (Knirel, 1990 ; Knirel & Kochetkov, 1994 ) indicated that the O antigen of IATS serotype O6 is a repeating linear tetrasaccharide with the structure shown in Fig. 1



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Fig. 1. Structure of the O antigen of IATS serotype O6.

 
Our laboratory previously described the cloning and characterization of the complete wbpO5 (new nomenclature based on Reeves et al., 1996 ) cluster of 16 genes involved in synthesis of P. aeruginosa PAO1 (serotype O5) O antigen (Lightfoot & Lam, 1993 ; Burrows et al., 1996 ). The cloning and partial sequence of a cluster involved in biosynthesis of B-band in P. aeruginosa serotype O11 was previously reported (Goldberg et al., 1992 ; Goldberg & Pier, 1996 ) and the complete sequencing of the O11 O antigen cluster has recently been completed (Dean et al. , 1999 ). Analysis of B-band O antigen synthesis in the serotype O5 strain showed that assembly proceeds via the Wzy-dependent pathway characteristic of heteropolymers (Burrows et al., 1996 ; de Kievit et al., 1995 ). The individual O-repeat units are assembled through the activities of specific glycosyltransferases at the cytoplasmic face of the inner membrane. O antigen units are translocated to the periplasm by the action of Wzx (Burrows & Lam, 1999 ) and then polymerized by Wzy (de Kievit et al., 1995 ). wzz, which is the first gene in the wbpO5 cluster (Burrows et al. , 1997 ), controls the modal/chain-length distribution of O antigen polymer. In the work described here, we isolated and characterized the O antigen biosynthetic cluster of serotype O6, and demonstrated that the gene encoding Wzy is not present in this cluster. We present evidence that WbpO and WbpP proteins are required for synthesis of UDP- GalNAcA and the formation of O6 B-band O antigen. We also demonstrate the requirement for wbpM and wbpV in LPS biosynthesis. Our results suggest that UDP-QuiNAc is the first sugar residue to be transferred to undecaprenol phosphate by the glycosyltransferase WbpL. Based on these results, a putative biosynthetic pathway for O6 B-band LPS synthesis is also presented.

Parts of this study were presented at the 12th Annual North American Cystic Fibrosis Conference (Bélanger & Lam, 1998 ).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. Bacterial strains were routinely propagated in Luria broth (Gibco-BRL) or on agar plates at 37 °C. Pseudomonas Isolation Agar (PIA; Difco) was used for selecting transconjugants following mating experiments. The following antibiotics (Sigma) were used in selective media at the indicated concentrations: ampicillin at 100 µg ml-1 for Escherichia coli; carbenicillin at 500 µg ml-1 or 650 µg ml-1 for P. aeruginosa O5 or O6, respectively, gentamicin at 15 µg ml-1 for E. coli and at 300 µg ml-1 or 200 µg ml-1 for P. aeruginosa O5 or O6, respectively; and tetracycline at 15 µg ml-1 and 90 µg ml-1 for E. coli and P. aeruginosa, respectively. IPTG (20 mM) and X-Gal (40 µg ml-1) (Gibco-BRL) were added to solid media to detect the loss of lacZ {alpha}-complementation in cloning experiments utilizing appropriate vector–host combinations.


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Table 1. Bacterial strains and plasmids

 
DNA procedures
DNA preparation.
Chromosomal DNA was isolated from P. aeruginosa using the method of Goldberg & Ohman (1984) . Small-scale plasmid DNA preparations were obtained by the alkaline lysis method of Birnboim & Doly (1979) , whereas plasmid DNA of sequencing quality was prepared with the QIAprep Spin Miniprep kit (Qiagen). Restriction enzymes were purchased from Gibco-BRL, Boehringer Mannheim and Pharmacia. Klenow enzyme and alkaline phosphatase were purchased from Boehringer Mannheim. T4 DNA ligase was from Gibco-BRL. All enzymes were used according to the suppliers’ specifications.

DNA transformation.
Plasmids were introduced into E. coli by CaCl2 transformation (Huff et al., 1990 ) and into P. aeruginosa by MgCl2 transformation (Berry & Kropinski, 1986 ). Plasmids were also mobilized into P. aeruginosa by biparental mating with E. coli SM10 carrying the plasmid of interest (Simon et al., 1983 ).

Southern and colony blot analysis.
For Southern blot analysis, restriction-enzyme-digested DNA was separated on agarose gels and transferred to Zeta-probe membranes (Bio- Rad) by the method of Ford et al. (1989) . For colony-blot analysis, bacteria were grown overnight on agar plates, transferred onto Zeta-probe membrane, and prepared as described by Sambrook et al. (1989) prior to hybridization. Hybridizations were carried out overnight at 42 °C under high- stringency conditions with a solution of 5xSSC, 2% blocking reagent (Boehringer Mannheim), 50% formamide, 0·1% N- lauroylsarcosine and 0·02% SDS. The washes were done twice for 5 min at room temperature in 2xSSC/0·1% SDS and twice for 12 min at 68 °C in 0·1% SSC/0·1% SDS. PCR amplification was used to generate gene-specific probes from various genes in the partial O6 LPS cluster. DNA fragments for gene probes were purified as described below and labelled with dUTP conjugated to digoxigenin (Boehringer Mannheim). Subsequent washes, incubations and detection steps were carried out according to the manufacturer’s recommendations (Boehringer Mannheim).

