Syringomycin Action Gene SYR2 Is Essential for Sphingolipid 4-Hydroxylation in Saccharomyces cerevisiae*

Michelle M. GrilleyDagger , Stephen D. StockDagger , Robert C. Dickson§, Robert L. Lester§, and Jon Y. TakemotoDagger

From the Dagger  Department of Biology, Utah State University, Logan, Utah 84322 and the § Department of Biochemistry and the Lucille P. Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The Saccharomyces cerevisiae gene SYR2, necessary for growth inhibition by the cyclic lipodepsipeptide syringomycin E, is shown to be required for 4-hydroxylation of long chain bases in sphingolipid biosynthesis. Four lines of support for this conclusion are presented: (a) the predicted Syr2p shows sequence similarity to diiron-binding membrane enzymes involved in oxygen-dependent modifications of hydrocarbon substrates, (b) yeast strains carrying a disrupted SYR2 allele produced sphingoid long chain bases lacking the 4-hydroxyl group present in wild type strains, (c) 4-hydroxylase activity was increased in microsomes prepared from a SYR2 overexpression strain, and (d) the syringomycin E resistance phenotype of a syr2 mutant strain was suppressed when grown under conditions in which exogenous 4-hydroxysphingoid long chain bases were incorporated into sphingolipids. The syr2 strain produced wild type levels of sphingolipids, substantial levels of hydroxylated very long chain fatty acids, and the full complement of normal yeast sphingolipid head groups. These results show that the SYR2 gene is required for the 4-hydroxylation reaction of sphingolipid long chain bases, that this hydroxylation is not essential for growth, and that the 4-hydroxyl group of sphingolipids is necessary for syringomycin E action on yeast.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Syringomycin E is a member of a family of cyclic lipodepsipeptides produced by strains of the plant bacterium Pseudomonas syringae pv. syringae (1). Traditionally regarded as a virulence factor in a variety of bacterial necrotic diseases of plants (2), syringomycin E and its analogs also possess antifungal properties, and it has been suggested that these metabolites are fungal antagonists that aid survival of the producing bacteria on plants (3, 4).

How these compounds produce their toxic effects is unknown, but past physiological studies have shown that syringomycin E targets primarily the plasma membrane (1, 5, 6). To further investigate the molecular mechanisms of action of this bioactive compound, resistant mutants of Saccharomyces cerevisiae were isolated to identify genes that encode proteins necessary for growth inhibition by syringomycin E (7). Several of the mutants were deficient in sterols, and one group was complemented by the gene SYR1 (identical to ERG3), which encodes sterol C-5,6 desaturase of the ergosterol biosynthetic pathway (8). These findings, when combined with results from binding (9) and lipid bilayer (10) studies, indicate that sterols influence the interaction of syringomycin E with the target plasma membrane.

Syringomycin E action in yeast was more recently shown to require a second, nonsterol biosynthetic gene, SYR2 (11). SYR2 is identical to SUR2, which was identified in a screen for mutants that suppress the impaired recovery of rvs161 strains from nutritional starvation (12). Syringomycin E-resistant syr2 mutants showed altered glycerophospholipid levels, and the SYR2 gene product was localized to the endoplasmic reticulum (11). Nevertheless, the precise function of Syr2p was unclear from these studies.

In addition to sterols and glycerophospholipids, sphingolipids are major lipid components of the plasma membrane (13). Ubiquitous in eukaryotic cells, sphingolipids all possess a sphingoid long chain base with mainly, in fungi and plants, a hydroxyl group at the C-4 position (phytosphingosine) or, in animals, a double bond at the C-4,5 position (sphingosine). Sphingolipids serve numerous roles, including mediating cell-cell interactions, anchoring membrane proteins, acting as enzyme co-factors (14), and serving as receptors for Escherichia coli verotoxin (15-17). In addition, sphingolipids are becoming recognized as significant players in the control of cell growth, differentiation, and response to stress through the second messenger action of sphingolipid metabolites sphingosine, sphingosine-1-phosphate, and ceramide (18-20). Despite their importance, numerous gaps remain in the knowledge of sphingolipid metabolism, including the nature of the enzymes directly responsible for phytoceramide or ceramide formation from the presumed immediate precursor dihydroceramide (Fig. 1) (21).


