INTRODUCTION

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In gram-negative bacteria, the lipopolysaccharide (LPS) is one of the major structural and immunodominant molecules of the outer membrane. It consists of three moieties: lipid A, core oligosaccharide, and O-specific antigen or O side chain. The O antigen is the most external component of LPS, and its structure consists in a polymer of oligosaccharide repeating units. Another interesting feature is the high chemical variability shown by the O antigen of the LPS, leading to a similar genetic variation in the genes involved in O-antigen biosynthesis, the so-called wb (rfb) cluster (for a review, see reference 45). In this work, a recently proposed nomenclature system for genes involved in expression of bacterial surface polysaccharides is followed (39); for clarity, the old gene names are also given in parentheses. The genetics of O-antigen biosynthesis have been intensively studied in members of the family Enterobacteriaceae, and it has been shown that the wb clusters usually contain genes involved in biosynthesis of activated sugars, glycosyltransferases, O-antigen polymerases, and O-antigen export (45). Despite heterogeneity in the structures of O antigens, only three pathways for assembly of O antigens have been recognized (55).

Serratia marcescens strains, as well as those of other species of enteric bacteria, can be grouped in O-antigen serogroups, and some of them have been chemically characterized (38). S. marcescens N28b (O4) produces a bacteriocin (8, 9, 51) that has been shown to be useful to identify recombinant clones harboring genes encoding small outer membrane proteins (13) and enzymes involved in core LPS biosynthesis (12). Few studies have been carried out regarding the genetics of O-antigen biosynthesis in S. marcescens, but three genes involved in S. marcescens O16-antigen biosynthesis have been characterized (47). The S. marcescens O4-antigen repeating unit consists of L-rhamnose linked by an 1-4 bond to D-glucose (38), and recently we described S. marcescens N28b wbbL and rmlD genes (42). Strains with mutations in both wbbL and rmlD genes were shown to be impaired in O4-antigen production (42), suggesting that they belong to the S. marcescens O4 wb cluster. Furthermore, expression of these two genes in Escherichia coli DH5 conferred serum resistance and bacteriocin 28b resistance, allowing an easily screenable phenotype (42). In this work, we present the first complete genetic analysis of an S. marcescens wb cluster, containing genes involved in S. marcescens O4-antigen biosynthesis.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. All strains were grown in Luria-Bertani (LB) Miller broth and LB Miller agar (33). LB media were supplemented with ampicillin (50 µg/ml), chloramphenicol (50 µg/ml), kanamycin (30 µg/ml), or rifampin (50 µg/ml), when needed. The physical maps of the plasmids used in this study are shown in Fig. 3.

General DNA methods. DNA manipulations were carried out essentially as previously described (43). DNA restriction endonucleases, T4 DNA ligase, E. coli DNA polymerase (Klenow fragment), and alkaline phosphatase were used as recommended by the suppliers. Recombinant clones were selected on LB Miller agar plates containing the appropriate antibiotics. To construct plasmid pSUB6, recombinant cosmid FGR20 was partially digested with HindIII, and the resulting DNA fragments were self-ligated and transformed into E. coli DH5. Plasmid pSUB7 was constructed by PstI partial digestion of FGR20, self-ligation, and transformation into E. coli XL1-Blue.

Construction of mutant strains N28b-3 (wbbA) and N28b-4 (wzm wzt). Two different mutant strains of S. marcescens N28b were constructed. To obtain the N28b-3 mutant (insertion in the wbbA gene), a method based on suicide plasmid pSF100 was used (42). Plasmid pSUB6 was EcoRV digested, and a wbbA internal DNA fragment (1,848 bp) was isolated, ligated to EcoRV-digested and dephosphorylated pFS100, and transformed into E. coli MC1061(pir) to generate plasmid pSF103. Plasmid pSF103 was isolated, transformed into E. coli SM10(pir), and transferred by conjugation to an S. marcescens N28b Rif mutant (from our laboratory collection) as previously described (42).