PCR amplification and cloning.
PCR amplification was carried out on a GeneAmp PCR System 2400 (Perkin-Elmer) using 50 pmol of each primer (Table 2 ), 50 ng of DNA template per 100 µl reaction and Taq DNA polymerase (Gibco-BRL) according to the manufacturer’s instructions. Following amplification, the PCR products (Table 2) corresponding to wbpO, wbpP, wbpR, wbpS, wbpT, wbpU, wbpL, wzz and the wbpQwzx region were purified using the High Pure PCR Product Purification Kit (Boehringer Mannheim), and DIG-dUTP-labelled.


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Table 2. Oligonucleotide primers used in PCR amplification for the generation of gene probes and insert for cloning

 
Since wbpMO6 was truncated near the 3' end during cloning of pFV600-26, a complete version obtained from strain IATS O6, which was subsequently used for sequencing, and for complementation experiments, was generated using a PCR approach. The downstream primer was based on the corresponding sequence from O5, since this region was previously shown to be conserved between serotypes O5 and O6 (Burrows et al., 1996 ). The high-fidelity thermal polymerase PwoI (Boehringer Mannheim) was used to amplify a functional product. The 2·65 kb PCR product was purified using the High Pure PCR Product Purification Kit, digested with BspEI, and the BspEI fragment was gel purified using the GENECLEAN II kit (Bio/Can Scientifica). The 2·10 kb BspEI fragment was cloned into the Ava I site of the vector pUCP26 (pFV610-26) for complementation.

The remaining portion of the cluster was obtained by using the Expand Long Template PCR System (Boehringer Mannheim) to amplify a 10·9 kb product from the IATS O6 strain. This product was subsequently digested with BamHI and the three resulting fragments were subcloned into pBluescript II SK and named pFV612, pFV613 and pFV614.

DNA sequencing and analysis.
The DNA sequences of both strands of the 6·4 kb Sst I–SstI insert of pFV600-26, the 3' end of the pFV609 insert, and the BamHI-BamHI inserts from pFV612, pFV613 and pFV614 were obtained from strain IATS O6 by primer walking and determined by the Guelph Molecular Super Centre (University of Guelph, Guelph, Ontario, Canada) using the d-Rhodamine Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer) and a GeneAmp PCR system 9600 thermal cycler (Perkin-Elmer). Oligonucleotide primers were synthesized on an Applied Biosystems model 394 DNA synthesizer and purified on OPC purification columns (Perkin-Elmer). Samples were run and analysed using a model 377 DNA sequencer (Perkin-Elmer).

DNA sequences were collated and analysed by using GENE RUNNER for Windows (Hastings Software), DNASIS for Windows (Hitachi Software) and PC/GENE (IntelliGenetics). Sequence homologies were determined by using the GenBank DNA and protein sequence databases through the National Center for Biotechnology Information BLAST network server (Altschul et al., 1997 ).

LPS analysis.
LPS was prepared as described by Hitchcock & Brown (1983) from overnight broth cultures. The LPS preparations were separated on SDS-PAGE gels and LPS profiles were detected by silver staining using the technique of Dubray & Bezard (1982) . Alternatively, LPS was transferred to Biotrace nitrocellulose (Gelman) and visualized by immunoblotting as described previously (Burrows et al., 1996 ). The LPS blots were incubated with hybridoma-culture supernatants containing mAb N1F10 (specific for A- band LPS), MF15-4 (specific for O5 B-band LPS) or O25-G3-D6 (specific for O6 B-band LPS). These antibodies have been described (Emara et al., 1995 ).

Mutagenesis of P. aeruginosa wbpKO5 , wbpOO6, wbpPO6, wbpV O6, wbpLO6, wbpMO6 , wbpNO5/O6 and tyrBO5/O6.
Chromosomal mutants were generated in P. aeruginosa strains IATS O6 (serotype O6) (wbpV, wbpM, wbpN , tyrB), PAK (serotype O6) (wbpO, wbpP, wbpL) or PAO1 (serotype O5) (wbpK, wbpN, tyrB) using the gene replacement strategy of Schweizer & Hoang (1995) as described by de Kievit et al. (1995) with slight modifications. Briefly, DNA containing the gene of interest was cloned into the gene replacement vector pEX100T except for wbpO, which was cloned into pEX18Ap (Hoang et al., 1998 ). A gentamicin-resistance (GmR) cassette from pUCGM was inserted into a unique site within the gene of interest. The resulting constructs were mobilized into P. aeruginosa through biparental mating with E. coli SM10, and GmR , sucroseS and carbenicillinS recombinants were selected on appropriate media. In the cases where merodiploids required resolution, colonies were plated on modified Luria medium containing 5% sucrose and no NaCl (Ma et al., 1998 ). This improved selection of a double-crossover event, which generated true recombinants by selecting for loss of the sacB-containing vector DNA. Gene replacement was ascertained by Southern blot analysis.

