We have cloned a gene from a Salmonella typhimu- rium with the ability to ...... complementation (Austin et al., 1990; Parker et al., 1992). Thus, a region with ...
Vol. 267, No.26, Issue of September 15, PP. 18874-188&L, 1992 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
@ 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
The rfaC Gene of Salmonella typhimurium CLONING, SEQUENCING, AND ENZYMATIC FUNCTION INHEPTOSE LIPOPOLYSACCHARIDE*
TRANSFER TO
(Received for publication, December 18,1990, and in revised form, June 11, 1992)
Dassanayake M. SirisenaS, KathrynA. Brozekj, P. Ronald MacLachlanSlI, Kenneth E. Sanderson$, and ChristianR. H.RaetzQII From the $Department of Biological Sciences, University of Calgary, Calgary,Alberta T2N lN4, Canada and the §Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
We have cloned a gene from a Salmonella typhimu- membrane of Gram-negative bacteria, is composed of three rium with the ability complement to the rfaCmutation domains: a glucosamine-based phospholipid known as lipid A (heptose-deficientlipopolysaccharide,sensitivity to (Fig. l),a nonrepeating core oligosaccharide consisting of an rough-specificbacteriophages, and susceptibilityto inner and an outer region(Fig. 2), and a distal repeating 1018-base pair EcoRV- oligosaccharide known as 0-antigen (Miikela and Stocker, hydrophobicantibiotics).A !lVhlllI fragment, subcloned into the pBluescriptKS+ 1984; Raetz, 1987; Raetz, 1990; Rick, 1987). Mutants lacking vector to yield pKZlO3, retains complementing activ- 0-antigen and most core sugars are viable (Fig. 2), but muity. Nucleotide sequencing revealed an open reading frame corresponding to a protein of 317 amino acids tants defective in lipid A or 3-deoxy-~-manno-octulosonic (Mr 36,100).TheplasmidpKZlO3,whichhas a acid (KDO) biosynthesis must be isolated as conditional leproperly aligned T7 promoter, can overexpress a pro- thals. Lipid A of Escherichia coli and Salmonella typhimurium tein of M, = 31,000 when T 7 RNA polymerase is (Raetz, 1990) is a P,1-6-linked disaccharide of glucosamine, supplied. An in vitro system was established for analysis of phosphorylated at positions 1 and 4’, and acylated with R-3heptose addition to the precursor [4’-3ap](KD0)2-IVA hydroxymyristate, laurate, and myristate (Fig. 1).At least 2 (Brozek, K.A., Hosaka, K., Robertson, A.D., and KDO residues are attached at position 6’ (Fig. 2). The inner Raetz, C. R. H. (1989) J. Biol. Chem. 264, 6966- core region also contains at least two L-glyCerO-D-mannO6966). Soluble fractions from wild-type or heptose- heptose units, linked to KDO residue I (Fig. 2). Phosphate deficient rfa mutants were tested for their ability to and phosphoethanolamine moieties may be present in subconvert [4’-32P](KD0)2-IVAto more polar substances. stoichiometric amounts (Rick, 1987). Mutants lacking heptose I n wild-type extracts, these conversions required ad- in their LPS are viable but have a “deep rough” phenotype, dition of ATP or ADP-heptose. In extracts of rfaC-, characterized by hypersensitivity to detergents and hydrophorfaD-,orrfaE-deficientstrains, no polarproducts bic antibiotics (Nikaido andVaara, 1987). were observed with ATP. ADP-heptose restored synMost of