LPS isolation and analysis. LPS was purified by the method of Westphal and Jann (53). For screening purposes, LPS was obtained after proteinase K digestion of whole cells according to the procedure of Hitchcock and Brown (17). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the procedure of Laemmli (26), and LPS bands were detected by the silver-staining method of Tsai and Frasch (50).

Antisera. Anti-E. coli O16 LPS serum and anti-S. marcescens O4 LPS serum were obtained and assayed as previously described (2) for LPS specificity against purified LPS and whole cells.

Western immunoblotting. After SDS-PAGE, immunoblotting was carried out by transfer to polyvinylidene fluoride membranes (Millipore Corp., Bedford, Mass.) at 1.3 Å for 1 h in the buffer of Towbin et al. (49). The membranes were then incubated sequentially with 1% bovine serum albumin, specific anti-O serum (1:500), alkaline-phosphatase-labeled goat anti-rabbit immunoglobulin G (1:1,000) (Boehringer Mannheim), and 5-bromo-4-chloro-3-indolylphosphate disodium-nitroblue tetrazolium (Boehringer Mannheim). Incubations were carried out for 1 h, and washing steps with 0.05% Tween 20 in phosphate-buffered saline were included after each incubation step. Colony blotting was performed with S. marcescens O4 antiserum as indicated above.

ELISA. Cytosol, whole membrane, and inner and outer membrane fractions were analyzed by enzyme-linked immunosorbent assay (ELISA). ELISAs were performed by dispensing standardized suspensions of each fraction in coating buffer (pH 9.6), into 96-well microtiter plates. The trays were left standing overnight at 4°C. The wells were blocked with 1% bovine serum albumin in phosphate-buffered saline for 2 h at 37°C. Anti-O4 polyclonal serum (1:200) was added and incubated for 2 h at 37°C. Detection was achieved by using peroxidase-labeled sheep anti-rabbit immunoglobulin G (1:1,000) (Boehringer Mannheim) and 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate] (Boehringer Mannheim) as substrate. Cytosol, whole, and inner and outer membrane fractions were prepared as previously described (37).

DNA sequencing. Double-stranded DNA sequencing was performed by the Sanger dideoxy-chain termination method (44) with the ABI Prism dye terminator cycle sequencing kit (Perkin-Elmer). Primers used for DNA sequencing were purchased from Pharmacia LKB Biotechnology.

DNA and protein sequence analysis. The DNA sequence was translated in all six frames, and all open reading frames (ORFs) greater than 100 bp were inspected. Deduced amino acid sequences were compared with those of DNA translated in all six frames from nonredundant GenBank and EMBL databases by using the BLAST network service at the National Center for Biotechnology Information (1). Multiple sequence alignments were carried out with the Clustal W program (48). Determinations of possible terminator sequences were done by using the Terminator program from the Genetics Computer Group package (Madison, Wis.) in a VAX 4300 computer. Hydropathy profiles were calculated according to the method of Kyte and Doolittle (25).

Nucleotide sequence accession number. The nucleotide sequence of the O4 wb cluster from S. marcescens N28b has been deposited in GenBank under accession no. AFO38816.

	RESULTS AND DISCUSSION

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Isolation of a 10-kb fragment conferring O4-antigen production in E. coli DH5. We have previously reported the isolation of S. marcescens N28b rmlD and wbbL genes, coding for dTDP-L-rhamnose synthase and rhamnosyltransferase, respectively, from recombinant cosmid FGR20 (42). The plasmid containing only the wbbL gene induced E. coli O16-antigen biosynthesis in E. coli DH5, by complementation of the wb-50 mutation. Analysis of LPS isolated from E. coli DH5 (FGR20) by SDS-PAGE and Western immunoblotting revealed that this strain's LPS reacted with S. marcescens O4 antiserum, suggesting that this recombinant cosmid harbored other genes involved in O4-antigen biosynthesis. To localize the genes required for O4-antigen biosynthesis, several subclones were constructed from cosmid FGR20. Among the different subclones obtained, plasmid pSUB6 was chosen for further study because it was the smallest subclone obtained still able to direct O4-antigen biosynthesis (Fig. 1, lane 2; Fig. 2, lane 2). Restriction enzyme analysis and Southern blotting experiments showed that both rmlD and wbbL genes were present in the approximately 10-kb plasmid pSUB6 DNA insert. Taken together, these results suggest that pSUB6 contains the wb genes required for S. marcescens O4-antigen production in E. coli DH5.