Analysis of potential amino acid auxotrophs.
P. aeruginosa O5 and O6 and their respective wbpNtyrB mutants were streaked on either Luria agar or Davis minimal agar medium containing glucose as a carbon source, and growth was scored as abundant, poor or no visible growth. The requirement of the mutants for phenylalanine, tyrosine, aspartic acid, and/or leucine was assessed using Davis minimal agar medium containing glucose and supplemented with the appropriate amino acids at 20 µg ml-1 each.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and sequence analysis of pFV600-26 DNA
In a previous study (Burrows et al., 1996 ), Southern hybridization analysis of chromosomal DNA isolated from the 20 serotypes of P. aeruginosa using high-stringency conditions showed that wbpM was conserved. Therefore, a wbpM O5 probe was used to screen an O6 Sstl plasmid library. One clone containing a 6·4 kb insert hybridized to the wbpM probe and was subsequently shown by DNA sequencing to contain the 3' region of the O antigen biosynthetic cluster. To clone the 5' region of the cluster, a PCR approach was used. We hypothesized that the rpsA and ihfß genes would be located upstream of the cluster in serotype O6 as they are in serotype O5. A primer based on rpsAO5 and a primer designed from the complementary strand of the 5' end of the insert of the 6·4 kb clone were used to amplify a 10·9 kb PCR product from the IATS O6 strain. DNA sequence analysis of pFV600-26, pFV612, pFV613 and pFV614, all obtained from strain IATS O6, revealed 12 ORFs, all transcribed in the same direction (Fig. 2), and sharing homology to LPS as well as to bacterial capsular biosynthetic genes (Table 3 ). No ORFs thought to be involved with LPS synthesis were identified on the opposite strand.



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Fig. 2. Genetic organization and physical map of the wbp gene cluster encoding proteins involved in synthesis of B-band lipopolysaccharide of P. aeruginosa serotype O6. The genes and ORFs are shown as arrows with their direction of transcription. The various regions subcloned and used in this study are shown. Plasmids pFV609-TG, pFV610-TG, pFV615-26a,b, pFV616-AG, pFV616-26a,b, pFV617-TG and pFV617-26a,b were generated by PCR amplification of IATS O6 chromosomal DNA. pFV131-TG and pFV131-26 (white arrows) were subcloned from serotype O5 DNA. Locations of the GmR cassette in various insertion mutants are shown (black arrowheads).

 

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Table 3. Similarities of P. aeruginosa O6 Wbp proteins to those in the databases

 
Genes involved in GalNAcA biosynthesis: wbpO and wbpP
P. aeruginosa WbpO and WbpP proteins showed significant homology to ORF1 and ORF2 of Shigella sonnei as well as to WcdA and WcdB of Salmonella typhi. Houng & Venkatesen (1998) proposed that Sh. sonnei ORF1 and ORF2 are involved in the biosynthesis of form I (the O antigen) and of the Vi antigen (the capsular antigen). The form I O antigen is composed of disaccharide repeating-units of 2-amino-2-deoxy- L-altruronic acid and 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose; the Vi antigen is a homopolymer of {alpha}-1,4 2-deoxy-2-N-acetylgalacturonic acid.

In S. typhi, WcdA and WcdB are NAD- and NADP-dependent enzymes required for synthesis of the Vi polysaccharide, a homopolymer of {alpha}-1,4 2-deoxy-2-N-acetylgalactosaminuronic acid (GalNAcA), variably O-acetylated at the C3 position. This sugar residue is found within the O antigen of O6 B-band LPS. To determine the role of wbpO and wbpP of P. aeruginosa serotype O6 in GalNAcA formation, knockout mutants of both genes were generated in strain PAK. wbpO::GmR (data not shown) and wbpP::GmR (Fig. 3) mutants were B-band deficient. The wbpO and wbpP mutants could be complemented by their respective wild-type alleles cloned in either orientation in pUCP26 (pFV616-26a,b and pFV617-26a,b: Fig. 2), suggesting that a functional promoter is present upstream of each of these genes. Furthermore, cross-complementation of the wbpO mutant with wcdA from S. typhi cloned in both orientations (pVT29-26 and pVT40-26) (data not shown) or of the wbpP mutant with pVT30-2 containing wcdB (Fig. 3) also restored production of P. aeruginosa O6 B-band LPS. These results indicate that wbpO and wbpP are participating in the biosynthesis of UDP-GalNAcA. Finally, the S. typhi promoter upstream of wcdA was found to be active in P. aeruginosa .