the genes responsible for core biosynthesis (Fig. 2) thesis in rfaD and rfaE not butrfaC extracts, indicating that rfaD and rfaEare involved in ADP-heptose are located in therfa cluster at min 79 between cysE andpyrE formation. When the cloned rfaC gene was introduced (Sanderson and Roth, 1988; Schnaitman et al., 1991; Roncero into anrfaC-deficient mutant, extracts from suchcells and Casadaban, 1992). Mutants have been used to identify at regained the ability to metabolize [4’-32P](KD0)2-IV~,least five genes of S. typhimurium involved in the synthesis showing that rfuC encodes the enzyme that attaches of the inner core; these are rfaC (Sanderson et al., 1974), rfaD the proximal heptose to lipopolysaccharide. (Lehmann et al., 1973), rfaE and -F (Wilkinson et al., 1972), and rfaP (Helander et al. 1989). All but rfaE, which maps at min 76 (Wilkinson et al., 1972), are located at min 79 (Kuo Lipopolysaccharide (LPS),’ amajor component of the outer and Stocker, 1972; Sanderson and Saeed, 1972). Addition of heptose to KDO requires the functions deter* This work was supported by an Operating Grant of the Natural mined by the genes rfaC, -D, and -E. In E. coli, the rfaD gene Sciences and Engineering Research Council of Canada (to K. E. S.) is thought to encode ADP-L-glycero-~-manm-heptose-6-epiand by National Institutes of Health Grant DK-19551 (to merase, which converts ADP-D-glycero-D-manno-heptoseinto C. R. H. R.). The costs of publication of this article were defrayed in (Coleman, 1983; Pegues et part by the payment of page charges. This article must therefore be ADP-L-glycero-D-manno-heptose hereby marked “advertisement” in accordance with 18 U.S.C. Section al., 1990; Raetz, 1990), the predominant epimer found in LPS (Fig. 2). However, a leaky rfaD mutant of S. typhimurium is 1734 solely to indicate this fact. The nucleotide sequence(s)reported in thispaper has been submitted thought to produce LPS containing some D-glycero-D-mannoto the GenBankTM/EMBL DataBank withaccessionnumber(s) heptose, which is not normally present (Lehmannet al.,1973). M95927. An rfaC mutant produces heptoseless LPS of chemotype Re 1Supported by a Fellowship from the Alberta Heritage Foundation for Medical Research. Present address: Dept. of Microbiology, Uni- (Sanderson et al., 1974), as does an rfaE mutant (Wilkinson et al., 1972). It has not been determined whether rfaC and versity of Guelph, Guelph, Ontario N1G 2W1, Canada. 11 To whom correspondence and reprint requests should be ad- rfaE, like rfaD, affect the synthesis of the nucleotide forms of dressed. Present address: Dept. of Biochemistry, Merck Research heptose, or whether they affect the enzyme ADP-hepLaboratories, Rahway, NJ 07065. Tel.: 908-594-7551. tose:lipopolysaccharide heptosyltransferase I (hereaftercalled The abbreviations used are: LPS, lipopolysaccharide; KDO, 3deoxy-D-manno-octulosonicacid; kb, kilobase(s); bp, base pair(s); heptosyltransferase I) required for the addition of the first acid PAGE, heptose unit (residue I11 in Fig. 2). In vitro systems for heptose HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic addition to LPSprecursors have not been described. polyacrylamide gel electrophoresis; ORF, open reading frame.
