Genetic and Structural Characterization of the Core Region of the Lipopolysaccharide from Serratia marcescens N28b (Serovar O4)

JOURNAL OF BACTERIOLOGY, Feb. 2004, p. 978–988 Vol. 186, No. 40021-9193/04/$08.00�0 DOI: 10.1128/JB.186.4.978–988.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Genetic and Structural Characterization of the Core Region of theLipopolysaccharide from Serratia marcescens N28b (Serovar O4)

Nuria Coderch,1 Nuria Pique,1 Buko Lindner,2 Nihal Abitiu,1 Susana Merino,3 Luis Izquierdo,3Natalia Jimenez,3 Juan M. Tomas,3 Otto Holst,4 and Miguel Regue1*

Departamento de Microbiología y Parasitología Sanitarias, Facultad de Farmacia, Universidada de Barcelona, 08028 Barcelona,1

and Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, 08071 Barcelona,3 Spain, and Divisionof Biophysics2 and Division of Structural Biochemistry,4 Research Center Borstel, Leibnitz Center for Medicine and

Biosciences, D-23845 Leibnitz, Germany

Received 2 September 2003/Accepted 6 November 2003

The gene cluster (waa) involved in Serratia marcescens N28b core lipopolysaccharide (LPS) biosynthesis wasidentified, cloned, and sequenced. Complementation analysis of known waa mutants from Escherichia coli K-12,Salmonella enterica, and Klebsiella pneumoniae led to the identification of five genes coding for products involvedin the biosynthesis of a shared inner core structure: [L,D-HeppIII�(137)-L,D-HeppII�(133)-L,D-HeppI�(135)-KdopI(442)�KdopII] (L,D-Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid). Complementation and/or chemical analysis of several nonpolar mutants within the S. marc-escens waa gene cluster suggested that in addition, three waa genes were shared by S. marcescens and K.pneumoniae, indicating that the core region of the LPS of S. marcescens and K. pneumoniae possesses additionalcommon features. Chemical and structural analysis of the major oligosaccharide from the core region of LPSof an O-antigen-deficient mutant of S. marcescens N28b as well as complementation analysis led to the followingproposed structure: �-Glc-(136)-�-Glc-(134))-�-D-GlcN-(134)-�-D-GalA-[(241)-�-D,D-Hep-(241)-�-Hep]-(133)-�-L,D-Hep[(741)-�-L,D-Hep]-(133)-�-L,D-Hep-[(441)-�-D-Glc]-(135)-Kdo. The D configura-tion of the �-Glc, �-GclN, and �-GalA residues was deduced from genetic data and thus is tentative.Furthermore, other oligosaccharides were identified by ion cyclotron resonance–Fourier-transformed electro-spray ionization mass spectrometry, which presumably contained in addition one residue of D-glycero-D-talo-oct-2-ulosonic acid (Ko) or of a hexuronic acid. Several ions were identified that differed from others by a massof �80 Da, suggesting a nonstoichiometric substitution by a monophosphate residue. However, none of thesemolecular species could be isolated in substantial amounts and structurally analyzed. On the basis of thestructure shown above and the analysis of nonpolar mutants, functions are suggested for the genes involved incore biosynthesis.

In gram-negative bacteria, the lipopolysaccharide (LPS) isone of the major structural and immunodominant molecules ofthe outer membrane. It consists of three domains: lipid A, coreoligosaccharide, and O-specific polysaccharide, or O antigen.The genetics of O-antigen biosynthesis has been intensivelystudied in members of the family Enterobacteriaceae and othergram-negative bacteria. Studies on characterization of thegenes involved in LPS core biosynthesis in Escherichia coli,Salmonella enterica, and Klebsiella pneumoniae have shownthat these genes are usually found clustered in a region of thechromosome, the waa (rfa) gene cluster (19, 38). This genearrangement is not always present in other gram-negative bac-teria (35): e.g., in Bordetella, it was shown that genes involvedin the biosynthesis of the O antigen and core region arepresent in the same gene cluster (2). (The nomenclature pro-posed in 1996 by Reeves et al. [37] for proteins and genesinvolved in core LPS biosynthesis is used in this work, with thenames originally reported given in parentheses.)

All core regions identified (23, 24) contain at least oneresidue of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), which

links this region to the lipid A moiety (Kdo I). The secondcharacteristic sugar of the core region is L-glycero-D-manno-heptose (L,D-Hep), in addition to which, D-glycero-D-manno-heptopyranose (D,D-Hep) is present in a few LPSs. This sugarwas identified as the biosynthetic precursor of L,D-Hep. Also,there are few LPSs that contain only D,D-Hep and others thatlack heptose completely. Either Kdo I (in Acinetobacter) orKdo II (in Burkholderia cepacia and Yersinia pestis) (23, 24) canbe replaced by D-glycero-D-talo-oct-2-ulopyranosonic acid (Ko).Its biosynthesis and the regulation of the exchange betweenKdo and Ko are still unknown.

In those cases in which L,D-Hep is present, the presence ofone Hep-�-(135)-Kdo moiety is a characteristic feature. KdoI may further be substituted at O-4 by a second Kdo residue(Kdo II; e.g., in S. enterica and E. coli).

Serratia marcescens is a recognized nosocomial pathogenthat causes pneumonia, septicemia, meningitis, and urinarytract infections (1, 7). S. marcescens N28b (O4) produces abacteriocin able to kill E. coli K-12 (48); this bacteriocin bindsto the core of LPS and to the outer membrane proteins OmpAand OmpF of sensitive E. coli cells (11). It was expected thatexpression of foreign genes in E. coli K-12 leading to alter-ations of the relative amounts or composition of the outermembrane molecules that interact with bacteriocin 28b would

* Corresponding author. Mailing address: Departamento de Micro-biología y Parasitología Sanitarias, Facultad de Farmacia, Univer-sidada de Barcelona, Av. Joan XXIII, 08028 Barcelona, Spain. Phone:34-93-4024496. Fax: 34-93-4024498. E-mail: [email protected].

978

confer a bacteriocin-resistant phenotype. We have shown thatbacteriocin 28b is a useful tool to identify recombinant plas-mids or cosmids harboring structural genes for small Ail-likeouter membrane proteins that, when expressed in E. coli K-12,lead to a decrease in the outer membrane proteins OmpA andOmpF (16). Similarly, we have shown that expression in E. coliK-12 of genes coding for enzymes involved in S. marcescensO-antigen (40, 41) and core region (17, 38) biosyntheses confera bacteriocin-resistant phenotype. This approach allowed theidentification and characterization of the S. marcescens waaA(kdtA) gene, coding for Kdo transferase, and the adjacentwaaE (kdtX) gene (17, 25).

In this work, the characterization of the complete waa genecluster involved in S. marcescens LPS core biosynthesis and astructural investigation of the core region are presented.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. Bacterial strains (Table 1)were grown in Luria-Bertani (LB) broth and on LB agar (30). LB medium wassupplemented with kanamycin (50 �g ml�1), ampicillin (100 �g ml�1), chloram-phenicol (30 �g ml�1), and tetracycline (25 �g ml�1) when needed. The physicalmaps of the plasmids used in this study are shown in Fig. 1.

Bacteriocin 28b production and sensitivity assay. Bacteriocin 28b was pre-pared as previously described (48). The overlay test for qualitative bacteriocinsensitivity assays and quantitative bacteriocin sensitivity assays were performedas previously described (11).

Southern blot hybridization. The DNA fragment containing the waaA andwaaE genes from S. marcescens was labeled with digoxigenin as described by themanufacturer (Boehringer Mannheim). BamHI-digested CosFGR16 DNA waselectrophoresed, denatured, and transferred to Hybond B membrane. Afterbaking, the membrane was prehybridized and hybridized in 5� SSC (1� SSC is0.15 M NaCl plus 0.015 M sodium citrate)–0.5% blocking reagent (BoehringerMannheim)–0.1% Sarkosyl–0.02% sodium dodecyl sulfate (SDS). Washing, an-tibody incubation, and signal detection with p-nitroblue tetrazolium chloride and5-bromo-4-chloro-3-indolylphosphate were done as recommended by the man-ufacturer (Boehringer Mannheim).