View larger version (60K): [in this window] [in a new window]   FIG. 1.   Silver-stained PAGE of LPS samples from E. coli DH5 (lane 1), E. coli DH5(pSUB6) (lane 2), and E. coli DH5(pSUB7) (lane 3).

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Western immunoblot of LPS reacted with S. marcescens O4 antiserum. LPS was from E. coli DH5 (lane 1), E. coli DH5(pSUB6) (lane 2), and E. coli DH5(pSUB7) (lane 3).

Sequencing of the DNA conferring O4-antigen production. The nucleotide sequence of the plasmid pSUB6 insert was determined in order to identify the S. marcescens genes conferring O4-antigen production on E. coli DH5. A nucleotide sequence of 9,917 bp was determined in both directions by using oligonucleotides T3 (5'-AATTAACCCTCACTAAAGGG-3') and H1 (5'-GTGTTCCGCTTCCTTTAG-3') complementary to vector SuperCos 1 sequences flanking the pSUB6 DNA insert. Other sequence-derived oligonucleotides were purchased (Pharmacia LKB) and used to complete the nucleotide sequence. Analysis of the sequenced region showed the 3' end of a potential ORF (ORF1) and six complete ORFs (Table 2 and Fig. 3). These ORFs were apparently transcribed in the same direction. In all cases, putative ribosome binding sequences were found at appropriate distances from the initial codon of each ORF. No sequences strongly similar to the 35 and 10 regions of E. coli promoters and properly spaced were found. The Terminator program from the Genetics Computer Group package allowed the identification of an inverted repeat followed by a run of T's 98 nucleotides downstream from the end of ORF7, similar to the rho-independent transcription termination sequence. Another putative rho-independent transcription termination sequence was previously identified between the rmlD (ORF3) and wbbL (ORF4) genes (42).

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Physical and genetic map of the S. marcescens O4 wb genes. To construct the map, the whole nucleotide sequence of the 9,917-bp plasmid pSUB6 insert was determined. Only two of the PstI restriction sites are shown. The physical maps of the plasmid used in this study are also shown.

Analysis of the ORF's deduced amino acid sequence. The DNA sequence was translated in all six frames, and all ORFs were inspected. Computer database searching was carried out to tentatively identify the sequenced genes. Proteins similar to each ORF gene product were analyzed to determine the levels of similarity and identity. This analysis showed that the 35-amino-acid peptide encoded by 5'-truncated ORF1 had high levels of amino acid identity (67 and 54%) and similarity (87 and 78%) to the carboxy-terminal regions of GalE proteins from Erwinia amylovora (32) and Haemophilus influenzae (10), respectively. This result suggested that this ORF could correspond to the S. marcescens galE gene. The deduced 177-amino-acid protein encoded by ORF2 showed high levels of both amino acid identity and similarity to dTDP-4-dehydrorhamnose 3,5-epimerases, involved in O-antigen or capsule biosynthesis, or similar proteins from different gram-negative bacteria, including members of the Enterobacteriaceae, Synechocystis sp., Neisseria meningitidis, and Neisseria gonorrhoeae (Table 3). ORF2 was named rmlC as proposed for genes encoding dTDP-4-dehydrorhamnose 3,5-epimerases (39). ORF3 and ORF4 were previously identified as rmlD and wbbL genes, putatively coding for dTDP-rhamnose synthase and rhamnosyltransferase, respectively (42). The deduced 277- and 441-amino-acid proteins encoded by ORF5 and ORF6 were found to be similar to ATP-binding-cassette-2-(ABC-2)-type transport system integral membrane and ATP-binding proteins, respectively (Table 3). Exporter systems similar to the ORF5-ORF6 system are involved in export of O antigen, except for ATP-binding protein AbcA involved in A-protein expression (5) and a Synechocystis protein of unknown function (19). The putative exporter component (ORF5) showed a 33.7% level of amino acid similarity to the corresponding Wzm protein involved in S. marcescens O16-antigen export, while the putative ATP-binding component showed a higher level of similarity (53.6%) to its O16 counterpart Wzt protein. ORF5 and ORF6 are named accordingly wzm and wzt, respectively. Hydrophobicity analysis and identification of putative transmembrane domains of Wzm protein (amino acid residues 49 to 69, 79 to 99, 128 to 148, 155 to 175, and 191 to 211), by the method of Klein et al. (24), suggest that this protein is indeed an integral membrane protein. On the other hand, the sequence GRNGAGKS (residues 76 to 82) from Wzt was found to correspond to box A, a motif present in ATP-binding proteins.