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Fig. 1.   Sphingolipid biosynthesis in S. cerevisiae. Shown is the likely pathway for de novo sphingolipid biosynthesis in yeast, along with the genes, when known, involved at each step. The two possible pathways for long chain base 4-hydroxylation, proposed to be catalyzed by Syr2p, and long chain base acylation are shown. Hydroxylation of the very long fatty acid chain is thought to occur at some point after acylation. Very long fatty acid chains of 24 and 26 carbon length and with zero, one, or two hydroxyl groups have been reported in S. cerevisiae (39). The predominant monohydroxy-C-26 fatty acid is pictured. R can be phosphoinositol, phosphoinositol-mannose, or diphosphoinositol-mannose. AUR1 is necessary for inositol phosphorylceramide synthase activity (26), whereas SYR4/IPT1 catalyzes addition of the terminal phosphoinositol (Ref. 57 and S. D. Stock, J. A. Radding, D. A. Young, and J. Y. Takemoto, unpublished results). Additional genes involved in head group assembly have not yet been identified.

In this report we present evidence that S. cerevisiae SYR2 is required for 4-hydroxylation of sphingoid bases and that this activity is necessary for syringomycin E action. We show that strains mutant in SYR2 produce sphingolipids missing the hydroxyl group at the C-4 position of the long chain base moiety, that supplying such cells with C-4 hydroxylated long chain base suppresses the syringomycin E-resistant phenotype of syr2 strains, and that strains that overexpress Syr2p are enriched in 4-hydroxylase activity.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Yeast Strains and Growth Conditions-- Yeast strains used in this study are listed in Table I. Yeast were grown at 28-30 °C with shaking. YPD, SC-ura, SG-ura (as SC-ura with glucose replaced by galactose), and SC-trp were as described by Kaiser et al. (22). The lcb1 mutants were grown in modified YPD (1% yeast extract, 1% peptone, 4% glucose, 50 mM sodium succinate, pH 5.0, 0.05% Tergitol (U. S. Biochemical Corp.), 25-50 µM phytosphingosine·HCl (PHS)1 or DL-erythro-dihydrosphingosine (DHS) (both from Sigma) or synthetic medium as described by Pinto et al. (23) minus either uracil or tryptophan. Stock solutions (100 mM) of PHS and DHS in 95% ethanol were diluted into 0.5% Tergitol and then added to the media to yield the indicated final long chain base and Tergitol concentrations.

                              
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Table I
S. cerevisiae strains used

Long Chain Base Analysis-- Yeast were grown in YPD and harvested at 1.5 × 108 cells/ml. Methanol-HCl hydrolysates of wild type (W303C and KZ1-1C) and syr2 cells (WDelta SYR2alpha and 13N-F2), as well as C-18 DHS and C-18 PHS standards were derivatized with the UV-absorbing 4-biphenylcarbonyl chloride by the method of Jungalwala et al. (24) as adapted by Dickson et al. (25). Samples were resolved by HPLC on a 250 × 4.6-mm Alltech Econosil C18 column with a 10-mm guard column. Elution was isocratic with methanol/water (90:10, v/v) as the solvent at a flow rate of 1.0 ml/min. Effluent was monitored by absorbance at 280 nm. Electrospray ionization mass spectrometry was performed by the Utah State University Biotechnology Center.

Sphingolipid Analysis-- W303C and WDelta SYR2alpha cells were inoculated to 5 × 106 cells/ml in modified YPD containing 0.5 mCi of [3H]inositol (New England Nuclear Co., 20 mCi/µmol) or 0.5 mCi of [4,5-3H]DHS (2.56 mCi/µmol) derived by hydrolysis of [3H]N-acetyl-DHS prepared as described previously (26). After culture for 18 h at 30 °C the cells reached a density of 1.9-2.4 × 108 cells/ml, and the reaction was stopped by adding trichloroacetic acid to a final concentration of 5%. The cells were processed to deacylate the ester lipids followed by extraction of the sphingolipids as described previously (27). To further purify the acidic sphingolipids, the sphingolipid extract was bound to and eluted from AG4 resin (Bio-Rad) as described previously (28). The AG4 eluate was dried, dissolved in 1 ml of chloroform/methanol/water (16:16:5, v/v/v), and 3-7-µl aliquots were subjected to thin layer chromatography on 20-cm silica gel plates (Whatman HP-K) with the solvent chloroform/methanol/4.2 N aqueous NH4OH (9:7:2, v/v/v). Each lane contained a mixture of yeast PHS-containing sphingolipids: 2 nmol each of inositolphosphoryl ceramide (IPC) and mannosylinositolphosphoryl ceramide (MIPC) species containing mono- and di-OH fatty acids and mannosyl-di(inositolphosphoryl) ceramide (M(IP)2C) with a mono-OH fatty acid. Radioactivity was measured with a BioScan apparatus. The standards were located by charring after spraying with 10% (w/v) CuSO4·5H2O in 8% H3PO4 followed by heating at 160 °C for 30 min (29). The mannosylated sphingolipids in the deacylated lipid extract were also detected after thin layer chromatography of larger aliquots (125 µl) on 20-cm Whatman K5 plates developed with the same solvent as above. The plate was first treated with orcinol reagent (30) to detect the carbohydrate containing sphingolipids, MIPC and M(IP)2C, and then treated with the CuSO4/phosphoric acid reagent as above.