-
18874
Structure and Function of the rfaC Gene Mutants in which the phosphorylation of the inner core region and the addition of the outer core region are reduced have been obtained in S. typhimurium (Helander et al., 1989), in S. minnesota (Droge et al., 1968; Muhlradt et al., 1968; Muhlradt, 1969; Jousimies and Makela, 1974), and in E. coli K-12 (Parker et al., 1992). Although these mutants may not all be in the same gene, they are termed rfaP. Interestingly, rfaF mutants produce LPS of chemotype Rd, (Fig. 2) with only 1 heptose unit attached (Wilkinson et aL, 1972). Since all heptose units of the LPS core appear to be derived from the same sugar nucleotide, the rfaF gene product (Makela and Stocker, 1984) is postulated to be the structural gene for the enzyme (hereafter called heptosyltransferase 11) that attaches the second heptose unit (residue IV in Fig. 2), but an in vitro system to demonstrate this has not been available. From a library of DNA fragments of S. typhimurium in X1059, we have now subcloned a 1.1-kb segment that complements the rfaC mutation of S. typhimurium. Expression of a protein of 31 kDa was demonstrated using a T7 promoterdriven system. Nucleotide sequencing revealed a corresponding open reading frame. We also report an in vitro system for analysis of heptose addition to LPS, based on the suggestion that underacylated LPS precursors can acquire a complete core in vivo (Walenga and Osborn, 1980). We show that [4'32P](KD0)2-IVA(Fig. 1) (Brozek et al., 1989), a precursor lacking laurate and myristate, can function as the acceptor for putative heptose units in vitro. We provide thefirst enzymologicalevidence that the cloned rfaC geneencodes heptosyltransferase I, that rfaF encodes heptosyltransferase 11, and that rfaE is involved in the synthesis of ADP-heptose. EXPERIMENTAL PROCEDURES
Bacterial Strains, Media, and Materials The bacterial strains and plasmids used are listed in Table I. In work done in Calgary, all strains weregrown in L-broth (10 g of tryptone, 5 g of yeast extract, 10 g of NaCI, and 20 g of glucose per liter) (Lennox, 1955) or on L-agar, which is L-broth with 1.5% agar (Difco). In work done in Wisconsin, cells were grown in LB broth (Lbroth without glucose). The growth temperature was 37 "C unless otherwise stated. Strains were stored in 15% glycerol at -70 "C and repurified as single colonies prior to use. SOC broth (Dower et al., 1988) was used to grow electrotransformed cells before plating. For X library screening, cells were grown in X agar (Davis et al., 1980). M9 medium (Miller, 1972) supplemented with 1%glucose plus 1%methionine assay medium (Difco) was used for protein radiolabeling. The following antibiotics were used with L-agar when required ampicillin (100 pg/ml), tetracycline (25 pg/ml), kanamycin (50 pg/ ml), andchloramphenicol (10-40 pglrnl). [Y-~*P]ATP was obtained from Amersham International; [cx-~*P] dCTP and [w3*P]dATPwere from Du Pont-New England Nuclear. ATP and KDO were purchased from Sigma. Other items were purchased from the following companies: Triton X-100 (Research Products International); yeast extract and tryptone (Difco Laboratories); Silica Gel 60 thin layer chromatography plates, 0.25 mm (Merck); bicinchoninic acid (Pierce). (KDO)*-IVA and [4'-32P](KDO)2-IVA were prepared as described previously (Brozek et al. 1989). Partially purified ADP-D-glycero-D-manno-heptose and ADP-L-glycero-Drnanno-heptose, the generous gifts of Dr. T. Kontrohr (Pecs, Hungary), were prepared as described (Kontrohr andKocsis, 1986). Screening of a Genomic Library
A genomic library, consisting of S. typhimurium LT2 DNA partially digested with Sau3A and ligated into the BamHI site of the X1059 vector, was the source of DNA for cloning the rfaC gene and was obtained from R. Maurer (Maurer et al., 1984). The host E. coli strain, LE392, was infected and plated on X agar to yield about 1500 plaques per plate. After incubation at 37 "C for 12 h, plaques were transferred onto Colony/Plaque Screen filters (Du Pont-New England Nuclear) as recommended by the supplier. Prehybridization was carried out at 60 "C for 12 h, and hybridization was at 68 "C for 16 h as described in Silhavy et al. (1984) without using formamide in the
18875
Function Structure and
18876 A
Inner Care
Outer Core
of the rfaC Gene
-
Plasmid Isolation and Cloning Methods
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Plasmid DNA was isolated by a modification of the alkaline lysis method of Birnboim and Doly (1979), as described in Maniatis etal. (1982). Large scale plasmid isolation was according to Portnoy et al. (1981). Bacterial chromosomal DNA was isolated as described in Silhavy et al. (1984). Restriction endonucleases (obtained from Pharmacia LKB Biotechnology Inc., Boehringer Mannheim, New England Biolabs, or Bethesda Research Laboratories) wereused as recommended by the supplier. DNA fragments for subcloning were excised from 0.7% agarose gels and purified with Geneclean kits (Bio 101 Inc.). Before ligation, vector DNA was dephosphorylated with calf intestinal alkaline phosphatase obtained from Boehringer Mannheim, and T4 DNA ligase from Bethesda Research Laboratories was used for ligation. The end filling reactions were carried out according to Maniatis et al. (1982), using Klenow fragment of E. coli DNA polymerase I for 5' overhangs and T4 DNA polymerase for 3' overhangs. Methods used for labeling of DNA fragments (Feinberg and Vogelstein, 1983) and for Southern transfer (Reed and Mann, 1985) have been described. Nested deletions were generated using exonuclease I11 and mung bean nuclease, according to the protocol of Stratagene Inc.
