molecular analysis of rfad gene, for heptose synthesis, and rfaf analysis ofthe rfad gene, for...

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  • JOURNAL OF BACrERIOLOGY, Apr. 1994, p. 2379-23850021-9193/94/$04.00+0Copyright C) 1994, American Society for Microbiology

    Molecular Analysis of the rfaD Gene, for Heptose Synthesis, and therfaF Gene, for Heptose Transfer, in Lipopolysaccharide

    Synthesis in Salmonella typhimuriumDASSANAYAKE M. SIRISENA,t P. RONALD MAcLACHLAN,t SHU-LIN LIU,

    ANDREW HESSEL, AND KENNETH E. SANDERSON*Salmonella Genetic Stock Centre, Department of Biological Sciences, University of Calgary,

    Calgary, Alberta, Canada T2N 1N4

    Received 15 November 1993/Accepted 15 February 1994

    We report the analysis of three open reading frames of Salmonella typhimurium LT2 which we identified asrfaF, the structural gene for ADP-heptose:LPS heptosyltransferase II; rfaD, the structural gene for ADP-L-glycero-D-manno-heptose-6-epimerase; and part of kbl, the structural gene for 2-amino-3-ketobutyrate CoAligase. A plasmid carrying rfaF complements an rfaF mutant of S. typhimurium; rfaD and kbl are homologousto and in the same location as the equivalent genes in Escherichia coli K-12. The RfaF (heptosyl transferase II)protein shares regions of amino acid homology with RfaC (heptosyltransferase I), RfaQ (postulated to beheptosyltransferase III), and KdtA (ketodeoxyoctonate transferase), suggesting that these regions function inheptose binding. E. coli contains a block of DNA of about 1,200 bp between kbl and rfaD which is missing fromS. typhimurium. This DNA includes yibB, which is an open reading frame of unknown function, and twopromoters upstream of rfaD (P3, a heat-shock promoter, and P2). Both S. typhimurium and E. coli rfaD genesshare a normal consensus promoter (P1). We postulate that theyibB segment is an insertion into the line leadingto E. coli from the common ancestor of the two genera, though it could be a deletion from the line leading to S.typhimurium. The G+C content of the rfaLKZYJI genes of both S. typhimurium LT2 and E. coli K-12 is about 35%,much lower than the average for enteric bacteria; if this low G+C content is due to lateral transfer from a sourceof low G+C content, it must have occurred prior to evolutionary divergence of the two genera.

    Lipopolysaccharide (LPS), a major component of the outermembrane of gram-negative bacteria, is composed of threedomains: lipid A; the core, which is an oligosaccharide consist-ing of an inner and an outer region; and a distal repeating unitknown as 0 antigen (25, 31, 35, 45). Lipid A of Escherichia coliand Salmonella typhimurium is a beta-1,6-linked glucosaminedisaccharide. To this is attached the inner core composed of atleast two 3-deoxy-D-manno-octulosonic acid (also called ke-todeoxyoctonate [KDO]) units followed by two units of hep-tose; the outer core region and the 0 antigen are attached toone of the heptose units (Fig. 1).

    Mutants which are lacking the 0 antigen and the outer corecomponents are viable and not much reduced in growth rate inculture, though they are nonvirulent. However, deep-roughmutants affected in the heptose region of the inner core oftenshow reduced growth rate, sensitivity to elevated temperature(7), and hypersensitivity to detergents and hydrophobic anti-biotics (28). Mutants defective in lipid A or KDO synthesishave been isolated only as conditional lethals.

    Several genes for synthesis of the inner core region havebeen identified. Two genes, rfaC and rfaF, are identified forheptosyl transferases for transfer of heptose from ADP-hep-tose to the inner core. ADP-heptose:LPS heptosyltransferase I

    * Corresponding author. Mailing address: Department of BiologicalSciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4.Phone: (403) 220-6792. Fax: (403) 289-9311. Electronic mail

    t Present address: Department of Botany, University of Kelaniya,Kelaniya, Sri Lanka.

    t Present address: Veterinary Infectious Disease Organization, Uni-versity of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N-OWO.

    (hereafter called heptosyltransferase I) is encoded by the rfaCgene; an rfaC mutant of S. typhimurium, which does nottransfer heptose from ADP-heptose to lipid-(KDO)2-IVA, wascomplemented by the cloned gene (47). rfaC was also clonedfrom E. coli (8). The gene rfaF was inferred to determineheptosyltransferase II because rfaF mutants of S. typhimuriummake LPS with only one heptose unit attached to KDO (25,50) and because the same rfaF mutants synthesize ADP-heptose (47), but detailed molecular studies have not beenreported. A third gene may be required for transfer of the thirdheptose to the LPS; rfaQ has been postulated for this function,on the basis of nucleotide sequence homology with rfaC andrfaF (45).Two genes for synthesis of ADP-L-glycero-D-manno-heptose

    (rfaD and rfaE) have been identified. The rfaD gene of E. coliencodes ADP-L-glycero-D-manno-heptose-6-epimerase, whichconverts ADP-D-glycero-D-manno-heptose to ADP-L-glycero-D-manno-heptose (9, 30); rfaD is also known as htrM and isrequired for viability of E. coli at high temperature (32).(Hereafter we refer to this gene as rfaD and to the enzyme asheptose epimerase.) A mutant of S. typhimurium produces LPScontaining some D-glycero-D-manno-heptose and was inferredto be an rfaD mutant (20). The second proposed gene forsynthesis of ADP-heptose is rfaE in S. typhimurium (50). AnrfaE mutant (50) was unable to synthesize ADP-heptose (47),but the specific step in synthesis controlled by rfaE is notknown.