To examine the nature of the long chain bases in the sphingolipid fractions, a portion of the [4,5-3H]DHS-labeled AG4 eluates were dried and hydrolyzed in 1 N HCl in methanol/water (82:18) at 80 °C for 18 h. The hydrolysates were dried, dissolved in chloroform/methanol/water (16:16:5, v/v/v), and spotted on Whatman LK5 plates along with 20 nmol of PHS and DHS standards in each lane. The plates were developed with chloroform/methanol/2 N aqueous NH4OH (40:10:1). Radioactivity was measured with a BioScan apparatus followed by detection of the standards with ninhydrin reagent.

Fatty Acid Analysis-- Fatty acids from an acidic sphingolipid fraction (prepared as described above without the addition of radioisotopes) or whole cells were liberated by saponification and converted to UV-absorbing phenacyl derivatives that were resolved and quantitated by reverse phase HPLC as described previously (25).

Assay of 4-Hydroxylase Activity-- The previously constructed SYR2 overexpression plasmid, pYSYR2, placed a 5'-truncated SYR2 gene under the control of the galactose-inducible promoter GAL1 (11). For this work the truncated SYR2 insert was removed and replaced with an AccI-SphI fragment containing the entire SYR2 coding region. Expression of Syr2p from this construct, pYSYR2a, was confirmed by observation of galactose-inducible complementation of syringomycin E resistance of a syr2 strain.

W303C containing pYSYR2a or the control plasmid pYES2 was grown overnight in SC-ura. Cells were washed with sterile water and diluted into 300 ml of SG-ura (8 × 106 cells/ml) to induce Syr2p expression. Cells were harvested after an additional 16 h of growth and washed with water. WDelta SYR2alpha cells were grown similarly except the medium was SC-ura at each step. Microsome preparation was modified from published procedures (31). The washed cell pellet was resuspended in 25 ml of 100 mM Tris-sulfate, pH 9.4, 10 mM dithiothreitol and incubated at room temperature 15 min, followed by a wash with 10 mM Tris-HCl, pH 7.5, 0.6 M sorbitol, 0.1 mM dithiothreitol, 0.1 mM EDTA. Cells were then incubated 1 h at 30 °C in 7.5 ml of 10 mM Tris-HCl, pH 7.5, 2 M sorbitol, 0.1 mM dithiothreitol, 0.1 mM EDTA, 0.1 mg/ml Zymolyase 100T (Seikagaku Corp., Tokyo). After washing with 10 mM Tris-HCl, pH 7.5, 2 M sorbitol, cells were disrupted with glass beads in 1 ml of cold 10 mM Tris-HCl, pH 7.5, 0.65 M sorbitol, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin. Glass beads and cell debris were removed by centrifugation. Microsomal membranes were collected by centrifugation at 4 °C for 90 min at 100,000 × g. Pellets were homogenized in 0.6 ml of cold 10 mM Tris, pH 7.5, 20% glycerol. Protein concentrations of the microsomal preparations were determined using Pierce Coomassie protein assay reagent with bovine serum albumin as standard.