I Smooth LPS. Wlld Type I
DNA Sequencing
B
HO
1
FIG. 2. Proposed structure of the core of S. typhimurium LPS, including compositions of various chemotypes. a, the genes correlated with specific chemotypes are indicated (adapted from Raetz, 1990). b, proposed stereochemistry of the inner core of E. coli K-12 and S. typhimurium (Raetz, 1990). The a,l-5 bond between the innermost heptose (residue 111) and KDO (residue I) is formed by heptosyltransferase I, the putative product of the rfaC gene. Possible partial substitions are indicated with dashed bonds. hybridization solution. Filters were autoradiographed at -70 "C with Kodak X-OMAT AR film using Cronex (Du Pont) intensifying screens. The positive plaques identified in the primary screening were propagated and used for the secondary screening. The positive clones identified in secondary screening were re-propagated, and bacteriophage DNA was isolated using Lambdasorb adsorbent (Promega). Gel Electrophoresis of LPS LPS isolation, proteinase K digestion, and silver staining of gels were done according to Hitchcock and Brown (1983). LPS was separated in 12% acrylamide gels containing sodium dodecyl sulfate (Laemmli, 1970) using Tris glycine buffer, pH 8.3. Sensitivity Tests Bacteriophage sensitivity tests were done by the method of Wilkinson etal. (1972) using a batteryof bacteriophages originally assembled by B. A. D. Stocker (Stanford University). Sensitivity to antibiotics and other agents was tested using discs placed on bacterial lawns on L-agar; discs were from Becton-Dickinson and Co. or were made by dispensing chemicals onto paper discs. Transformation Transformation with plasmid DNA was done either by a calcium chloride heat shock method (MacLachlan and Sanderson, 1985) or by high voltageelectrotransformation using the Bio-Rad Gene Pulser and Pulse Controller and themethods of Dower et al. (1988).
DNA sequence was determined by the dideoxy chain termination method (Sanger and Coulson, 1975). The strains, constructed by transforming theE. coli strain JM109 with appropriate deletion plasmids (pBluescriptM13+ derivatives), were infected with helper phage R408 to prepare single-stranded DNA templates according to a protocol of Stratagene Inc. Sequenase (modified T7 polymerase) and reagents from U. S. Biochemical Corp. were used for sequencing reactions. Sequence reactions were done using dITP in addition to the usual dGTP to reduce anomalous migration due to secondary structure in the synthesized strand (compressions). The DNA sequence was compiled using The Eyeball Sequence Editor software program written by E. Cabot, Burnaby, British Columbia. DNA and protein sequence analysis program version 5.09 from Genetic Software Center, Dept. of Molecular and Cellular Biology, University of Arizona, Tucson was used to analyze the DNA sequence. Protein Labeling The method of Tabor and Richardson (1985) was used to identify the proteins encoded by the DNA fragment carried on aplasmid. The plasmid was transformed into the E. coli strain D L T l l l bearing the plasmid pGP1-2 that carries the T7 RNA polymerase gene. After inducing the T7polymerase gene at 42 "C, rifampicin was added to a final