General DNA methods. General DNA manipulations were done essentially asdescribed previously(42). DNA restriction endonucleases, T4 DNA ligase, E. coliDNA polymerase (Klenow fragment), and alkaline phosphatase were used asrecommended by the suppliers. The S. marcescens N28b genomic DNA used wasdescribed previously (41). The following plasmids subcloned in vector pGEMTwere constructed by ligation to the vector of PCR-amplified products: pGEMT-WaaCSm (5�-GTTTAATGCACGTTGCCGCA-3� and 5�-CCCAGGTTGATAATGTGCAG-3�), pGEMT-WaaFSm (5�-ACAAAAAAGGCAGCATCGAGTA-3� and 5�-TGTCGCTGCCGAACCAGTTT-3�), pGEMT-WaaLSm (5�-GCTGTTGTCGCATATCGACT-3� and 5�-TGCATGCTGCAGGCCGACATT-3�),pGEMT-WaaQSm (5�-GCGAACTCGACGTAAGCC-3� and 5�-TGCACGCCCATAAAGTGAA-3�), pGEMT-orf9-10Sm (5�-TCAAATGCTGGAGCGAAGAG-3� and 5�-TGTTCTTTGGCGATACCGATA-3�), pGEMT-orf11 (5�-AATCCGCCGCAGATAAATCA-3� and 5�-GATCACCAGCTTGGGATTCA-3�),pGEMT-WaaASm (5�-AGGCGTGGTGCAAACAAGAT-3� and 5�-AAGACTTTGGCGCCCAGACT-3�), and pGEMT-WaaESm (5�-ACCTTCAACTTTAAAGACA-3� and 5�-AAAGTCAGACACCGCCCG-3�).

DNA sequencing and computer analysis of sequence data. Double-strandedDNA sequencing was performed by using the Sanger dideoxy-chain terminationmethod (43) with the Abi Prism dye terminator cycle sequencing kit (Perkin-Elmer). The relevant parts of CosFGR2 and CosFGR16 inserts were sequencedwith oligonucleotide T3 (5�-AATTAACCCTCACTAAAGGG-3�), which bindsto the T3 promoter region on vector SuperCos1; oligonucleotide F412 (5�-TTTGCACCACGCCTCTGA-3�) to extend the sequence from the previously re-ported waaA gene (17); and oligonucleotide FB1 (5�-CGGCTTCCTCGACGGTAAA-3�) to obtain sequence data downstream of the known waaE genesequence (17). Other sequence-derived oligonucleotides were used to extend thenucleotide sequence. Primers used for DNA sequencing were purchased fromAmersham Pharmacia Biotech. The DNA sequence was translated in all sixframes, and all open reading frames (ORFs) greater than 100 bp were inspected.Deduced amino acid sequences were compared with those of DNA translated inall six frames from nonredundant GenBank and EMBL databases by using theBLAST (3, 4) and FASTA (33) network service at the National Center for

Biotechnology Information and the European Biotechnology Information, re-spectively. The Genetics Computer Group (Madison, Wis.) package Terminatorprogram was used for prediction of possible terminator sequences. Clustal W(46) was used for multiple sequence alignments. Hydropathy profiles were cal-culated according to Kyte and Doolitle (26). The TopPredII program (9) wasused to identify predicted protein transmembrane domains.

LPS isolation and electrophoresis. Cultures for analysis of LPS were grown inTrypticase soy broth at 37°C. LPSs were extracted with either hot phenol-water(51) or phenol-chloroform-light petroleum (13). For screening purposes, LPSwas obtained after proteinase K digestion of whole cells (21). LPS samples wereseparated by SDS-polyacrylamide gel electrophoresis (PAGE) or SDS-Tricine-PAGE and visualized by silver staining as previously described (34, 47).

Isolation of oligosaccharide 3. The LPS was hydrolyzed in 1% acetic acid(100°C for 90 min), and the precipitate was removed by centrifugation (2,500 �g for 1 h). The supernatant was evaporated to dryness, dissolved in water,centrifuged (100,000 � g at 4°C for 4 h), and separated by gel permeationchromatography on a column (2.5 by 70 cm) of Sephadex G-50. The coreoligosaccharides were then separated by high-performance anion-exchange chro-matography (HPAEC) on a column (4 by 250 mm; Dionex Corp.) of CarboPackPA100, which was eluted at 1 ml min�1 with a linear gradient program of 15 to40% 1 M sodium acetate in 0.1 M NaOH over 70 min, and isolated fractions weredesalted by gel-permeation chromatography on a column (1 by 70 cm) of Seph-adex G-10 in 10 mM aqueous NH4HCO3.

Compositional analyses. Neutral sugar and uronic acid (as neutral sugars,after reduction of the carboxyl group) analyses, fatty acid analyses, determinationof organic bound phosphate, and Kdo and GlcN quantification were performedas described previously (31).

NMR spectroscopy. For structural assignments, NMR spectra were recordedon a solution (0.5 ml) of oligosaccharide 3 (2 mg) in 2H2O with a Bruker AMX600 spectrometer (1H NMR, 600.13 MHz; 13C NMR, 125.77 MHz) and a BrukerDigital Avance 800 instrument at 27 or 47°C. The resonances were measuredrelative to internal acetone: (CH3)2CO �H, 2.225; �C, 31.07. The correlation(COSY), total correlation spectroscopy (TOCSY), double-quantum-filteredCOSY (DQFCOSY), as well as the 1H-13C-heteronuclear multiple-quantumcoherence (HMQC) and nuclear Overhauser enhancement spectroscopy(NOESY) experiments were all measured with standard Bruker software.

Mass spectrometry. Ion cyclotron resonance Fourier-transformed electrosprayionization mass spectrometry (ESI FT-ICR-MS) was performed in the negative-ion mode with an APEX II instrument (Bruker Daltonics) equipped with a 7-Tactively shielded magnet and an Apollo ion source. Mass spectra were acquiredwith standard experimental sequences as provided by the manufacturer. Sampleswere dissolved at a concentration of �10 ng � �l�1 in a 50:50:0.001 (vol/vol/vol)mixture of 2-propanol, water, and triethylamine and sprayed at a flow rate of 2�l � min�1. Capillary entrance voltage was set to 3.8 kV, and the dry gas tem-perature was set to 150°C. To facilitate the interpretation, mass spectra, whichshowed several charge states for each component, were charge deconvoluted,and the mass numbers given refer exclusively to the monoisotopic molecularmasses.

S. marcescens orf7, orf9-10, and waaE mutant construction. To obtain S. marc-escens mutant strains N28b30, N28b20, and N28b16, the method of Link et al.(28) was used to create chromosomal in-frame waa deletions. Briefly, CosFGR16and primer pairs A (5�-CGCGGATCCCCGTTGGGCGTTCAACGAAT-3�),B (5�-CCCATCCACTAAACTTAAACAGAACCAGTCGGCAACCTTAAT-3�), and C (5�-TGTTTAAGTTTAGTGGATGGGATTCAGCCGCAGCGGATTTAT-3�), D (5�-CGCGGATCCGCAGGGGAAACGTTCGAAGA-3�) wereused in two sets of asymmetric PCRs to amplify DNA fragments of 602 (AB) and546 (CD) bp, respectively. DNA fragment AB contains from nucleotide 7302(corresponding to the third base of the 13th codon of orf7) to nucleotide 7904.DNA fragment CD contains from nucleotide 5756 (inside orf6) to nucleotide6302 (corresponding to the first base of codon 346 of orf7). DNA fragments ABand CD were annealed at their overlapping region (underlined letters in primersB and C) and amplified by PCR as a single fragment with primers A and D. Thefusion product was purified, BamHI digested (BamHI site shown as double-underlined letters in primers A and D), ligated into BamHI-digested and phos-phatase-treated pKO3Km vector, electroporated into E. coli DH5�, and platedon kanamycin plates at 30°C to obtain plasmid pKO3Kmorf7. Plasmid pSKF41(17) and primer pairs A1 (5�-CGCGGATCCCACCGCAAGCTGCTGGAAAA-3�) and B1 (5�-CCCATCCACTAAACTTAAACAGCTTTTGCGGCTGCTCATTC-3�) and C1 (5�-TGTTTAAGTTTAGTGGATGGGGTGGTCAACGCGCAATATAC-3�) and D1 (5�-CGCGGATCCTCCTTCACCAGTGATGAGGA-3�) were used to obtain plasmid pKO3KmwaaE, which contains an internallydeleted waaE gene (the first 6 codons, a 7-codon tag, and the last 24 codons)flanked by 541 bp upstream and 409 bp downstream. Plasmid CosFGR16 and

VOL. 186, 2004 SERRATIA MARCESCENS N28b LIPOPOLYSACCHARIDE 979

primer pairs A2 (5�-CGCGGATCCAAATGCTGGAGCGAAGAGA-3�) andB2 (5�-CCCATCCACTAAACTTAAACACGCCAAAAGAAATGCTTTC-3�)and C2 (5�-TGTTTAAGTTTAGTGGATGGGAATCCGCCGCAGATAAATCA-3�) and D2 (5�-CGCGGATCCTTGGGCACGAAAGATATTCA-3�) (BamHIsite shown as double-underlined letters in primers A2 and D2) were used toobtain plasmid pKO3Kmorf9-10, which contains a double-deleted orf9-orf10pair (the first 7 codons of orf9, a 7-codon tag, and the last 20 codons of orf10)flanked by 480 bp upstream and 787 bp downstream. Plasmids pKO3Kmorf7,pKO3KmwaaE, and pKO3Kmorf9-10 were used to construct mutantsN28b30, N28b16, and N28b20, respectively.