To assay 4-hydroxylase activity, 50 nmol of either DHS or dihydroceramide in chloroform was dried in a stream of nitrogen and then resuspended by sonication in 0.1 ml 0.3% CHAPS. This was combined with 0.1 ml of 100 mM Tris-HCl, pH 7.5, 0.2 mM NADPH, 0.2 mM NADH, and microsomes (0.7 mg of protein) to initiate the reaction. Incubation was at 25 °C for 90 min, followed by methanol-HCl hydrolysis, 4-biphenylcarbonyl chloride derivatization, and reverse phase HPLC analysis of long chain bases as described above. Chromatographs were integrated using Beckman System Gold Nouveau software.

Construction of lcb1 Disruptant Strains-- A disrupted LCB1 allele, lcb1-Delta 3, was constructed by replacing in pTZ18-LCB1 (32), a 1.5-kilobase pair SalI/BamHI fragment that contains the C-terminal 80% of the LCB1 gene, with a 1.4-kilobase pair TRP1-containing fragment to yield plasmid pLCB1-Delta 3. Plasmids were propagated in E. coli DH5alpha . 3 µg of pLCB1-Delta 3 were digested with restriction endonuclease NdeI, the fragments were separated on a 0.8% agarose gel, and the 3.1-kilobase pair fragment containing the lcb1-Delta 3 allele was isolated using an Elu-Quik DNA Purification Kit as directed by the manufacturer (Schleicher & Schuell). The DNA fragment was transformed into diploid strain W303-1A by electroporation and plated onto SC-trp medium. Trp+ colonies were isolated and sporulated, and the resultant tetrads were dissected onto modified YPD plates containing 25 µM PHS. One Trp+ long chain base auxotrophic colony was selected and designated WDelta LCB1. Replacement of the LCB1 allele with lcb1-Delta 3 was confirmed by Southern blotting using enhanced chemiluminescence detection (Amersham Pharmacia Biotech). The double disruptant, WDelta LCB1Delta SYR2, was produced by crossing WDelta LCB1 with WDelta SYR2a and selecting for Trp+ Ura+ long chain base auxotroph progeny. Standard genetic procedures were as described by Kaiser et al. (22).

Syringomycin E Treatment-- WDelta LCB1Delta SYR2 cells were grown overnight in modified YPD containing either 50 µM DHS or 50 µM PHS. WDelta SYR2alpha and W303C were grown in modified YPD with no long chain base addition. Cells were washed once with sterile water and then transferred to modified YPD minus Tergitol and long chain base and with the indicated amounts of syringomycin E, prepared as described previously (33). Final cell densities were A600 = 0.2 as measured in a Shimadzu UV-1201 spectrophotometer (1 A600 unit equals approximately 3.7 × 107 cells/ml). Incubation was continued at 28 °C. After 1 h Tergitol was restored to 0.05%, and DHS or PHS was restored to 50 µM as appropriate to prevent starvation for long chain base. Syringomycin E was found to be ineffective if added directly to medium containing working concentrations of Tergitol and long chain base. 16 h after syringomycin E addition, aliquots were removed and diluted 10-fold to measure growth by turbidity at A600.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Sequence Analysis Suggests That Syr2p Is a Diiron Binding Lipid Hydroxylase or Desaturase-- BLAST algorithm (34) comparisons of the deduced amino acid sequence of Syr2p with those in protein data bases showed significant similarities to endoplasmic reticulum proteins associated with lipid metabolism. In particular, close similarities were found with S. cerevisiae SYR1/ERG3 (C-5 sterol desaturase, score 51, p = 0.0098, 33% identities, 56% positives), Arabidopsis thaliana ERG3 (C-5 sterol desaturase, score 77, p = 0.014, 44% identities, 68% positives), and yeast ERG25 (C-4 sterol methyl oxidase, score 65, p = 4.5 × 10-8, 52% identities, 65% positives). Similar findings were reported by Bard et al. (35) and Li and Kaplan (36). Despite the similarity of Syr2p to enzymes of sterol biosynthesis, an involvement for Syr2p in sterol metabolism could not be found. Comparisons of sterol profiles of syr2 mutants with similarly grown wild type strains revealed no differences (11),2 indicating that Syr2p functions in some other metabolic pathway.