concentration of 400 pg/ml to inhibit E. coli RNA polymerase. Labeling of the protein products with [35S]methioninewas performed a t 32 "C for 15 min. The protein samples were separated on a 12% polyacrylamide gel and exposed to Kodak X-OMAT AR film at -70 "C. Assay of [44"32PJ(KD0)2-IVA Metabolism Method A-The reaction mixture contained 50 mM HEPES, pH 7.5, 10 mM MgC12, 5 mM ATP, 0.1% Triton x-100, 25 p M [4'-32P](KD0)2-IVA(1-2 X IO4cpm/nmol), and 10 mg/ml soluble fraction of S. typhimurium or E. coli extract (Brozek and Raetz, 1990) in a final volume of 20 pl. Reactions were incubated at 30 "C for the indicated time periods and were stopped by applying 5J samples directly to a silica gel thin layer plate. The plate was developed in chloroform, pyridine, 88%formic acid, water (15:35:85, v/v) and was exposed to x-ray film for 12-15 h at 70 "C using an enhancing screen. No exogenous heptose donor was added. Method B-To test the effect of ADP-heptose on the observed metabolism, the following changes were made. Only1mg/ml cytosolic protein was used, and approximately 200 pM ADP-D-glycero-Dmanno-heptose, 200 p~ ADP-L-glycero-D-manno-heptose, or 5 mM ATP was included as indicated. TLC analysis of the reaction products was carried out exactly as described for Method A. RESULTS
A Chromosomal Map of the rfa Region-Plasmids carrying some of the rfa genes that have been cloned previously are shown in Fig. 3a. pKZ26 carries a 7.5-kb H i d 1 1 fragment with the r f d , -I, -B, and -G genes, while pKZ15 carries the rfaG gene in a 4-kb EcoRI fragment (Kadamet al., 1985). The
Structure and Function of the rfaC Gene
Reference Strain S . typhiumurium DB9005 DS166 DS167 DS310 DS311 DS313 LB5010 SA1355 SA1377 SA2635 SA3548 SA3617 SA3624 SA3669 SA3671 SA3673 SA3674 SA3675 SA3689 SA3694 SA3698 SL1102 SL3600 SL3749 SL3769 SL3770 SL3789 SA4010 E . coli K-12 D21e7 DLTlll JM109 LE392 SAB3684 SAB3686 SAB3688 SAB3699 SAB3700 SAB3701 XLlBlue Plasmids pBluescript K S pGP1-2
descriution
18877
TABLE I Bacterial strains and plasmids Genotwe or
rfa+/F' thyA de0 (cured of pSLT and one of the "Fels" prophages) (P22 gene 17-)+ Same as SL3749, with pKZ72 Same as SA1377, with pKZ72 Same as SL3789, with pKZ72 Same as SL3600, with pKZ72 Same as SA1377, with pKZ76 metA22 metE551 trpC2 leu-3121 ilu-452 rpsLl2O nml- galE856 hsdL.6 hsdSA29 hsdSB121 xyl-404 H2-b H2-e,n,x fla-66 (Fels2)rfa+ (P22)+ rfaC630 (P22)' A his(hisDCBHAF1E) 712 rfaG474 A his(hisDCBHAF1E) 712 rfaG474/pSLT-/F' proAB lacIq lacZAM15 zzf::TnlO" Same as SL3789, with F' hcIq Same as SA1377, with pKZ84 rfaC630 recAl srl-202:TnlO' (P22)+ Same as SA3669, with pKZ84 Same as SA3669, with pKZ98 Same as SL3789, with F' h Z q and pKZ98 Same as SA3689, with pKZ98 rfaC630 (P22)+/F' proAB lacIqlacZAM15 zzf:TnlOd Same as SA3669, with pKZ103 Same as SA3689, with pKZ84 metA22 trpC2 HI -b H2-e,n,x(cured of Fels 2) flu-66 rfaE543 rfaD657 metA22 metE551 trpC2 xyl-404flu-66 rpsLl20 HI-b H2-e,n,x(cured of Fels 2) rfal.446 rfaG rfa+ rfaF511 recAl derivative of SL3789 with pKZ98 pro trp his lac rpsL ampA tsx rfa-I HfrC X+ sr1::TnlOrecA/pGP1-2+ recAl gyrA96 thi hsdRl7supE44 relAl Alac proAB X-/F' traD36 proA+B+ladq lacZAM15 hsdR514 (r; mi) supE44 supF58 lacy1 galK2galT22 metBl trpR55 XSame as JM109, with pKZ99 Same as JM109, with pSLl00 Same as JM109, with pKZlOl Same as DLT111, with pBluescript M13(+) Same as DLT111, with pKZ98 Same as DLT111, with pKZ103 recA1 endAl thigyrA96 hsdR17 supE44 relAl (X)- A(lac-proAB)/F' proA+B+ladq lacZAM15 TnlO