Nucleotide sequence accession number. The nucleotide sequence of the S.marcescens N28b waa gene cluster containing the gmhD, waaF, waaC, orf4, waaL,

orf6, orf7, waaQ, orf9, orf10, orf11, waaA, waaE, coaD, and fpg genes has beendeposited in GenBank under accession no. U 52844 (Fig. 1).

RESULTS

Cloning and sequence determination of the S. marcescenswaa gene cluster. We have previously reported the isolationand characterization of S. marcescens N28b waaA, coding forKdo transferase, and waaE (kdtX) genes from recombinantCosFGR2 (17). DNA fragments containing waaQ (rfaQ) or

TABLE 1. Bacterial strains and plasmids used in this study

Strain, cosmid, or plasmid Relevant characteristic(s)a Source orreference

StrainsS. marcescens Serotype O:4 14

N28bN28b4 N28b-derived wbbA mutant (lacks O:4 antigen) 41N28b16 N28b-derived waaE mutant This workN28b20 N28b-derived double orf9 orf10 mutant This workN28b30 N28b-derived orf7 mutant This work

K. pneumoniae 3252145 O1:K2NC16 52145-derived waaE mutant 38NC19 52145-derived waaQ mutant 38

E. coli 36NM554 recA13 araD139 (ara-leu)7696 (lac)X74 galE15 galK16 hsdR2 rpsL

mcrA mcrBDH5� F� endA hsdR17(rK

� mK�) supE44 thi-1 recA1 gyrA96 80lacZM15

(argF lacZYA)U16918

X11 Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac (F� proABlacI�ZM15 Tn10)

Stratagene

CJB26 waaA::kan recA-harboring plasmid pJSC2 5

S. enterica serovar TyphimuriumSA1377 waaC630 8SL3789 waaF511 39SL3749 waaL 29

Cosmids and plasmidsSuperCos1 Tetr Kmr cosmid vector StratageneCosFGR2 Recombinant SuperCos1 harboring the S. marcescens orf10, orf11, and

waaA, waaE, coaD, and fpg genes17

CosFGR16 Recombinant SuperCos1 harboring S. marcescens waaC, orf4, waaL,orf6, orf7, waaQ, orf9, orf10, orf11, waaA, waaE, and coaD

This work

pKO3 Cmr sacB temperature-sensitive replication 28pKO3Km Kmr sacB temperature-sensitive replicon This workpKO3orf7 pKO3 containing the engineered orf7 deletion This workpKO3orf9–10 pKO3 containing the engineered double orf9 orf10 deletion This workpKO3waaE pKO3 containing the engineered waaE deletion This workpGEMT Ampr plasmid vector PromegapGEMT-WaaCSm S. marcescens waaC gene in pGEMT This workpGEMT-WaaFSm S. marcescens waaF gene in pGEMT This workpGEMT-WaaASm S. marcescens waaA gene in pGEMT This workpGEMT-WaaESm S. marcescens waaE gene in pGEMT This workpGEMT-WaaEKp K. pneumoniae waaE gene in pGEMT 38pGEMT-WaaLSM S. marcescens waaL gene in pGEMT This workpGEMT-WaaQSm S. marcescens waaQ gene in pGEMT This workpGEMT-Orf9–10Sm S. marcescens orf9 and �10 in pGEMT This workpGEMT-Orf8–9Kp K. pneumoniae orf8 and �9 in pGEMT 38pGEMT-Orf7Sm S. marcescens orf7 in pGEMT This workpGLU LgtF from H. ducreyi 12pJSC2 Cmr temperature-sensitive replication plasmid containing E. coli waaA 5

a Ampr, ampicillin resistance; Cmr, chloramphenicol resistance; Kmr, kanamycin resistance; Tetr, tetracycline resistance.

980 CODERCH ET AL. J. BACTERIOL.

waaGPSBI (rfaGPSBI) genes from E. coli K-12 were found tohybridize to recombinant CosFGR2, suggesting that other coreLPS biosynthesis-encoding genes were located in CosFGR2besides waaA and waaE. The nucleotide sequence of the wholeinsert in CosFGR2 revealed only two putative complete openreading frames (ORFs) upstream of the waaA gene (Fig. 1). Tofind further upstream genes, an S. marcescens N28b genomiclibrary was screened again by a previously described approach(17). A recombinant cosmid was isolated (CosFGR16) thatoverlapped with CosFGR2 and contained six additional com-plete ORFs (Fig. 1). In all of the waa gene clusters character-ized so far in the Enterobacteriaceae, the cluster begins with agmhD gene (19, 38). To characterize this region, two oligonu-cleotides were used. The GMHD1 oligonucleotide (5�-TGAAAGSCGGCACCAAGTTT-3�) was designed from the knownK. pneumoniae gmhD gene sequence (38), and the WAAF1oligonucleotide (5�-GTGTCGCTGCCGAACCAGTTT-3�)was designed from the sequence determined from the insert ofCosFGR16. Using genomic DNA from the N28b strain as atemplate, these oligonucleotides generated a PCR-amplifiedDNA fragment of about 2 kbp. The nucleotide sequence de-termined for this fragment overlapped that of the CosFGR16insert (Fig. 1).

Analysis of the S. marcescens waa gene sequence. Analysis ofthe 20,693-bp nucleotide sequence revealed 14 ORFs. Se-quences corresponding to putative ribosome binding sites werefound upstream of each of the ORF start codons. Data sum-

marizing the location of the ORFs and the characteristics ofthe putative encoded proteins are shown in Table 2. The anal-ysis of the intergenic regions between the successive ORF pairsrevealed distances of 10 (orf1-orf2), 3 (orf2-orf3), and 29 (orf4-orf5) bp and overlapping stop and start codons for the orf3-orf4and orf5-orf6 pairs. Since no sequences similar to Rho-inde-pendent transcription termination sequences were found be-tween orf4 and orf5, this organization suggests that the first sixORFs constitute a transcriptional unit. A similar analysis of theintergenic regions between the other ORFs suggests that orf8,-9, and -10 and orf12, -13, and -14 constitute two additionaltranscription units, while orf7 and -11 apparently correspond tomonocistronic genes transcribed in the opposite direction.

S. marcescens waa genes shared by all known Enterobacteri-aceae. In the LPSs of the Enterobacteriaceae studied so far, theinner core region contains one to three Kdo residues and atleast three residues of L-glycero-D-manno-heptopyranose (L,D-HeppI, L,D-HeppII, and L,D-HeppIII). These residues consti-tute the structure L-�-D-HeppIII-(137)-L-�-D-HeppII-(133)-L-�-D-HeppI-(135)-[�-KdopII-(234)-]-�-KdopI (23, 24). The5�-truncated orf1 and orf2, orf3, orf8, and orf12 had high levelsof amino acid identity to the known enterobacterial GmhD(ADP-D-glycero-D-manno-heptose epimerase) (95 to 96%),WaaF (ADP-heptose-LPS heptosyltransferase II) (82 to 88%),WaaC (ADP-heptose-LPS heptosyltransferase I) (82 to 88%),WaaQ (ADP-heptose-LPS heptosyltransferase III) (82 to88%), and WaaA (bifunctional CMP-Kdo:lipidA Kdo trans-

FIG. 1. Diagram of the S. marcescens N28b waa region and comparison of this cluster with those of E. coli K-12, S. enterica serovarTyphimurium, and K. pneumoniae C3. The physical maps of plasmids used in this study showing only insert DNA are shown. Small arrows denoteprimers used to amplify and characterize the gmhD-waaF region. Inner core genes common to all known Enterobacteriaceae (black arrows), innercore genes common to E. coli and S. enterica serovar Typhimurium (arrows with horizontal bars), core genes common to S. marcescens N28b (O4)and K. pneumoniae (striped arrows), hypothetical lipooligosaccharide or capsule-related genes (checkerboard arrows), and the O-antigen ligasegene (gray arrows) are also shown.


ferase (83 to 88%) proteins, respectively (10, 19, 20, 38) (Fig.1).