View larger version (98K): [in this window] [in a new window]   FIG. 4.   Alignment of S. marcescens N28b WbbA (WbbA-Sm) and N. meningitidis LgtD (LGTD-Nm) (P96946), N. gonorrhoeae LgtD (LGTD-Ng) (Q50949), E. coli YcdQ (YcdQ-Ec) (P75905), H. influenzae HI0868 (HI0868-Hi) (P96336), H. influenzae LgtD (LgtD-Hi) (Q57287), Arquaeoglobus fulgidus AF0321 (AF0321-Af) (AE001082), Actinobacillus actinomycetemcomitans UN1 (UN1-Aa) (D1020407), and A. actinomycetemcomitans UN2 (UN2-Aa) (D1020408). Basic residues (white letters on black background), hydrophobic residues (black letters on gray background), acid residues (boldface), and cluster-breaking prolines (underlined boldface) are shown. The asterisks denote regions similar to domain A catalytic sites 1 and 2 of the ExoU family of glycosyltransferases.

E. coli DH5 rmlAB genes are required for S. marcescens O4-antigen production. The S. marcescens O4-antigen repeating unit consists of L-rhamnose linked by an 1-4 bond to D-glucose (38), but no genes encoding glucose-1-phosphate thymidylyl transferase (RmlA) and dTDP-D-glucose 4,6-dehydratase (RmlB), required for the two initial steps in dDTP-rhamnose biosynthesis, were found in plasmid pSUB6. E. coli DH5 rmlAB genes should provide these functions to explain the pSUB6-directed biosynthesis of the O4 antigen in this strain. In order to test if plasmid pSUB6-directed O4-antigen production in E. coli DH5 required some other E. coli wb genes, plasmid pSUB6 was introduced into E. coli CLM4, containing the (sbcB-rfb) deletion. E. coli CLM4(pSUB6) was found to produce O antigen recognized by S. marcescens O4 antiserum (Fig. 5, lane 5). The E. coli K-12 wec cluster contains genes involved in the biosynthesis of the enterobacterial common antigen (31). Marolda and Valvano (30) showed that the rffHG genes, in the wec cluster, are homologous to the rmlAB genes and encode proteins with activities identical to those of RmlA and RmlB proteins. Thus, in the E. coli CLM4 background rffHG gene products should catalyze the two initial steps in dTDP-rhamnose biosynthesis. Although most enterobacterial wb clusters involved in biosynthesis of rhamnose-containing O antigens present the four rmlABCD genes (45), our results suggest that the rmlAB genes involved in S. marcescens O4-antigen biosynthesis are not physically linked to the O4 wb cluster carried by plasmid pSUB6.