Sequence comparisons also pointed to the occurrence in Syr2p of an eight-histidine motif grouped into three characteristic clusters (Fig. 2). Shanklin et al. (37) have recently reported that 75 proteins of known function contain this eight-histidine motif, which is thought to bind a catalytically active diiron cluster. Of these proteins, 66 are integral membrane proteins. All 66 catalyze 1 of 11 distinct O2-dependent modifications of hydrocarbon substrates, acting as either desaturases, hydroxylases, oxidases, or decarbonylases (37). Syr2p contains the eight-histidine motif and, based on sequence analysis and subcellular fractionation studies, is an integral membrane protein (11) and thus was predicted to also catalyze an O2-dependent modification of a hydrocarbon substrate. Because SYR2 did not appear to play a role in sterol biosynthesis and also differed from the yeast gene, OLE1, required for glyceride fatty acid desaturation (38), we were prompted to investigate the potential involvement of SYR2 in sphingolipid synthesis. In the yeast sphingolipid biosynthetic pathway (Fig. 1), two uncharacterized processes were deemed potential candidates for catalysis by eight-histidine motif hydroxylases: C-4 hydroxylation of the sphingoid long chain base portion of the sphingolipid and hydroxylation of the very long chain fatty acid.


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Fig. 2.   Putative metal binding domains of Syr2p and similarity to sterol biosynthetic enzymes. The sequences were obtained as part of a BLAST analysis and are from S. cerevisiae (S.c.) or A. thaliana (A.t.). Histidine residues identified by Shanklin et al. (58) as being involved in metal binding are indicated in bold type. Histidine residues identified by Li and Kaplan (36) as a fourth histidine cluster motif specific to the ERG3/ERG25/SYR2 family of sequences are underlined.

Yeast Strains Deficient in SYR2 Lack the Sphingoid Long Chain Base Phytosphingosine-- Sphingolipids in yeast are normally composed of the sphingoid long chain base PHS (D-4-hydroxysphinganine), typically of 18 or 20 carbon chain length (C-18 and C-20, respectively), an amide-linked very long chain fatty acid, primarily mono-hydroxy-C-26 chains with lesser amounts of di- and nonhydroxyl forms, and a phosphoinositol-containing head group (39) (Fig. 1). To test the involvement of Syr2p in the sphingoid base 4-hydroxylation, the sphingoid base compositions of mutant strain WDelta syr2alpha and the isogenic wild type strain W303C were determined (25). Reverse phase HPLC separation of biphenylcarbonyl-derivatized long chain bases derived from the wild type strain W303C revealed two peaks of UV-absorbing material, as expected, with retention times of 15 and 25 min (Fig. 3B). Coelution with a derivatized C-18 PHS standard and electrospray mass spectral analysis of the material collected from the two peaks verified their identities as C-18 PHS at 15 min and mainly C-20 PHS at 25 min. The derivatized long chain bases from the Delta syr2 mutant strain also separated into two peaks, with one again eluting at 25 min but the other eluting at 42 min (Fig. 3C). Little material with a retention time of 15 min was apparent (<0.3% of total long chain base). Authentic C-18 DHS treated in the same manner as the lipid extracts eluted with a retention time of 25 min (Fig. 3A). It was not possible to distinguish between the C-20 PHS and C-18 DHS derivatives by the chromatography system used, but mass spectral analysis of the Delta syr2 material eluting at 25 min revealed a molecular ion mass of 482.3 Da, consistent with its assignment as the N-biphenylcarbonyl derivative of C-18 DHS. The mass of the material eluting at 42 min was 510.4 Da, as expected for N-biphenylcarbonyl-C-20 DHS. Similar results were obtained with an independently isolated syr2 mutant strain in a different genetic background (13N-F2 (syr2) and KZ1-1C (SYR2); data not shown).


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Fig. 3.   Sphingoid long chain bases produced by wild type and Delta syr2 strains. Methanol-HCl hydrolysates of wild type strain W303 (B) and WDelta syr2 cells (C), as well as C-18 DHS and C-18 PHS standards (A), were derivatized with the UV-absorbing 4-biphenylcarbonyl chloride and resolved by reverse phase HPLC as described under "Experimental Procedures." Effluent was monitored by absorbance at 280 nm.

Thin layer chromatography of acid-hydrolyzed, radiolabeled sphingolipids from Delta syr2 and wild type cells confirmed that the wild type produced sphingolipids containing primarily PHS (85%) with 15% DHS, whereas the Delta syr2 mutant produced solely DHS (data not shown). We conclude from these data that Syr2p is necessary for the 4-hydroxylation of the DHS component of yeast sphingolipids. These results are consistent with Syr2p functioning as dihydroceramide or DHS hydroxylase.