Complementation analyses of known inner core backbonemutants were performed to confirm the functions of thesegenes. E. coli strain CJB26 harbors a kanamycin resistancedeterminant inserted in the waaA gene and a wild-type waaAgene in a temperature-sensitive plasmid (pJSC2), leading to atemperature-sensitive phenotype. A plasmid containing the S.marcescens waaA gene (pGEMT-WaaASm) was found to re-store growth at 44°C in the E. coli CJB26 mutant.

S. enterica serovar Typhimurium mutant strains SA1377(waaC630) and SL3789 (waaF511) were complemented byplasmids pGEMT-WaaCSm and pGEMT-WaaFSm, respec-tively, as judged by analysis of LPS by SDS-Tricine-PAGE.These results strongly suggested that orf3 and orf2 coded forthe ADP-heptose-LPS heptosyltransferase I and ADP-hep-tose-LPS heptosyltransferase II, respectively.

To identify the gene encoding ADP-heptose-LPS heptosyl-transferase III, the K. pneumoniae waaQ mutant strain NC19was used. LPS obtained from mutant NC19 contained O anti-gen and migrated slightly faster than that of the parent 52145strain. Strain NC19 was transformed with a plasmid containingthe S. marcescens orf8 (pGEMT-WaaQSm). LPS from thetransformed strain showed an electrophoretic banding patternidentical to that of the wild-type strain (data not shown). Theseresults suggested that orf8 codes for ADP-heptose-LPS hepto-syltransferase III.

Three other waa genes shared by S. marcescens N28b and K.pneumoniae C3. We have shown that in both S. marcescens andK. pneumoniae, a similar gene (waaE) is present downstreamfrom the waaA gene (17, 38) (Fig. 1). Recently, we presentedevidence suggesting that the K. pneumoniae waaE gene is in-volved in the addition of a branched D-Glc residue to L,D-HeppI by a �-(134) linkage (25). Both WaaE proteins sharehigh levels of similarity and identity (70 and 80%), suggestingthat they perform the same function. To test this hypothesis, anS. marcescens waaE nonpolar mutant was constructed essen-tially as previously described (28, 38). LPSs from strains N28b(wild type) and N28b16 (waaE) were extracted and analyzed bySDS-Tricine-PAGE. The result obtained (Fig. 2, lanes 1 and 2)showed that the core LPS from strain N28b16 migrated fasterthan that of the wild-type strain, and it appeared that themutant LPS still contained O antigen, although in smalleramounts than wild-type LPS. Plasmids containing either the S.marcescens waaE gene (pGEMT-WaaESm) or the K. pneu-moniae waaE homologue (pGEMT-WaaEKp) were trans-formed into NC16 and N28b16 mutants. Analysis of LPS bySDS-Tricine-PAGE showed that both waaE homologues wereable to complement waaE mutations in both strains (Fig. 2,lane 3). In addition, the lgtF gene from Haemophilus ducreyi(Fig. 2, line 4) complemented strain N28b16. These resultssuggested that, similarly to K. pneumoniae and H. ducreyi, asubstitution of position O-4 of L,D-HeppI by a �-D-glucopyr-anose [�-D-Glcp-(134)-L,D-HeppI] should be present in the S.marcescens LPS inner core.

The deduced 375-amino-acid protein encoded by orf9 wasfound to share high levels of similarity (80%) and identity(70%) to the protein encoded by orf8Kp in the K. pneumoniaewaa gene cluster (38) (Fig. 1). Furthermore, both proteinsshared the same number of residues and also showed limited

similarity to WaaG proteins from Pseudomonas aeruginosa (ac-cession no. O33426) and S. enterica (19) and E. coli core typesK-12 (44) and R2, R3, and R4 (19, 20). WaaG protein wasreported to be a glucosyltransferase involved in the �133linkage of D-GlcpI to L,D-HeppII in E. coli and S. enterica (19).The deduced 366-amino-acid protein encoded by orf10Sm

showed similarity (68%) and identity (58%) to the proteinencoded by orf9Kp in the K. pneumoniae waa gene cluster (38)(Fig. 1). The proteins encoded by orf8Kp, orf9Kp, orf9Sm, andorf10Sm belong to the retaining glycosyltransferase family 4(http://afmb.cnrs-mrs.fr/�pedro/CAZY/db.html).

The above analyses suggested that S. marcescens orf9 andorf10 were involved in biosynthesis of the core region. To testthis hypothesis, a double nonpolar orf9 orf10 mutant, strainN28b20, was constructed (see Materials and Methods). LPSwas extracted from the mutant and wild-type strains, and anal-ysis of LPS preparations by SDS-Tricine-PAGE showed a fast-er-migrating band for the mutant strain N28b20 LPS (Fig. 3,lanes 1 and 2). As expected, the mutant phenotype was com-plemented by introduction of plasmid pGEMT-orf9Sm-10Sm

(Fig. 3, lane 3), while neither orf9 nor orf10 alone was able tocomplement the double mutation. The similarity of S. marc-escens orf9 and orf10 to K. pneumoniae orf8 and orf9 indicatedthat these ORFs could perform similar functions in the innercore biosynthesis in both species. Further support for this sug-gestion was obtained by complementation of the N28b20 mu-tant by pGEMT-orf8Kp-9Kp, as judged by SDS-Tricine-PAGEanalysis (Fig. 3, lane 4).

Core structure of LPS from S. marcescens strain N28b4.These genetic data indicated that the core region of the LPSfrom S. marcescens N28b should share with the LPS from K.pneumoniae three residues in addition to the common innercore features shared by all known cores from LPS of Enter-obacteriaceae. To test this hypothesis, the structure of the coreregion from LPS of the O-antigen-deficient mutant N28b4 wasinvestigated. Sugar analyses of the LPS revealed the presence

TABLE 2. S. marcescens N28b waa gene cluster and downstreamcoaD (kdtB) and fpg genes

Locus Basepositionsd

No. ofaminoacids

Molecularmass

(kDa)pIa GRAVYb

orf1 (gmhD)c 2–775 257 29.3 4.87 �0.444orf2 (waaF) 785–1831 348 38.7 9.11 �0.123orf3 (waaC) 1834–2799 321 35.9 8.44 �0.318orf4 2796–3962 388 43.4 8.39 �0.104orf5 (waaL) 3991–5232 413 46.0 9.40 0.586orf6 5229–6209 326 37.4 8.89 �0.277orf7 6246–7340c 364 41.2 9.02 �0.161orf8 (waaQ) 8516–9598 360 40.1 7.22 �0.037orf9 9595–10722 375 42.3 9.47 �0.201orf10 10725–11825 366 40.3 9.14 �0.114orf11 11870–12883c 337 38.2 8.97 �0.239orf12 (waaA) 13009–14286 425 47.6 9.53 �0.055orf13 (waaE) 14287–15060 257 29.2 8.82 �0.283orf14 (coaD) (15) 15064–15549 161 17.6 5.55 0.242orf15 (fpg) (44) 15553–16368c 271 30.5 8.56 �0.339

a Isoelectric point of the protein calculated using ProtParam at the ExPassyserver.

b Grand average hydropathicity of the protein calculated by the Kyte andDoolitle method (26).

c Truncated ORF.d c indicates that base positions correspond to the complementary DNA

strand.


of Glc, GlcN, GalA, Kdo, and L,D- and D,D-Hep residues (Fig.4). In addition, small amounts of Ko were detected in Glc-MSanalysis.

The core oligosaccharide fraction was isolated from LPSafter acetic acid hydrolysis and gel permeation chromatogra-phy on Sephadex G-50 (see Materials and Methods) and fur-ther separated by preparative HPAEC, yielding a complexmixture of oligosaccharides, from which oligosaccharides 1, 2,and 3 were obtained. Monosaccharide analysis of these threeisolates revealed that all contained Glc, GlcN, GalA, L,D-Hep,and D,D-Hep; however, the latter was present in oligosaccha-ride 2 only in traces. Since 1H NMR analyses (not shown) ofoligosaccharide 2 suggested that this was not a pure compoundand since 1H NMR analyses (not shown) of oligosaccharide 1suggested that it represented a smaller variant of oligosaccha-ride 3, only the structure of oligosaccharide 3 was studied indetail by 1H and 13C NMR spectroscopy.