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Fig. 3. Analysis of LPS from knockout mutations and complementation experiments in wbpPO6. (a) Silver- stained SDS-PAGE gel. (b) Western immunoblot using mAb O25-G3-D6 specific for O6 B-band LPS. wbpP(-), mutant of wbpP .

 
Involvement of WbpV and WbpM in UDP-QuiNAc synthesis
WbpV has homology to WbfT of V. cholerae O139, which contains QuiNAc in its O antigen. WbfT has been putatively assigned the UDP-galactose-4-epimerase function (Comstock et al., 1996 ; Stroeher & Manning, 1997 ). WbpV also has 34·4% identity to P. aeruginosa WbpKO5, which has been proposed to be required for the biosynthesis of UDP- Fuc2NAc (Burrows et al., 1996 ).

To examine the role of wbpV in O6 B-band biosynthesis, and to determine if wbpVO6 and wbpKO5 are functionally interchangeable, null mutants were created in both genes. Both wbpVO6 and wbpKO5 null mutants were deficient in B-band LPS production (Fig. 4). Complementation experiments using wbpKO5 (pFV165-26) or wbpVO6 (pFV611-26) restored the homologous mutant to a phenotype identical to that of the parent. Although wbpVO6 has homology to wbpKO5, they could not cross-complement, showing that they are not functionally interchangeable (Fig. 4). Similar to many dehydratases/epimerases, both WbpKO5 and WbpV O6 possess consensus NAD-binding domains (GXXGXXG; Liu et al., 1996 ) near their N-termini at amino acids 8 to 14 and 10 to 16, respectively. These NAD-binding domains are believed to be important for dehydratase activity (Wyk & Reeves, 1989 ). A homologue of WbpKO5/WbpVO6, WbjF, was recently identified in P. aeruginosa O11 (Dean et al., 1999 ; Table 3). The O antigens of serotypes O5 and O11 contain Fuc2NAc (6-deoxy-N- acetylgalactosamine). In serotype O6, WbpV could be involved in the pathway for synthesis of UDP-QuiNAc (6-deoxy-N- acetylglucosamine).



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Fig. 4. Analysis of LPS from knockout mutations and complementation experiments in wbpKO5 and wbpV O6. (a) Silver-stained SDS-PAGE gels. (b) Western immunoblots using (1, 3) mAbs specific for B-band LPS [MF15-4 for serotype O5 (1) and O25-G3-D6 for serotype O6 (3)] and (2, 4) mAb N1F10 specific for A-band LPS.

 
The protein product encoded by wbpMO6 is 97·6% identical to P. aeruginosa WbpMO5, thought to be necessary for the synthesis of the Fuc2NAc residue of B- band (Burrows et al., 1996 ). The differences between WbpMO5 and WbpMO6 lie mainly in the N- terminus. Similar to WbpMO5, WbpMO6 has two consensus NAD-binding domains, one in the middle of the protein and the other is near the carboxy terminus. Burrows et al. (1996) demonstrated that the insertion of a GmR cassette into the NruI site of wbpMO5 abolished the production of B-band lipopolysaccharide. A similar mutant created in wbpMO6 also showed a B-band LPS-minus phenotype. Cross-complementation experiments showed that wbpM genes from serotypes O5 and O6 are functionally interchangeable. In serotype O6, WbpM is likely involved in the formation of UDP-QuiNAc.