View larger version (68K): [in this window] [in a new window]   FIG. 5.   Silver-stained PAGE of LPS samples from E. coli 21548 (lane 1), E. coli 21548(pSUB6) (lane 2), E. coli 21548(pSUB7) (lane 3), E. coli CLM4 (lane 4), E. coli CLM4(pSUB6) (lane 5), and E. coli CLM4(pSUB7) (lane 6).

S. marcescens O4-antigen production is wecA dependent. E. coli CLM4 is a recA derivative of strain S874, and it has been previously shown (36) that the (sbcB-rfb) deletion extends from sbcB to udk genes; thus, strains S874 and CLM4 are devoid of the whole E. coli K-12 wb gene cluster including the wzy (rfc) gene. The results obtained with E. coli CLM4(pSUB6) suggest that biosynthesis of the O4 antigen is wzy independent, although it cannot be ruled out that the wecF (rffT) gene, a wzy analog (35), could be involved in sequential assembly of O4-antigen repeating units. It has been previously shown that WecA (Rfe), an N-acetylglucosamine-1-phosphatetransferase involved in biosynthesis of the enterobacterial common antigen (31), is also involved in the biosynthesis of both wzy-dependent and -independent O antigens in members of the Enterobacteriaceae (46, 56). To test the role of WecA protein in the S. marcescens O4-antigen biosynthesis, E. coli 21548, a wecA::Tn10 mutant, was transformed with plasmid pSUB6. E. coli 21548(pSUB6) did not produce O antigen, as judged by SDS-PAGE (Fig. 5, lane 2). These results suggest that, although N-acetylglucosamine is not found in the O4 repeating unit (38), the wecA gene product is essential for O4-antigen production. It has been shown that WecA protein primes the synthesis of some homopolysaccharide O antigens (6, 21); our results suggest that WecA could play a similar role in S. marcescens O4 biosynthesis.

wbbA is essential for S. marcescens O4-antigen biosynthesis. E. coli XL1-Blue and E. coli DH5 harboring pSUB7 (Fig. 3) produced an O antigen (Fig. 1, lane 3) that reacted with E. coli O16 antiserum but not with S. marcescens O4 antiserum in Western blotting experiments (Fig. 2, lane 3). As expected, no O antigen was produced in E. coli CLM4 and E. coli 21548 harboring pSUB7 (Fig. 5, lanes 6 and 3). Restriction analysis, Southern blotting experiments, and nucleotide sequence showed that pSUB7 insert DNA harbored the O4 wb gene cluster with a Tn1000 element inserted (3) in the wbbA gene (nucleotide 8120). This phenotype is similar to the one conferred on E. coli by the plasmid containing only the rmlD and wbbL genes (42). These results suggest that the wbbA gene is essential for O4-antigen production. To further prove the role of WbbA in O4-antigen biosynthesis, an S. marcescens wbbA insertion mutant was constructed by using pir-dependent plasmid pSF103. Plasmid pSF103 was transferred to S. marcescens N28b by conjugation, and 10 colonies were screened by Southern hybridization with the internal wbbA EcoRV fragment (1,848 bp) labeled with digoxigenin as the probe. Genomic DNAs from the wild-type strain and candidate mutants were doubly digested with BamHI and HindIII, separated in agarose gels, and transferred to nitrocellulose membranes. Two DNA fragments of 4.5 and 2.1 kb from mutant strain N28b-3 were found to hybridize. As expected, only a 5.0-kb fragment from the wild-type strain hybridized with the DNA probe. Since there are two HindIII sites flanking the EcoRV insert in plasmid pSF103, these results are consistent with the expected wbbA insertion mutation. As expected, S. marcescens N28b-3 did not show O4 antigen in Western blotting experiments (Fig. 6, lane 1). The E. coli O16 wb cluster contains a glucosyltransferase (wbbK) (46), but a similar gene was not found in the characterized O4 S. marcescens wb cluster, despite glucose being present in the S. marcescens O4 antigen (38). The E. coli wbbK gene product, able to transfer the glucose moiety to the E. coli O16 antigen, is apparently not necessary for S. marcescens O4-antigen production, since E. coli CLM4(pSUB6) is able to produce the O4 antigen. The similarity between WbbA region A and the ExoU family domain A suggests a glycosyltransferase activity, able to transfer glucose to the S. marcescens O4 antigen, for the WbbA protein. Most members of the ExoU family, in wb clusters, are about one-third shorter than the WbbA protein, suggesting that this protein could have an additional unknown function. In other wb clusters, genes coding for large proteins have been found, and some of them have been proposed to code for bifunctional proteins (45).