The anomeric region of the 1H NMR spectrum region ofoligosaccharide 3 contained 10 signals (Fig. 5 and Table 3).Additionally, signals of a deoxy compound (residue K) wereidentified between 2.34 and 3.96 ppm. The 11 residues could beidentified as 5 heptose residues, 3 hexose residues, 1 aminohex-ose residue, 1 hexosuronic acid residue, and, presumably, 1Kdo derivative. Their identification was possible by the assign-ment of most chemical shifts, utilizing two-dimensional homo-nuclear 1H, 1H COSY, TOCSY, and NOESY and hetero-nuclear 1H, 13C HMQC experiments. Two hexose residues (Jand I, Fig. 5) possessed the �-gluco-configuration, as identifiedby their chemical shift data and J2,3 coupling constants. An-other hexose (residue A) and the hexosamine (F) possessedthe �-gluco configuration. The hexuronic acid was �-galactoconfigured, and the heptoses (C, D, E, G, and H) were�-manno configured, which in the latter cases was establishedby the chemical shifts of the anomeric protons and their smallJ2,3 coupling constants of about 1 Hz. According to the ano-meric proton shifts and the coupling constants of H-1, H-2,H-3, and H-4, the heptose residues could be classified intothree groups. Residues G and H gave very similar chemicalshifts and possessed anomeric proton resonances at high field,in agreement with their proposed terminal nature. The ano-meric proton of the proposed trisubstituted heptose C was theone possessing a chemical shift at relative low field. In heptoses

D and E, the chemical shifts of the anomeric protons werefound in intermediate positions. Finally, residue K resembledKdo: however, with some unidentified modification. Probably,it represented an artifact that was formed from Kdo duringalkaline HPAEC. The chemical shifts at 2.34 and 2.81 ppm(probably of protons H-3ax and H-3eq) were not consistent withthose published for pyranosidic Kdo (6). In the 13C NMR

FIG. 2. SDS-Tricine-PAGE analysis of LPS from S. marcescens.N28b (wild type) (lane 1), N28b16 (waaE) (lane 2), N28b16(pGEMT-WaaEKp) (lane 3), and N28b16(pGLU) (lane 4).

FIG. 3. SDS-Tricine-PAGE analysis of LPS from S. marcescensN28b (lane 1), N28b20 (double orf9 orf10 mutant) (lane 2),N28b20(pGEMT-Orf9-10Sm) (lane 3), and N28b20(pGEMT-Orf8-9Kp) (lane 4).

FIG. 4. Proposed structure of oligosaccharide 3 isolated from theLPS from S. marcescens N28b4. The D configuration of residues B, F,and I was deduced from genetic data and thus is tentative, as is thepartial structure D-(I-)-C. In oligosaccharide 1, residues H and Kcould not be identified. The proposed functions for the S. marcescenswaa genes and the effect of the double orf9 orf10 mutant (N28b20) arealso shown.


spectrum, the chemical shift of a carbonyl group was identifiedat 180.35 ppm. Chemical shifts at 2.34 and 2.81 ppm possessedintensities similar to those of the anomeric proton signals ofthe other residues. Thus, it was supposed that residue K waspart of oligosaccharide 3. Other proton chemical shifts of Kwere at 3.64, 3.87, and 3.96 ppm.

The 13C NMR chemical shifts were assigned by hetero-nuclear 1H, 13C HMQC experiments using the interpreted 1HNMR spectrum. Nine anomeric signals were identified in the13C NMR spectrum of oligosaccharide 3. There was no ano-meric signal, but a chemical shift (probably of C-3 of themodified Kdo residue) could be detected. Low-field shiftedsignals indicated substitutions at O-2 (residue E), O-4 (residueF), O-6 (residue A), O-2 and O-4 (residue B), O-3 and O-4(residue C), and O-3 and O-7 (residue D). Residues J, G, I,and H were terminal sugars.

The sequence of the monosaccharide residues was deter-mined by using data obtained from a NOESY experiment(Table 4). Interresidual NOE contacts were identified fromH-1 of GalA residue B to protons H-3 (strong) and H-2 ofheptose D and to H-1 of heptose E; H-1 of residue A and H-4(strong) of residue F; H-1 of residue D and H-3 (strong), H-4,and H-2 of residue C; H-1 of residue E, H-1 (strong) and H-2(strong) of residue B, and H-1 (weak) of residue G; H-1 ofresidue F and H-4 (weak) and H-5 (strong) of residue B; H-1of residue G and H-2 and H-3 (both strong) of residue E; H-1of residue H and H-7 (strong) and H-6 of residue D; H-1 ofresidue I and H-2 (weak) and H-6 (strong) of residue C; andH-1 of residue J and H-6 (strong) of residue A. Finally, a NOE

contact could be determined between H-1 of heptose residue Cand the putative deoxy protons of K. With regard to residue I,no NOE connectivity could be identified between its H-1 andH-4 of residue C. However, C was substituted at O-3 and O-4(13C chemical shifts of C-3 and C-4 at 73.0 and 75.2 ppm,respectively). According to genetic data and in analogy toother enterobacterial core structures, it is highly likely thatresidue C was substituted at O-3 and O-4 by residues D and I,respectively. C itself should be linked to Kdo, probably at O-5.

Although the absolute configuration of the monosaccharideresidues was not determined, the successful complementationof the N28b16 and N28b20 mutants by the K. pneumoniaewaaE and orf8 and orf9 genes, respectively, and the known K.pneumoniae core structure suggested that residues F, B, and Iare D configured. The L,D configuration of the inner coreheptose residues C, D, and H was deduced from compositionalanalysis of this and the LPS of strain N28b20 (see below).

ESI FT-ICR-MS. In order to check whether the chemicalstructure determined from the isolated oligosaccharide 3 wasrepresentative, ESI FT-ICR-MS was performed from the coreoligosaccharide fraction (see above). The charge-deconvolutedmass spectrum (Fig. 6) comprised a complex pattern of masspeaks. The peaks with the highest intensity (2,021.67 and2,003.66 Da) were in excellent agreement with a moleculeconsisting of KdoHep5Hex3HexAHexN and its anhydro form(calculated masses of 2,021.65 and 2,003.63 Da), respectively.Thus, core oligosaccharide 3 represented the most abundantcompound in the mixture of core oligosaccharides. Additionalpeaks (summarized in Table 5) could be attributed to molec-

FIG. 5. Anomeric regions of the 1H NMR spectra of oligosaccharide 3. The letters refer to the carbohydrate residues and denote the anomericproton of each residue.


ular species comprising (i) oligosaccharide 3 substituted by anadditional Ko residue (peak 2 in Fig. 6 [2,257.72 Da]), (ii)oligosaccharide 3 substituted by an additional HexA (peak 5,2,197.70 Da), and (iii) oligosaccharide 3 lacking two Hep res-idues but containing an additional HexA (peak 4, 1,813.56 Da).Of peaks 1, 2, and 4, species with an 80-Da-higher mass wereidentified (peaks 3, 7, and 6, respectively), which could repre-sent monophosphorylated derivatives. None of these molecu-lar species representing variants of oligosaccharide 3 could beisolated.

Charge-deconvoluted ESI FT-ICR-MS analysis of the corefraction of the LPS from the wild-type strain N28b gave apattern of peaks comprising those of the O-antigen-deficientcore, described above, plus one peak of low intensity thatrepresented oligosaccharide 3 lacking two Hep residues(1,637.53 Da). Thus, the LPS from both the wild-type strainand the O-antigen-deficient mutant share the same core struc-tures.

Proposed waa gene functions. The genetic complementationexperiments allowed the identification of the S. marcescensN28b genes coding for the enzymes involved in the transfer ofthe Kdo (waaA), Hep I (waaC), Hep II (waaF), Hep III(waaQ), and Glc (waaE) inner core residues. The elucidation

of the S. marcescens N28b LPS core structure allowed us tohypothesize functions for the remaining genes of the cluster.