Characterization of the glycosyltransferase function of WbpL O6
WbpL is highly hydrophobic and is 64% identical to WbpLO5 (Burrows et al., 1996 ) (Table 3). WbpL O5 was shown by Rocchetta et al. (1998) to be a bi-functional glycosyltransferase required to initiate synthesis of both A-band and B-band LPS. Therefore, WbpLO5 is the first transferase initiating A-band synthesis with a GlcNAc or GalNAc residue and B-band synthesis with a Fuc2NAc residue (Rocchetta et al., 1998 ). Based on such homology, WbpL is likely the initiating enzyme for B-band biosynthesis in P. aeruginosa O6. However, the O antigen repeat of O6 does not contain Fuc2NAc; therefore, we assessed the role of wbpLO6 in A- and B-band polysaccharide synthesis by generating an O6 chromosomal mutant. IATS O6 produces only small amounts of A-band, making its detection difficult. Instead we chose to use another O6 strain, PAK, which produces larger amounts of A-band, to generate the wbpLO6 knockout. This facilitated the examination of the effect of a wbpLO6 null mutation on both A- band and B-band synthesis in serotype O6. LPS isolated from the parental strain and from the isogenic mutant were compared on SDS-PAGE and Western immunoblots. The wbpLO6 mutant is devoid of both A-band and B-band LPS. Complementation of this mutant with the appropriate wild-type allele (wbpLO6 in pFV605-26) restored A-band and B-band LPS synthesis (Fig. 5). Cross-complementation using pFV110 ( wbpLO5) also restored the synthesis of both A- and B-band LPS. It has been shown that WecAE.coli has a broad specificity by being able to initiate LPS synthesis with various sugar residues including GalNAc (Zhang et al., 1997 ; Rocchetta et al., 1998 ) and GlcNAc (Rochetta et al., 1998 ). To determine if WecAE.coli could initiate O6 B-band synthesis with a different sugar, QuiNAc, as well as being able to initiate A-band synthesis in O6, cross- complementation using the WecAE.coli (pMAV11-26) was performed. pMAV11-26 could restore A-band synthesis, but not B-band synthesis. It is apparent that the substrate specificity of WbpLO5, but not of WecAE.coli extends to UDP- QuiNAc. Finally, the wbpLO6 gene cloned in pUCP26 (pFV605-26) was used to complement a wbpL O5 knockout mutant which was deficient in A- and B-band LPS production. In this case, while A-band production was fully restored, production of O5 B-band LPS was only restored at a reduced level as compared to the O5 wild-type strain (Fig. 5). These results suggest that WbpL from O6 does not efficiently transfer Fuc2NAc. However, the O5 O unit can be completed once the first sugar is added.



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Fig. 5. Analysis of LPS from knockout mutations and complementation experiments in wbpLO5 and wbpL O6. (a, b) Silver-stained SDS-PAGE gels. (c–f) Western immunoblots using a mAb specific for A-band LPS, N1F10 (c, d), or mAbs specific for B-band LPS: O25-G3-D6 for serotype O6 (e) and MF15- 4 for serotype O5 (f).

 
WbpR, WbpT and WbpU are putative glycosyltransferases
WbpT and WbpU are similar to a number of glycosyltransferases (Table 3). WbpU is likely to be a galactosyltransferase, as it is similar to RfbFKpO8, which is an initiating galactosyltransferase capable of transferring one residue each of galactopyranose and galactofuranose to a primed lipid intermediate (Clarke et al., 1995 ). WbpU is a protein of 377 aa, which is consistent with the 377 aa, 380 aa and 377 aa sizes of WbfSVc, RfbFSmO16 and RfbF KpO1/KpO8, respectively.

WbpT has homology to TrsE of Yersinia enterocolitica. They share a motif common to {alpha}-D-galactosyl- and mannosyltransferases, suggesting their probable function as galactosyl- or N-acetylgalactosaminyltransferases (Skurnik et al. , 1995 ). WbpT also has homology to a Salmonella typhi galactosyltransferase, WcdD (Waxin et al., 1993 ; Bacterial Polysaccharide Gene Database at www.microbio.usyd.edu.au/BPGD/default.htm), which is involved in the biosynthesis of the Vi antigen, a homopolymer of {alpha}-1,4-D-N -acetylgalactosaminuronic acid (Hashimoto et al., 1993 ; Virlogeux et al., 1995 ). Therefore, WcdD recognizes a GalNAcA residue as an acceptor molecule and then transfers another GalNAcA residue to the growing homopolymer. In light of these similarities, we propose that WbpT is the galactosyltransferase required for addition of a UDP-GalNAcA residue to the previous GalNFmA in the growing O unit.

Alignments of WbpT, WbpU and their homologues revealed consensus motifs in the carboxy terminus which are presumably important for their enzymic function (Fig. 6). Geremia et al. (1996) recently reported a motif consisting of two invariant glutamic acid residues separated by seven amino acids (EX7E) which is strictly conserved among {alpha}-D-glycosyltransferases. This motif is present in WbpT and WbpU (Fig. 6), thus supporting the assignment of these proteins as {alpha}-D- galactosyltransferases.



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Fig. 6. Partial amino acid alignments of WbpT and WbpU of P. aeruginosa serotype O6 with other {alpha}-D- glycosyltransferases. Identical amino acids are marked by asterisks; similar amino acids are marked by dots. The motif associated with {alpha}-D-glycosyltransferases (Geremia et al., 1996 ) is indicated in bold. Alignments were performed using the CLUSTAL program (PC/GENE, IntelliGenetics).

 
The wbpR gene product has homology to a number of putative glycosyltransferases, including IcsA of Synechocystis sp. (Table 3). Therefore, P. aeruginosa WbpR is likely a glycosyltransferase involved in transferring a sugar residue of the B-band LPS.