Fatty Acid Analysis of Delta syr2 Strain-- The effect of the syr2 mutation on hydroxylation of the very long chain fatty acid component of sphingolipids was also examined as described under "Experimental Procedures." Hydroxylation of the very long chain fatty acids was observed in the absence of Syr2p activity. For example, the percentage of distribution of non-, mono-, and di-hydroxyl fatty acids for wild type cells was 8, 78, and 14%, respectively, and for Delta syr2 cells, it was 53, 47, and 0%, respectively. Clearly Delta syr2 cells hydroxylate the very long chain fatty acid in sphingolipids. Why the distribution of hydroxylated species differs from wild type is not known.

In Vitro Measurement of 4-Hydroxylase Activity-- Sphingoid base 4-hydroxylase activity was measured in microsomal fractions, because Syr2p has previously been localized to the endoplasmic reticulum (11). Microsomes, prepared from SYR2 wild type, overexpression, and deletion strains, were supplied with substrate, either DHS or dihydroceramide solubilized in CHAPS, along with NADH and NADPH. Both NADH and NADPH have been reported to be cofactors for activity of other putative diiron proteins involved in oxygen-dependent reactions of hydrocarbon substrates. After 90 min at 25 °C, sphingoid long chain bases were released by methanol-HCl hydrolysis and extracted, and their 4-biphenylcarbonyl derivatives were separated and quantitated by reverse phase HPLC. Hydroxylated product was apparent when DHS or dihydroceramide were supplied to microsomes from a SYR2 overexpressing strain, W303C(pYSYR2a). Using the same amount of protein, 3-4-fold less hydroxylated product was produced if NADH and NADPH were omitted from the reaction or if the source of microsomes was a wild type strain, W303C(pYES2), containing only the chromosomal copy of SYR2 and a control plasmid (Table II). No 4-hydroxyl products were detected if microsomes were from the deletion strain WDelta syr2alpha .

                              
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Table II
In vitro 4-hydroxylase activity
Microsomes were prepared from the indicated strains, then incubated with either 50 nmol DHS or dihydroceramide with or without 0.1 mM each NADH and NADPH. Sphingoid long chain bases were treated with methanol-HCl, extracted, derivatized, and analyzed by reverse phase HPLC as described under "Experimental Procedures." Values are the averages of two to three trials with standard deviations for 90-min reactions containing 0.7 mg of microsomal protein. ND, none detected. ---, not measured.

Growth in the Presence of PHS Suppresses the syr2 Phenotype-- To confirm that the syringomycin E resistance phenotype of the syr2 mutant is due to a loss of hydroxylase activity rather than an additional unknown activity of Syr2p, we wished to test whether syringomycin E sensitivity is restored by supplying syr2 cells with the product of the hydroxylase. It is still uncertain, however, if the hydroxylase substrate is phytoceramide, PHS, or both (see "Discussion"). As yeast do not readily utilize exogenous ceramides but will incorporate exogenous sphingoid long chain bases into the sphingolipid biosynthetic pathway (40), syringomycin E sensitivity was tested following growth in medium containing hydroxylated (PHS) or nonhydroxylated (DHS) long chain bases rather than hydroxylated or nonhydroxylated ceramides. Also, to ensure that all sphingolipids were built on the exogenous long chain base, the LCB1 gene was deleted in strain WDelta syr2a (see "Experimental Procedures"). LCB1 encodes a subunit of serine palmitoyltransferase, the first enzyme in sphingolipid biosynthesis (Fig. 1). An lcb1 mutation results in auxotrophy for long chain bases (32).

The Delta syr2 Delta lcb1 double mutant was grown several generations in medium containing either DHS or PHS to ensure that sphingolipids lacked or contained phytoceramide, respectively. Each culture was diluted into fresh medium and then challenged with increasing concentrations of syringomycin E. The effectiveness of syringomycin E was assessed after an additional 16 h of growth. For the Delta syr2 Delta lcb1 strain, syringomycin E had no noticeable effect on growth of the DHS-containing cultures. The PHS-containing cultures, on the other hand, showed essentially no growth at syringomycin E concentrations of 1 µg/ml and higher (Fig. 4). These results, with the Delta syr2 Delta lcb1 strain supplemented with either DHS or PHS, mimicked those obtained upon syringomycin E treatment of Delta syr2 and SYR2 strains, respectively (Fig. 4). Further, an isogenic SYR2 Delta lcb1 strain, which retains Syr2p function, also retained its syringomycin E sensitivity whether supplied with DHS or PHS in the growth medium (not shown). Thus, the Delta syr2 Delta lcb1 strain was restored to the wild type syringomycin E-sensitive phenotype by supplying it with hydroxylated long chain base, and the difference between Delta syr2 Delta lcb1 cultures grown on PHS or DHS was dependent on a lack of Syr2p function. These results support the conclusion that Syr2p functions as a 4-hydroxylase and indicate that this hydroxylation is essential for syringomycin E action.