    In this article we describe the cloning and sequencing of therfaD and rfaF genes of S. typhimurium for ADP-heptoseepimerase and for heptosyltransferase II, respectively. Thegenes are located in the rfa gene cluster at 79 min on thelinkage map (40) in the order rfaDFCL; on the basis of the


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  • 2380 SIRISENA ET AL.

    FIG. 1. Proposed stereochemistry of the inner core of LPS of E. coli and S. typhimurium, adapted from Raetz (31). The alpha,1-5 bond betweenthe innermost heptose (residue I) and KDO is formed by heptosyltransferase I (rfaC protein); the alpha,1-3 bond between the second heptose(residue II) and the innermost heptose is formed by heptosyltransferase II (rfaF protein). Possible partial substitutions are indicated with dashedbonds. The outer core region is attached through a glucose residue to heptose (residue II).

    orientation of the open reading frames (ORFs) they aretranscribed clockwise on the chromosome in the above order.In S. typhimurium the gene kbl, which determines glycineacetyltransferase, is adjacent to rfaD and independently tran-scribed counterclockwise. In E. coli K-12, gene organization issimilar except for a 1.2-kb insertion containing a 1-kb ORFcalled yibB or 26K between rfaD and kbl.


    Strains and bacteriological methods. The strains used in thisstudy are summarized in Table 1; all are S. typhimurium LT2unless indicated otherwise. The strains were maintained at- 70C in 15% glycerol as described previously (43). Cells wereroutinely cultured in L broth (21), Luria-Bertani broth, or2XYT medium (27). Antibiotics when required were added atthe following final concentrations (in micrograms per millili-ter): ampicillin, 50; tetracycline, 25; streptomycin, 200. Modi-fied minimal Davis medium was the defined medium (42). Forsolid media, Difco Bacto Agar was added to a final concentra-tion of 1.5%. Incubation was at 37C unless stated otherwise.For screening the lambda library, cells were grown in lambdaagar (11). M9 medium (27) supplemented with 1% glucose,and 1% methionine assay medium (Difco) was used for proteinradiolabelling.

    Genetic transformation. Transformation was by calciumchloride-heat shock methods of MacLachlan and Sanderson(24) or by electrotransformation methods of Dower et al. (12)as modified by Binotto et al. (2).The genomic library. The source of cloned genes was a

    genomic library, consisting of S. typhimurium LT2 DNA par-tially digested with Sau3A and ligated into the BamHI site ofthe vector X1059. The library was obtained from R. Maurer(26) and is distributed from the Salmonella Genetic Stock

    TABLE 1. Bacterial strains, plasmids, and bacteriophages

    Strain, plasmid, or Genotype or description Referencebacteriophage or source

    StrainsS. typhimuniumLB51010 metA22 metE551 trpC2 leu3121 5

    ilv412 rpsL120 galE856 hsdL6hsdA29 hsdB121 xyl404 Hi-b H2e,n,x fla-66 nml (Fels 2)

    SA3617 rfaF511/F'proA+B+ laclqlacZAM15 zzf::TnJO

    SA3670 rfaF511 recAl srl-202::TnlOSA3770 rfa +SL3789 rfaF511

    E. coli K-12JM109 recAI gyrA96 thi hsdR17 supE44

    relA1 X A(lac-proAB)/F'proA+B+ lacl4 lacZAM15

    DLT111 HfrC lambda srl::TnJO 49recA/pGP1-2

    Plasmid pBluescriptKS+ ApR lacZ (3 kb)a Stratagene

    BacteriophagesP22c2 Clear-plaque mutant of P22, 50

    which adsorbs to S. typhimuriumwith 0 antigen

    Felix 0 (FO) Virulent phage of S. typhimurium 50which requires a complete corefor adsorbtion

    Ffm Lyses E. coli K-12 and rough 50mutants of S. typhimurium

    a Also designated M13+.

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    5+i 110



    R Ev _=-() c s Nlr ; B |/H H B E E H B

    pT3 pT7 pKZ 106pT7 | p pT3 pKZ107pT3 _B pKZ108 (106)Nr_C pKZ1O9(106)H in: pKZ110(107)sacc pKZ113(107)Ev_B pKZ154(108)

    FIG. 2. Structure of the chromosome in the rfa region at 79 min on the linkage map of S. typhimunium and isolation of plasmids carrying genesin the rfaDF region. (A) The cysE genes are to the left (counterclockwise on the circular linkage map), and thepyrE genes are to the right (clockwiseon the linkage map) (40). The region is illustrated from the ClaI


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