Genes involved in the Wzy-dependent LPS assembly pathway
The first gene in the B-band O antigen cluster of P. aeruginosa O6 is homologous to wzzO5. To verify its function, complementation studies were performed in the O6 strain Fisher immunotype 1, which displays a random, non-modal distribution of O antigen chain lengths. Transformation of Fisher 1 with pFV401-26 ( wzzO5) (Burrows et al., 1997 ) or pFV615-26a,b (wzzO6) showed that each wzz conferred a characteristic O antigen modulation with a higher proportion of high-molecular-mass LPS being produced (Fig. 7). wzzO5 restored some modality of the B-band LPS O chains while wzzO6 increased the amount of high-molecular-mass B-band LPS even further. The banding pattern of A-band LPS remains unaltered (data not shown). A functional promoter is likely present upstream of wzzO6 , as the apparent wzz defect in Fisher 1 was complemented by pFV615-26b, which contained wzzO6 in the orientation opposite to the vector promoter.



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Fig. 7. Silver-stained SDS-PAGE analysis of LPS isolates from Fisher 1 strain and from Fisher 1 strain harbouring pFV401-26 ( wzzO5) or pFV615-26 (a, wzzO6 cloned downstream of lacZ promoter in pUCP26; b, wzz O6 cloned in the opposite orientation). Transformation of Fisher 1 with the various plasmids led to a higher proportion of high- molecular-mass LPS (arrow) being produced.

 
Translocation of the O-polysaccharide unit across the cytoplasmic membrane is catalysed by Wzx (Burrows & Lam, 1999 ; Liu et al. , 1996 ). In the wbpO6 cluster, Wzx has homology to the Methanobacterium thermoautotrophicum O antigen transporter and its hydropathic index indicated that the protein is hydrophobic. A probe derived from this gene was sero- specific and hybridized to chromosomal DNA of serotype O6 only.

In general, formation of heteropolymeric O chains requires an O antigen polymerase encoded by wzy, which maps adjacent to, or within, rfb clusters. Interestingly, we could not locate a wzy gene either within or immediately flanking the wbp cluster of P. aeruginosa serotype O6. However, the ability of IATS O6 to generate long-chain O antigen suggests that there is a functional version of Wzy encoded outside of the O antigen cluster.

Genetic organization of the O6 LPS gene cluster
Serotypes O5, O6 and O11 all have homologues of wbpKO5 , wbpLO5 and wbpMO5, in that order (Fig. 8). However, only wbpM appears to be highly conserved. In serotype O5, an insertion sequence, IS1209 , is present between wbpL and wbpM, at the junction between the serogroup-specific and non-specific regions. This insertion sequence is not present at the corresponding position in serotypes O6 and O11. Interestingly, WbpLO5 is only 303 aa in length, while the corresponding proteins in serotype O6 and O11 are 346 aa and 341 aa, respectively. It is possible that insertion of IS1209 in O5 led to truncation of wbpL O5. However, the decreased size of WbpLO5 does not appear to affect its glycosyltransferase activity (Rocchetta et al., 1998 ). In contrast to enteric O antigen clusters, the wbpO6 cluster is not flanked by the his or gnd genes. In wbpO5, two his genes, hisH and hisF, are located in the centre of the cluster (Burrows et al., 1996 ). No such genes are present within wbpO6. Similar to wbp O5, wbpO6 is flanked by rpsA and ihfß at the 5' end and by orf48.5 and uvrB at the 3' end. A recent report by Dean et al . (1999) also showed wzz as the first gene at the 5' end of the serotype O11 O antigen biosynthesis cluster, also preceded by ihfß (himD). These two sets of genes at the 5' and 3' end respectively may therefore act as markers for O antigen genes in P. aeruginosa, as do gnd and his in enterics.



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Fig. 8. Comparison of the gene organization of the wbp alleles located at the 3' end of the clusters of P. aeruginosa serotypes O5 (Burrows et al., 1996 , GenBank accession number: U50396), O6 (this study, AF035937) and O11 (Dean et al., 1999 , AF147795). Homologous genes are represented by similar arrows. The IS1209 element is located directly upstream of wbpM in serotype O5.

 
Based on Southern blot and dot blot analysis, within the wbp O6 cluster, five genes, wbpQ, wzx, wbpU , wbpV and wbpL are strictly O6 serospecific. In contrast, probes prepared from wzz, wbpO, wbpR and wbpT hybridized to serotypes O2, O6, O11, O17 and O19, which appear to form a related group. Probes from wbpP and wbpS hybridized to all serotypes (data not shown). Southern blot analysis using gene-specific probes showed that the wbp locus is the same in both serotype O6 strains, IATS O6 and PAK, used for mutagenesis (data not shown). PCR of individual wbp genes from IATS O6 and PAK was also performed. Identical corresponding size products were obtained for each gene, confirming the results of Southern hybridization (data not shown). Also, the identity between IATS O6 and PAK DNA sequences was confirmed when needed.