Maurer et al., 1984 This study This study This study This study This study Bullas and Ryu, 1983 Sanderson et al., 1974 Sanderson et al., 1974 K. Sanderson* This study This study This study This study This study This study This study This study This study This study This study Wilkinson et al.,1972 Lehmann et al., 1973 Roantree et al., 1977 Roantree et al., 1977 Roantree et al., 1977 Roantree et al., 1977 This study Boman and Monner, 1975 Tabor and Richardson 1985 Yanish-Perron et al., 1985 Murray et al., 1977 This study This study This study This study This study This study Stratagene, Inc.
Stratagene, Inc. Tabor and Richardson, 1985 DSL~OO Promoterless CAT. AD^ (5.1 kb) Li et al.. 1984 XL1 Blue was mated with SA2635 with selection for KmRand TcR.M13 sensitivity and Br60 resistance were observed, to confirm transfer of the F factor. Salmonella Genetic Stock Center, University of Calgary. Strain SA1377 was transduced with phage ES18hl propagated on TT521 (srl-:TnlOrecAl); TcR transductants were selected and screened for sensitivity to ultraviolet light to detect a recAl allele, to yield SA3669. Strain SA1377 was mated with SA3548, and transconjugants were selected for TcR. Presence of the F factor was confirmed by sensitivity t o male-specific phages M13 and f2. ApR,lacZ (3 kb); also designated M13' c1857 PLgene 1, T7 RNA polymerase gene
rfuL and -Kgenes have been cloned in the plasmid pKZ38 carrying a 4.3-kb HindIII fragment.' ES18- and P22-mediated transduction analysis place the rfuC and r f d ' genes closer to cysE than to pyrE at 79 to 80 min on the linkage map of S. typhimurium (Kuo and Stocker, 1972; Sanderson and Saeed, 1972). The restriction map of the rfu region was extended by Southern blot hybridization of genomic digests of the wildtype S. typhimurium strain DB9005 using the 0.8-kb NcoIHindIII fragment from the left end of pKZ38 as a probe (Figs. 3 and 4). The probe recognized the 4.3-kb Hind111 fragment from which it was derived,but 16-kb EcoRI, 11.8-kbPstI, 4.2P. R. MacLachlan and K. E. Sanderson, unpublished data.
kb BglII, and 6.2-kb NcoI genomic fragments were also seen (Fig. 4). By using these data and known restriction sites in the plasmids pKZ26,pKZ27, and pKZ38, the sites shown between the 0- and 10-kb region on the chromosomal map of Fig. 3a were established. Cloning the rfuC Gene-To clone DNA on the cysE side of rfaL, which might carry rfczC, a X1059 genomic library of S. typhimurium (Maurer et ul., 1984) was screened with the 0.8kb HindIII-NcoI probe from pKZ38 (shown as a hatched bur in Fig. 3u). Ten positive clones were identified. DNA isolated from some of these clones was digestedwith PstI or PstI plus EcoRI and separated by agarose gel electrophoresis. Southern transfers of these gelswerehybridized to the same probe to
18878
Function Structure and 0 1
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