The core fraction of the LPS from strain N28b20 (orf9Sm

orf10Sm double mutant) was obtained by mild acetic acid hy-drolysis and recovered by gel permeation chromatography, asdescribed for the fraction of the LPS from strain N28b4. Com-positional analysis of this core fraction revealed the absence ofGlcN, GalA, and D,D-Hep residues and a strong reduction inthe Glc content. Furthermore, charge-deconvoluted ESI FT-ICR-MS analysis gave major peaks at 622.19 and 604.17 Dathat corresponded to the oligosaccharide Hep2Kdo (calcu-lated, 622.20 Da) and its anhydro product (calculated, 604.19Da). Another peak at 858.25 Da corresponded to the oligo-saccharide Hep2KdoKo (calculated, 858.26 Da), of which the(Na-H) and (K-H) adducts were also identified (peaks at870.24 and 896.20 Da, respectively). Other peaks with smallerintensities were present but could not be attributed. Since noD,D-Hep was detected in the compositional analysis of this corefraction, it was concluded that, as in other Enterobacteriaceae,the first two inner core heptose residues were in the L,D con-figuration. According to the proposed structure of the coreregion from the strain N28b4 O-deficient mutant (Fig. 4), itcould be predicted that these two genes (orf9Sm orf10Sm)should code for the enzymatic activities involved in the transferof D-GlcN and D-GalA. However, further work will be neces-sary to identify the predicted D-GalA- and D-GlcN-transferasesbetween these two genes.

BLAST and position-iterated BLAST searches of the orf7-and orf11-encoded proteins showed that both proteins sharedsimilarities to known ADP-heptose-LPS heptosyltransferases,

TABLE 3. 1H NMR and 13C NMR chemical shift data foroligosaccharide 3 derived from LPS of the rough mutant S.

marcescens N28b4a

Residue

Chemical shift (ppm)

H-1 H-2 H-3 H-4 H-5 H-6a H-7a/H-6b H-7b

C-1 C-2 C-3 C-4 C-5 C-6 C-7

B, �-GalA 5.45 4.08 4.25 4.49 4.53100.1 74.0 68.8 80.3 71.96 175.7

A, �-Glc 5.49 3.64 3.76 3.57 3.96 3.93 4.23100.1 72.5 73.1 69.6 72.6 69.5

C, �-L,D-Hep 5.47 4.08 4.11 4.3 3.82 4.11101.6 70.7 73.0 75.2 72.5 69.6

D, �-L,D-Hep 5.39 4.2 4.03 3.81 3.67 4.24 3.85 3.76101.8 70.8 80.0 66.2 73.9 68.7 69.9

E, �-D,D-Hep 5.32 4.02 4.01 3.95 3.86 4.0596.6 81.2 71.2 70.9 71.5 73.0

F, �-GlcN 5.14 2.90 4.09 3.78 4.34 3.86 3.9898.3 52.0 70.6 77.0 71.4 61.2

G, �-Hep 5.08 4.07 3.89 3.88 3.73 4.07 3.78 3.75103.0 71.0 72.0 67.0 74.0 73.4 64.0

H, �-L,D-Hep 5.04 4.06 3.91 3.94 3.67 4.1 3.82 3.74101.0 70.5 72.2 67.2 72.2 71.0 64.0

I, �-Glc 4.61 3.34 3.56 3.4 3.44 3.77 3.93103.9 73.6 76.2 70.9 77.2 62.5

J, �-Glc 4.52 3.36 3.54 3.45 3.5 3.76 3.96103.8 74.4 76.2 70.7 76.5 62.0

K 2.34ax, 2.81eq 3.96 3.88 3.65180.4 42.0 74.6 75.6 78/76.8

a Monosaccharide residues are as shown in Fig. 5.

TABLE 4. NOE signals of oligosaccharide 3 observed in theNOESY spectruma

Residue ProtonSignal

Interunit Intraunit

B, �-GalA B1 D3(s), D2(w), E1(s) B5(m)B5 D2

A, �-Glc A1 F4(s) A3(s), A2(m)A6 J2(w)

D, �-L,D-Hep D1 C3/C6(s), C2(m), C4(m),B5(w)

D3(m), D2(w), D7(w)

E, �-D,D-Hep E1 B1(s), B2(s), B3(m), G1(s),G5(s)

E1(s), E2(m)

F, �-GlcN F1 B5(s), B4(w)

G, �-Hep G1 E2(s), E3(s) G2(s), G3(w)

H, �-L,D-Hep H1 D7(s), D6(s) H3(s), H2(s), H5(w)

I, �-Glc I1 C6/C3(s), C2(w) I5(s), I4(m), I3(m)

J, �-Glc J1 A6, A5 J5(s), J4(m), J3(m)

C, �-L,D-Hep C1 K 3.65 ppm(s), K3.88ppm(m)

Kdob 2.811 C2, C4

a Shown are signals that were important for the structural determination.Monosaccharide residues are as depicted in Fig. 5. w, weak; m, medium; s,strong.

b Unknown Kdo modification.


and both proteins contained a domain characteristic of glyco-syltransferase family 4. The orf7- and orf11-encoded proteinswere similar to known WaaF (22 to 23% identity and 39 to40% similarity) and WaaQ (23 to 25% identity and 39 to 40%similarity) proteins of the Enterobacteriaceae, respectively. Thisanalysis was in agreement with the presence of a �-Hep-(132)-D,D-Hep disaccharide in the outer core of the LPS fromS. marcescens N28b (Fig. 4). An S. marcescens nonpolar orf7mutant, strain N22b30, was constructed (see Materials andMethods). LPSs from S. marcescens N28b30 and from themutant complemented by the orf7 gene were isolated, and theirchemical compositions were determined. The results showed adrastic reduction in the content of D,D-Hepp in the core regionof the mutant LPS, suggesting that the orf7-encoded proteinwas involved in the transfer of the D,D-Hep residue. It may alsobe hypothesized that the orf11-encoded protein will be in-volved in the transfer of the terminal outer core Hep residue.

In the core region of the LPS from S. marcescens N28b, a�-Glc-(136)-�-Glc disaccharide attached to O-4 of GlcN wasidentified (Fig. 4). In addition, the deduced 326- and 388-amino-acid proteins encoded by orf6 and orf4, respectively,were similar to several putative glycosyltransferases of differentbacteria involved in the biosynthesis of capsule, O antigen, orcore region. Thus, it can be hypothesized that orf6- and orf4-

encoded products are involved in the transfer of the two outercore Glc residues.

Finally, the deduced 388-amino-acid protein encoded byorf5 showed significant levels of amino acid similarity (39 to40%) and identity (22 to 23%) to WaaL proteins from E.coli core types K-12, R1, R2, and R4. In addition, TopPred2analysis of the orf5-encoded protein predicted 10 mem-brane-spanning domains, suggesting an integral membranelocation. The distribution of these putative transmembranedomains along the protein sequence and the protein hydrop-athy profile were found to be very similar to those of WaaLproteins, suggesting that orf5 corresponds to the S. marc-escens N28b waaL gene.

DISCUSSION

In this work, the waa gene cluster and the structure of thecore region of the LPS from S. marcescens strain N28b wereinvestigated. The proposed structure for the major core oligo-saccharide (oligosaccharide 3) of S. marcescens N28b differsfrom that of K. pneumoniae by two main features: the substi-tution of the �-D-GlcNp at the O-4 position by the �-Glc-(136)-�-Glc disaccharide and the substitution of the �-D-GalpA at the O-2 position by the �-Hepp-(132)-�-D,D-Hepp

FIG. 6. ESI FT-ICR-mass spectrum of the core oligosaccharide fraction isolated from the LPS of strain N28b4 after acetic acid hydrolysis andgel permeation chromatography. The mass numbers refer to the monoisotopic neutral molecular peak.


disaccharide (Fig. 4). Although no residue of Ko could beidentified in the main isolated compound (oligosaccharide 3)obtained from LPS of an O-antigen-deficient mutant, smallamounts of this residue were detected in the mixture of coreoligosaccharides isolated from the LPS. Furthermore, ESI FT-ICR-MS analysis of similar core oligosaccharide fractions ob-tained from the N28b wild-type strain, an O-antigen-deficientmutant, and a double orf9 orf10 mutant strongly suggested thatKo is present in some of the strain N28b core oligosaccharides.The presence of Ko residues, linked in nonstoichiometricamounts to O-4 of the Kdo residue, has been described in thecore regions of the LPS from S. marcescens strains 111R (se-rotype O:29) and 3735 (E. V. Vinogradov, B. Lindner, G.Seltmann, J. Radziejewska-Lebrecht, and O. Holst, XXIst Int.Carbohydr. Symp., p. 279, 2002). Ko residues have not beenreported in the core region of LPS from K. pneumoniae.