View larger version (40K): [in this window] [in a new window]   FIG. 6.   Western immunoblot of LPS reacted with S. marcescens O4 antiserum. Samples were from S. marcescens N28b-3, wbbA insertion mutant (lane 1); S. marcescens N28b-4, wzm-wzt deletion mutant (lane 3); and S. marcescens N28b, wild type (lanes 2 and 4).

Role of the Wzm and Wzt proteins. To assess the role of the putative ABC-2-type transporter encoded by wzm and wzt genes, an S. marcescens strain (N28b-4) containing a deletion encompassing both genes was constructed. In order to avoid polar effects on the expression of the downstream wbbA gene, the wzm and wzt genes were replaced by an in-frame-tagged deletion containing the first six codons of wzm, seven tag codons, and the last 11 codons of wzy. Plasmid pSF104 was electroporated into S. marcescens N28b and plated in LB-chloramphenicol at 30 and 42°C. Chloramphenicol-resistant colonies grown at 42°C were picked (10 colonies), serially diluted, and plated on LB medium containing 5% sucrose. Sucrose-resistant colonies were replica plated on LB-chloramphenicol. Sucrose-resistant, chloramphenicol-sensitive colonies were screened for the presence of the wzm-wzt double deletion by PCR amplification with primers E (5'-TTTAGGGGTAAGATGGATG-3') and F (5'-AAGCAGTCGCCAAATATTCC-3'). As expected, a 3.4-kb DNA fragment was amplified from the wild-type strain. An amplification product of about 1.0 kb was found in only 1 of 60 sucrose-resistant, chloramphenicol-sensitive colonies assayed. Nucleotide sequence determination showed that the amplified fragment (1,108 bp) contains the wzm-wzt deletion. N28b-4 LPS did not react with O4-antigen antiserum (Fig. 6, lane 3). ELISAs (Table 4) with S. marcescens O4-antigen-specific antiserum were performed with different cellular fractions from S. marcescens N28b and N28b-4. As expected, the outer membrane fraction in wild-type S. marcescens N28b showed a high affinity for the specific antiserum. By contrast, the inner membrane fraction obtained from S. marcescens N28b-4 showed a high response to specific antiserum, while no response to the same antiserum was observed for the outer membrane fraction of this mutant strain. Furthermore, the outer membrane fraction from strain N28b-4 transformed with pSUB6 again showed affinity for the O4-specific antiserum. These results strongly suggest that these two proteins are indeed involved in O4-antigen export in S. marcescens N28b, and the N28b-4 mutant seems to accumulate the anti-O4-reactive products in its cytosol and inner membrane fraction.

Origin of the Tn1000 insertion. The Tn1000 insertion element in the wbbA gene in plasmid pSUB7 was an unexpected result. To our knowledge, no Tn1000 insertion has been found in S. marcescens (7), nor in the original recombinant cosmid FGR2; in plasmid pSUB6, a similar element could be detected by analysis of the vector and insert nucleotide sequence. E. coli XL1-Blue contains a Tn1000 element in its F' plasmid (7), and pSUB7 was obtained in this strain background. Then, most probably the Tn1000 element found in the wbbA gene resulted from a transposition event, originating in E. coli XL1-Blue. Since this strain is widely used in molecular genetic manipulations, researchers working with this strain should be aware of the transposition phenomenon (with the corresponding gene knockout) that we have found.