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Fig. 4.   Effect of PHS and DHS on the syringomycin E resistance syr2 phenotype. WDelta LCB1Delta SYR2, cultured in the presence of either PHS (open circle ) or DHS (bullet ), W303C (square ) and WDelta SYR2alpha (black-square) cells were diluted into medium with the indicated amounts of syringomycin E. Syringomycin E resistance, as measured by cell growth (turbidity (O.D.600)), was evaluated 16 h after syringomycin E addition. The example shown is representative of three independent experiments.

Sphingolipid Analysis of Delta syr2 Strain-- The fact that Delta syr2 mutants are viable, whereas sphingolipids are essential, raised questions about the mutant lipid composition. The loss of long chain base hydroxylation could result in the simple substitution of nonhydroxylated long chain base for 4-hydroxy-long chain base in the sphingolipid pools or, alternatively, in an alteration of the overall levels or types of sphingolipids. The recovery of similar quantities of long chain base from mutant and wild type strains (Fig. 3) would argue against a large change in total sphingolipid content, but in order to more directly investigate the quantity and nature of sphingolipids produced by the Delta syr2 mutant, wild type and Delta syr2 cells were cultured overnight with either [3H]inositol or [3H]DHS to label the sphingolipids. The cells were processed to deacylate the ester lipids, such as phosphatidylinositol (PI), and sphingolipids were extracted. Based on the radioactivities of [3H]inositol-labeled extracts before and after PI deacylation, the distribution and amounts of PI and inositol sphingolipids were about the same in wild type (25.7 × 106 cpm PI, 15.3 × 106 cpm sphingolipid) and Delta syr2 cells (30.6 × 106 cpm PI, 14.4 × 106 cpm sphingolipid), confirming that the mutant strain was able to make as much total sphingolipid as the wild type.

The sphingolipid extracts were further purified by binding to and elution from AG4 resin. Thin layer chromatography was carried out on the final preparations, and the results for the [3H]DHS-labeled preparations are shown in Fig. 5. The upper trace (Fig. 5) from wild type cells shows the location of PHS-containing sphingolipid standards, which are coincident with the radiolabeled sphingolipids. These include species G and H, which are IPCs, with mono- and dihydroxylated fatty acids, respectively; species I and J, which are MIPCs, with mono-and dihydroxylated fatty acids, respectively; and species K, which is M(IP)2C with a monohydroxyfatty acid. The sphingolipid preparation from the Delta syr2 mutant strain (Fig. 5, lower trace) shows peaks that are displaced to higher RF values for which we propose the following compositions, all containing DHS instead of PHS: A and B, IPCs with non- and mono-hydroxylated fatty acids, respectively; C and D, MIPCs with non- and mono-hydroxylated fatty acids, respectively; E and F, M(IP)2Cs with non- and mono-hydroxylated fatty acids, respectively.


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Fig. 5.   Thin layer chromatography of sphingolipids obtained by in vivo labeling of wild type and Delta syr2 cells with [3H]DHS. Wild type W303C and WDelta syr2 cells were grown in the presence of [4,5-3H]DHS and then processed to deacylate the ester lipids, followed by extraction and purification of the sphingolipids as described under "Experimental Procedures." Samples were subjected to thin layer chromatography on 20-cm silica gel plates (Whatman HP-K) with the solvent chloroform/methanol/4.2 N aqueous NH4OH (9:7:2, v/v). Each lane contained a mixture of yeast PHS-containing sphingolipids: 2 nmol each of IPC and MIPC species containing mono- and di-OH fatty acids and M(IP)2C with a mono-OH fatty acid. The standards were located by charring after spraying with 10% (w/v) CuSO4·5H2O in 8% H3PO4 followed by heating at 160 °C for 30 min (29). The radioactivity was evaluated with a BioScan apparatus (solid line, wild type; dotted line, mutant). The upper curve was displaced upward by 1500 counts for display purposes. G, H, I, J, and K refer to the locations of an unlabeled mixture of yeast PHS containing sphingolipids added to each lane and detected by charring. G and H, IPCs with mono- and di-OH fatty acids, respectively; I and J, MIPCs with mono- and di-OH fatty acids, respectively; K, M(IP)2C with a mono-OH fatty acid. A, B, C, D, E, and F are the locations of peaks in the mutant strain, all with DHS. A and B, IPCs with zero and mono-OH fatty acid, respectively; C and D, MIPCs with zero and mono-OH fatty acid, respectively; E and F, M(IP)2Cs with zero and mono-OH fatty acid, respectively.