A comparison of the known waa gene clusters from mem-bers of the Enterobacteriaceae revealed similarities as well asdifferences in their organization (Fig. 1). In all known cases,the genes gmhD and coaD are located at the 5� and 3� ends,respectively Four genes involved in epimerization (gmhD) andtransfer of L,D-Hepp I (waaC), L,D-Hepp II (waaF), and L,D-Hepp III (waaQ) and a fifth gene (waaA) coding for the trans-fer of the Kdo residue were identified in S. marcescens N28b.The presence of these genes correlated with the structure L-�-D-HeppIII-(137)-L-�-D-HeppII-(133)-L-�-D-HeppI-(135)-Kdop,found in the LPS inner core region of the Enterobacteriaceaestudied (23, 24) (Fig. 4). In agreement with the elucidated S.marcescens N28b major core structure, no genes similar to waaPand waaY involved in phosphoryl modification of L,D-HeppI andL,D-HepII, respectively, were found in the S. marcescens N28bwaa gene cluster. Instead, genes that could be involved in the

transfer of the inner core D-Glc (waaE) and outer core D-GalAand D-GlcN residues (orf9Sm orf10Sm and orf8Kp orf9Kp) wereidentified in both S. marcescens and K. pneumoniae. The presenceof orf9Sm orf10Sm homologues may be expected in Enterobacteri-aceae species containing the �-GlcN (134)-�-GalA disaccharide.Consistent with this, the core region of the LPS from Proteusmirabilis serotype O3 contains this structural feature (50), and italso contains orf9Sm orf10Sm homologues (M. Regue et al., un-published results). Two additional putative heptosyltransferaseswere found in the S. marcescens waa gene cluster (i.e., orf7 andorf11). The reduced content of D,D-HepII in the LPS of an S.marcescens orf7 mutant strongly suggested that the orf7-encodedprotein was involved in the transfer of one of the branching Heppresidues, probably that possessing the D,D configuration. The re-maining two genes (orf4 and orf6) from the S. marcescens waagene cluster encoded proteins that possessed characteristic fea-tures of glycosyltranferases. Since the core region of the LPS ofstrain N28b contained a �-Glc-(136)-�-Glc disaccharide at-tached to O-4 of the D-GlcN residue, it is tempting to speculatethat these two genes are involved in the sequential transfer of theouter core Glc residues.

The JUMPStart sequence (for just upstream of many po-lysaccharide-associated starts) (22, 27) was found to be locatedupstream of the waaQ operon in E. coli, containing 9 genes,and that in S. enterica, containing 10 genes (19). No suchsequence was found in the 126-bp intergenic region betweenorf11 and the waaA operon, as expected from the monocis-tronic nature of orf11. Furthermore, no JUMPStart similarsequences were found upstream of the three operons of the S.marcescens waa gene cluster, similarly to what has been foundin the K. pneumoniae waa gene cluster (38). The significance ofthis feature will require further studies.

Initial analysis using different strains from S. marcescensbelonging to several serovars seemed to indicate that the ge-netic organization of the waa gene cluster reported in this workwas shared by all of them. If further studies confirm this pre-liminary result, this means that a main core type is present inthe LPS of S. marcescens, as was described for LPS from K.pneumoniae (38, 45, 49).

ACKNOWLEDGMENTS

This work was supported by grants from the Plan Nacional de I�D(Ministerio de Ciencia y Tecnología, Spain) and Generalitat de Cata-lunya. N.C. and L.I. were supported by FPI fellowships from theGeneralitat de Catalunya and Ministerio de Ciencia y Tecnología,(Spain), respectively, and by the Deutsche Forschungsgemeinschaft(grant LI-448/1-1 to B.L.).

We thank Maite Polo, Regina Engel, and Sylvia Dupow for technicalassistance and Miguel Feliz for his contribution to NMR experimentsand their interpretation.

REFERENCES

1. Acar, J. F. 1986. Serratia marcescens infections. Infect. Control 7:273–276.2. Allen, A. G., and D. J. Maskell. 1996. The identification, cloning and mu-

tagenesis of a genetic locus required for lipopolysaccharide biosynthesis inBordetella pertussis. Mol. Microbiol. 19:37–52.

3. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.Basic local alignment search tool. J. Mol. Biol. 215:403–410.

4. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 25:3389–3402.

5. Belunis, C. J., T. Clementz, S. M. Carty, and C. H. R. Raetz. 1995. Inhibitionof lipopolysaccharide biosynthesis and cell growth following inactivation ofthe kdtA gene in Escherichia coli. J. Biol. Chem. 270:27646–27652.

6. Bock, K., J. U. Thomsen, P. Kosma, R. Christian, O. Holst, and H. Brade.

TABLE 5. Mass peaks identified in the charge-deconvoluted ESIFT-ICR mass spectrum of the core oligosaccharide fraction isolated

from the LPS of strain N28b4 after acetic acid hydrolysis and gelpermeation chromatographya

Peakno.

Mass (Da)Proposed structure

Identified Calculated

1 2,021.67 2,021.65 Oligosaccharide 32,003.66 2,003.63 Anhydro form

2 2,257.72 2,257.71 Oligosaccharide 3 plus one Koresidue

3 2,101.63 2,101.61 Oligosaccharide 3 plus onephosphate residue

2,083.69 2,083.60 Anhydro form

4 1,813.56 1,813.55 KdoHep3HexA2Hex3HexN1,795.55 1,795.54 Anhydro form

5 2,197.70 2,197.68 KdoHep5HexA2Hex3HexN2,179.68 2,179.67 Anhydro form

6 1,893.43 1,893.52 KdoHep3HexA2Hex3HexNP1,875.51 1,875.51 Anhydro form

7 2,337.69 2,337.68 Oligosaccharide 3 plus 1 Ko residueand P residue

a Listed are monoisotopic masses of the neutral molecule. The peaks are givenin descending order of intensity. The peak numbers are as depicted in Fig. 6.Additional sodium and potassium adducts (�22 and �38 Da, respectively) foundin the spectrum are not listed.


1992. A nuclear magnetic resonance spectroscopic investigation of Kdo-containing oligosaccharides related to the genus-specific epitope of Chla-mydia lipopolysaccharides. Carbohydr. Res. 229:213–224.

7. Bollman, R., E. Halle, W. Sokosllowska-Kohler, E. L. Grauel, P. Buchholz,L. Klare, H. Tschape, and W. Witte. 1989. Nosocomial infections due toSerratia marcescens: clinical findings, antibiotic susceptibility patterns andfine typing. Infection 17:294–300.

8. Chatterjee, A. K., H. Ross, and K. E. Sanderson. 1976. Leakage of periplas-mic enzymes from lipopolysaccharide-defective mutants of Salmonella typhi-murium. Can. J. Microbiol. 22:1549–1560.

9. Claros, M. G., and G. von Heijne. 1994. Prediction of transmembrane seg-ments in integral membrane proteins, and putative topologies, using severalalgorithms. Comput. Appl. Biosci. 10:685–686.

10. Clementz, T., and C. H. R. Raetz. 1991. A gene coding for 3-deoxy-D-manno-octulosonic-acid transferase in Escherichia coli: identification, mapping,cloning, and sequencing. J. Biol. Chem. 266:9687–9696.

11. Enfedaque, J., S. Ferrer, J. F. Guasch, J. M. Tomas, and M. Regue. 1996.Bacteriocin 28b from Serratia marcescens N28b: identification of Escherichiacoli surface components involved in bacteriocin binding and translocation.Can. J. Microbiol. 42:19–26.

12. Filiatrault, M. J., B. G. Gibson, B. Schilling, S. Sun, R. S. Munson, Jr., andA. A. Campagnari. 2000. Construction and characterization of Haemophilusducreyi lipooligosaccharide (LOS) mutants defective in expression of hepto-syltransferase III and �1,4-glucosyltransferase: identification of LOS glyco-forms containing lactosamine repeats. Infect. Immun. 68:3352–3361.

13. Galanos, C., O. Luderitz, and O. Westphal. 1969. A new method for theextraction of R lipopolysaccharides. Eur. J. Biochem. 9:245–249.