Generation of wbpN/tyrB mutants
Previously, we reported that the conserved gene downstream of wbpMO5, wbpNO5, could potentially play a role in LPS biosynthesis, based on its homology to proteins involved in antibiotic biosynthesis (Burrows et al., 1996 ). To determine whether wbpN was necessary for LPS biosynthesis, gene replacement knockout mutants of wbpN were made in serotypes O5 and O6. LPS from the mutants was analysed by silver- stained SDS-PAGE and Western immunoblot using the B-band specific mAbs MF15-4 (for O5) and O25-G3-D6 (for O6). The LPS prepared from wbpN ::GmR mutants of both serotypes exhibited banding patterns in SDS-PAGE identical to those of the parent strains (not shown), indicating that wbpN is not involved in LPS biosynthesis. This gene was therefore renamed orf48.5.

Interestingly, an ORF that encodes a 43·3 kDa protein was identified on the opposite DNA strand of orf48.5. This ORF has more than 65% amino acid similarity to the aromatic and aspartic amino acid transferases (tyrB and aat) of a variety of organisms (E. coli TyrB, GenBank accession number P04693; S. typhimurium TyrB, P74861; Haemophilus influenzae Aat, P44425). Generation of wbpN::GmR mutants led simultaneously to a knockout of this ORF. To determine whether this gene may be necessary for amino acid biosynthesis in P. aeruginosa , we grew both the O5 and O6 parent strains and their respective tyrB::GmR mutants on Davis minimal medium containing only glucose as a carbon source. The wild-type O5 strain grew well on the minimal medium while the O5 tyrB mutants grew very poorly (not shown). It was not possible to assess the effect of this mutation on serotype O6 since both the IATS O6 parent and the mutant strains grew poorly on minimal medium. The tyrB gene is required for the biosynthesis of phenylalanine, tyrosine, leucine and aspartic acid (Fotheringham et al., 1986 ; Nakai et al., 1996 ). Supplementation of the minimal medium with these amino acids permitted abundant growth of the O5 tyrB mutants (not shown).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
 
In this report, we describe the characterization of a 14·6 kb fragment containing P. aeruginosa serotype O6 genes responsible for B-band biosynthesis. We propose a putative pathway for O6 O antigen biosynthesis and assign a function to the majority of the proteins encoded by this gene cluster (Fig. 9).



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Fig. 9. Proposed pathway for the biosynthesis of the P. aeruginosa O6 O antigen. The functions of the Wbp proteins were predicted based on their homologies to known biosynthetic proteins. No function could be predicted for wbpQ and wbpS. wbpM and wbpV are implicated in the formation of UDP- QuiNAc but their exact function remains to be elucidated. *TDP-L-Rha may be synthesized by enzymes encoded outside of the O antigen biosynthetic cluster, as no genes within the wbpO6 cluster had homology to rmlA,B,C,D genes, which are responsible for the formation of L-Rha. The process for the addition of a formyl group1 or an acetyl group2 to the UDP-GalNAcA remains to be elucidated.

 
Based upon both homology and the successful complementation of wbpO and wbpP mutants by wcdAS.typhi and wcdBS.typhi, respectively, we have assigned wbpO and wbpP to the metabolic steps for the synthesis of UDP-GalNAcA. Investigation of this pathway by a biochemical approach is now under way in our laboratory. We have recently obtained preliminary evidence supporting this hypothesis (C. Creuzenet, M. Bélanger & J. S. Lam, unpublished data). To generate the sugar residue, GalNAc(OAc)AN (N-acetyl- D-galactosaminuronamide), as described in the O6 O antigen structure, an O-acetylase would be required to add an acetyl group on the hydroxyl of carbon 3 of the GalNAcA sugar while an amidation step would occur at carbon 6. The other amino sugar, GalNFmA, carries an acyl substituent, the formyl group, which occurs rarely in natural carbohydrates. The enzymic steps required to form the latter are presently unknown.

The P. aeruginosa O6 O-unit contains four sugar residues, and four putative sugar transferases are encoded by the O antigen gene cluster. In enteric O antigen clusters, as well as in the P. aeruginosa wbpO5 gene cluster, the transferase genes are arranged in the opposite order to which they act. In P. aeruginosa serotype O6, the hydrophobicity of WbpLO6, its position as the most distal transferase in the cluster, its homology to WbpLO5, as well as the A- B - phenotype observed in knockout mutants, indicates that it is responsible for initiating A-band and B-band synthesis in serotype O6. The initiating residue in O5 B-band synthesis is UDP-Fuc2NAc, whereas the initiating residue in O6 is likely UDP-QuiNAc.

Two other putative transferases, WbpT and WbpU, are encoded in the O6 LPS cluster. WbpUO6 is thought to add GalNFmA to the primed lipid intermediate 6-deoxy-GlcNAc (QuiNAc)-undecaprenol phosphate based upon similarity to RfbFKp which is required to transfer the disaccharide Galf-Galp (D-galactan I) to the primed lipid intermediate GlcNAc-undecaprenol phosphate (Clarke et al., 1995 ). WcdDSt is responsible for the transfer of GalNAcA residues to GalNAcA during formation of the Vi homopolymer antigen (Hashimoto et al., 1993 ). The homology of WbpT to WcdDSt supports its assignment as the transferase catalysing the transfer of the GalNAcA residue to GalNFmA in the O6 O antigen unit.

Finally, a fourth transferase, WbpR, likely adds the fourth residue, L-Rha, to the B-band unit. L-Rha is found both in the O antigen and in the core oligosaccharide of serotype O6 (Knirel, 1990 ; Masoud et al., 1995 ). In bacteria, L-Rha synthesis is encoded by the highly conserved rmlABCD genes (Macpherson et al., 1994 ; Reeves, 1993 ). No genes within the wbpO6 cluster have homology to rml genes but an rml gene cluster has been identified elsewhere on the serotype O6 chromosome (R. Rahim & J. S. Lam, unpublished data). Therefore, the enzymes for L-Rha synthesis are not encoded by genes within the O antigen cluster but may well be encoded by the rml cluster.

The structure of the repeating unit of the serotype O6 O antigen has been chemically elucidated (Knirel, 1990 ; Knirel & Kochetkov, 1994 ). However, the identity of the first sugar residue attached to the core has not been determined. Based on the order of the genes in the O6 cluster which encode the glycosyltransferases, and on the result of the wbpLO6 knockout, the ‘biological’ iteration for the O antigen unit is likely Rha- GalNAcA-GalNFmA-QuiNAc, with QuiNAc being the first sugar attached to the core. Ideally, this should be verified by performing structural elucidation of LPS from a semi-rough mutant of serotype O6. The established approach is to construct a null mutation in wzy (de Kievit et al., 1995 ). However, as discussed below, wzy in O6 has not yet been identified.

We have shown that the O6 B-band O antigen assembly likely follows the Wzy-dependent pathway characteristic of heteropolymers. Homologues of wzx and wzz were identified in the O6 cluster. Generally, the wzy gene is located within or adjacent to the O antigen cluster. However, Curd et al. (1998) found that the O antigen gene clusters of Salmonella enterica groups D1 and B contain only the remnant of a D3-like wzy gene and that the full-length wzy gene is located elsewhere on the chromosome (Naide et al., 1965 ). Although a complete wzy gene was not found either within or immediately flanking the IATS O6 cluster, SDS-PAGE analysis showed that there is synthesis of long-chain O antigen. These observations suggest that a functional wzy gene is located outside of the O6 B-band cluster.

The data presented allowed the comparison of the organization of the LPS genes among serotypes O5, O6 and O11 which is necessary to gain a full understanding of LPS biosynthesis in P. aeruginosa. We have now established that O antigen clusters of P. aeruginosa O5 and O6 begin with wzz and end with wbpM and that both clusters are flanked by rpsA and ihf ß at the 5' end and by tyrB and uvrB at the 3' end. Analysis of the flanking regions of the serotype O11 O antigen biosynthesis cluster by Dean et al. (1999) corroborated these observations. This organization is very different from that of the O antigen gene clusters in enteric organisms, which are usually flanked by the his operon and gnd (Schnaitman & Klena, 1993 ). It is also apparent that among all three serotypes examined, glycosyltransferase genes are localized near the 3' end of the sero-specific region of the clusters.

The evidence presented here, using chromosomal insertion mutants and complementation experiments, clearly shows that the genes currently designated wbpO, wbpP, wbpV, wbpL and wbpM in serotype O6, as well as wbpK in serotype O5, are essential for O antigen biosynthesis in P. aeruginosa. We have also demonstrated that wbpN, now renamed orf48.5 , does not have a role in O antigen biosynthesis, and that the tyrB gene is likely associated with biosynthesis of one or more of phenylalanine, tyrosine, leucine and/or aspartic acid. In this study, we have characterized important genes responsible for O antigen biosynthesis in the most clinically prevalent P. aeruginosa serotype, O6. Precise characterization of the P. aeruginosa O6 O antigen biosynthetic pathway will await biochemical confirmation of the proteins’ activities.


   ACKNOWLEDGEMENTS
 
This work was funded by an operating grant to J.S.L. from the Medical Research Council of Canada (#MT-14687). M.B. is the recipient of a fellowship from the Natural Sciences and Engineering Research Council of Canada Fellowship and the Canadian Cystic Fibrosis Foundation (CCFF). L.L.B. is the recipient of a Fellowship from the CCFF.

We thank J. Ng for valuable technical assistance and M. Y. Popoff (Institut Pasteur, Paris, France) for the kind gift of plasmids (pVT29, pVT30 and pVT40).


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Received 6 May 1999; revised 26 August 1999; accepted 15 September 1999.