14. Gargallo-Viola, D. V. 1989. Enzyme polymorphism, prodigiosin production,and plasmid fingerprints in clinical and naturally occurring isolates of Serratiamarcescens. J. Clin. Microbiol. 27:860–868.

15. Geerlof, A., A. Lewendon, and W. V. Shaw. 1999. Purification and charac-terization of phosphopantetheine adenylyltransferase from Escherichia coli.J. Biol. Chem. 274:27105–27111.

16. Guasch, J. F., S. Ferrer, J. Enfedaque, M. B. Viejo, and M. Regue. 1995. A17 kDa outer-membrane protein (Omp4) from Serratia marcescens conferspartial resistance to bacteriocin 28b when expressed in Escherichia coli.Microbiology 141:2535–2542.

17. Guasch, J. F., N. Pique, N. Climent, S. Ferrer, S. Merino, X. Rubires, J. M.Tomas, and M. Regue. 1996. Cloning and characterization of two Serratiamarcescens genes involved in core lipopolysaccharide biosynthesis. J. Bacte-riol. 178:5741–5747.

18. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plas-mids. J. Mol. Biol. 166:557–580.

19. Heinrichs, D. E., J. A. Yethon, and C. Whitfield. 1998. Molecular basis forstructural diversity in the core regions of the lipopolysaccharides of Esche-richia coli and Salmonella enterica. Mol. Microbiol. 30:221–232.

20. Heinrichs, D. E., M. A. Monteiro, M. B. Perry, and C. Whitfield. 1998. Theassembly system for the lipopolysaccharide R2 core-type of Escherichia coliis a hybrid of those found in Escherichia coli K-12 and Salmonella enterica.J. Biol. Chem. 273:8849–8859.

21. Hitchcock, P. J., and T. M. Brown. 1983. Morphological heterogeneityamong Salmonella lipopolysaccharide chemotypes in silver-stained polyacryl-amide gels. J. Bacteriol. 154:269–277.

22. Hobbs, M., and P. R. Reeves. 1994. The JUMPstart sequence: a 39 bpelement common to several polysaccharide gene clusters. Mol. Microbiol.12:855–856.

23. Holst, O. 1999. Chemical structure of the core region of lipopolysaccharides,p. 115–154. In H. Brade, S. M. Opal, S. N. Vogel, and D. C. Morrison (ed.),Endotoxins in health and disease. Marcel Dekker, Inc., New York, N.Y.

24. Holst, O. 2002. Chemical structure of the core region of lipopolysaccharides.An update. Trends Glycosci. Glycotechnol. 14:87–103.

25. Izquierdo, L., N. Abitiu, N. Coderch, B. Hita, S. Merino, R. Gavin, J. M.Tomas, and M. Regue. 2002. The inner-core lipopolysacharide biosyntheticwaaE gene: function and genetic distribution among some Enterobacteri-aceae. Microbiology 148:3485–3496.

26. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying thehydropathic character of a protein. J. Mol. Biol. 157:105–132.

27. Leeds, J. A., and R. A. Welch. 1997. Enhancing transcription through theEscherichia coli hemolysin operon, hlyCBAD: RfaH and upstream JUMP-Start DNA sequences function together via a postinitiation mechanism. J.Bacteriol. 179:3519–3527.

28. Link, A. J., D. Phillips, and G. M. Church. 1997. Methods for generatingprecise deletions and insertions in the genome of wild-type Escherichia coli:application to open reading frame characterization. J. Bacteriol. 179:6228–6237.

29. MacLachlan, P. R., and K. E. Sanderson. 1985. Transformation of Salmo-nella typhimurium with plasmid DNA: differences between rough and smoothstrains. J. Bacteriol. 161:442–445.

30. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

31. Molinaro, A., C. De Castro, R. Lanzetta, A. Evidente, M. Parrilli, and O.Holst. 2002. Lipopolysaccharides possesing two L-glycero-D-manno-heptopy-ranosyl-�-(135)-3-deoxy-D-manno-octulopyranosonic acid moieties in thecore region. The structure of the core region of the lipopolysaccharides fromBurkholderia caryophylli. J. Biol. Chem. 277:10058–10063.

32. Nassif, X., J.-M. Fournier, J. Arondel, and P. J. Sansonetti. 1989. Mucoidphenotype of Klebsiella pneumoniae is a plasmid-encoded virulence factor.Infect. Immun. 57:546–552.

33. Pearson, W. R., and D. J. Lipman. 1988. Improved tools for biologicalsequence comparison. Proc. Natl. Acad. Sci. USA 85:2444–2448.

34. Pradel, E., and C. A. Schnaitman. 1991. Effect of rfaH (sfrB) and tempera-ture on expression of rfa genes of Escherichia coli K-12. J. Bacteriol. 173:6428–6431.

35. Raetz, C. R. H., and C. Whitfield. 2002. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 71:635–700.

36. Raleigh, E. A., N. E. Murray, H. Revel, R. M. Blumenthal, D. Westaway,A. D. Reith, P. W. J. Rigby, J. Elhai, and D. Hanahan. 1988. McrA and McrBrestriction phenotypes of some E. coli strains and implications for genecloning. Nucleic Acids Res. 16:1563–1575.

37. Reeves, P. R., M. Hobbs, M. A. Valvano, M. Skurnik, C. Whitfield, D. Coplin,N. Kido, J. Klena, D. Maskell, C. R. Raetz, and P. D. Rick. 1996. Bacterialpolysaccharide synthesis and gene nomenclature. Trends Microbiol. 4:495–503.

38. Regue, M., N. Climent, N. Abitiu, N. Coderch, S. Merino, L. Izquierdo, M.Altarriba, and J. M. Tomas. 2001. Genetic characterization of the Klebsiellapneumoniae waa gene cluster, involved in core lipopolysaccharide biosynthe-sis. J. Bacteriol. 183:3564–3573.

39. Roantree, R. J., T. T. Kuo, and D. G. MacPhee. 1977. The effect of definedlipopolysaccharide core defects upon antibiotic resistances of Salmonellatyphimurium. J. Gen. Microbiol. 103:223–234.

40. Rubires, X., F. Saigí, N. Pique, N. Climent, S. Merino, S. Albertí, J. M.Tomas, and M. Regue. 1997. A gene (wbbL) from Serratia marcescens N28b(O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives. J.Bacteriol. 179:7581–7586.

41. Saigí, F., N. Climent, N. Pique, C. Sanchez, S. Merino, X. Rubires, A.Aguilar, J. M. Tomas, and M. Regue. 1999. Genetic analysis of the Serratiamarcescens N28b O4 antigen gene cluster. J. Bacteriol. 181:1883–1891.

42. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

43. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing withchain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.

44. Sofia, H. J., V. Burland, D. L. Daniels, G. Plunkert III, and F. R. Blattner.1994. Analysis of the Escherichia coli genome. V. DNA sequence of theregion from 76.0 to 81.5 minutes. Nucleic Acids Res. 22:2576–2586.

45. Susskind, M., L. Brade, H. Brade, and H. Holst. 1998. Identification of anovel heptoglycan of �132-linked D-glycero-D-manno-heptopyranose.Chemical and antigenic structure of lipopolysaccharides from Klebsiellapneumoniae ssp. pneumoniae rough strain R20 (O1�:K20�). J. Biol. Chem.273:7006–7017.

46. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment throughsequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 22:4673–4680.

47. Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain for detectinglipopolysaccharide in polyacrylamide gels. Anal. Biochem. 119:115–119.

48. Viejo, M. B., S. Ferrer, J. Enfedaque, and M. Regue. 1992. Cloning and DNAsequence analysis of a bacteriocin gene from Serratia marcescens. J. Gen.Microbiol. 138:1737–1743.

49. Vinogradov, E. V., and M. B. Perry. 2001. Structural analysis of the coreregion of the lipopolysaccharides from eight serotypes of Klebsiella pneu-moniae. Carbohydr. Res. 335:291–296.

50. Vinogradov, E. V., J. Radziejewska-Lebrecht, and W. Kaca. 2000. The struc-ture of the carbohydrate backbone of core-lipid A region of the lipopoly-saccharides from Proteus mirabilis wild-type strain S1959 (serotype O3) andits Ra mutant R110/1959. Eur. J. Biochem. 267:262–268.

51. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides: extractionwith phenol-water and further applications of the procedure. Methods Car-bohydr. Chem. 5:83–91.


Genetic and Structural Characterization of the Core Region of the Lipopolysaccharide from Serratia marcescens N28b (Serovar O4)

Documents