Several lines of evidence support these assignments. First, the results obtained with the [3H]inositol-labeled sphingolipid extracts (not shown) are identical to those obtained with [3H]DHS labeling (Fig. 5), showing that the lower trace in Fig. 5 consists of inositol-labeled sphingolipids. Second, as stated above, analysis of the HCl-methanol hydrolysate of the sphingolipid preparation from the mutant strain exhibits only DHS, not PHS, whereas the wild type shows mainly PHS and some DHS. Finally, when the sphingolipid preparations from unlabeled cells are subjected to thin layer chromatography followed by staining for mannose by spraying with orcinol-sulfuric acid, the mutant exhibits orcinol positive lipids in the locations of species C and D; species E and F, which are located at higher RF values than the orcinol-positive spots in wild type cells; species I and J (MIPCs); and species K (M(IP)2Cs). This result is consistent with the conclusions that species C and D and species E and F are MIPC and M(IP)2C species, respectively. We conclude that the Delta syr2 cells make sphingolipids with head groups similar to those found in wild type cells but that their ceramide moieties contain only DHS and no PHS.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The present study of yeast gene SYR2 reveals that sphingolipids play a key role in the action of the antifungal syringomycin E. It also uncovers a previously uncharacterized activity of the sphingolipid biosynthetic pathway. The lack of PHS-based sphingolipids and accumulation of DHS-based sphingolipids in syringomycin E-resistant syr2 mutants, the greater 4-hydroxylase activity in a SYR2 overexpression strain, the lack of activity in a syr2 mutant strain, and the ability to bypass the syr2 defect with exogenously added PHS provide support for a biosynthetic role of Syr2p in 4-hydroxylation of sphingoid bases. In addition, the sequence similarity of Syr2p to membrane hydroxylases and desaturases and its localization in the endoplasmic reticulum (11), the site of early sphingolipid biosynthetic steps (41), suggest that Syr2p is the enzyme that catalyzes this specific hydroxylation step. An essential catalytic role in fatty acid hydroxylation is not likely because very long chain fatty acid hydroxylation was still detected in the syr2 mutant despite a complete loss of SYR2 transcripts (11). While this report was in preparation, Haak et al. (42) reported that an independent sur2/syr2 mutant produces sphingolipids lacking long chain base 4-hydroxylation, whereas a second gene product, Scs7p, is required for sphingolipid very long chain fatty acid hydroxylation.

Identification of Syr2p as the sphingolipid 4-hydroxylase will pave the way for isolation and characterization of the enzyme. This will, in turn, permit clarification of several issues regarding sphingolipid biosynthesis. One important question concerns the identity of the lipid substrate of Syr2p. The analogous reaction in animal cells, desaturation at the C-4 position, is thought to occur at the level of dihydroceramide (43). In yeast, however, it is not known if 4-hydroxylation occurs before or after long chain base acylation, i.e. if Syr2p converts DHS to PHS, dihydroceramide to phytoceramide, or both (Fig. 1). Inhibition of ceramide synthesis leads to accumulation of both DHS and PHS (44, 45), suggesting a direct conversion from DHS to PHS is possible, at least under conditions of limited ceramide formation. We have shown that incubation of either DHS or dihydroceramide with microsomes from a SYR2 overexpressing strain leads to hydroxylation. With crude microsome preparations, however, enzymes capable of catalyzing acylation or deacylation of the added substrate before hydroxylation may be present and obscure the true nature of the substrate.

Confirmation that Syr2p is in fact an iron-containing oxidative enzyme as predicted from the sequence (Fig. 2) will also be afforded by biochemical analysis. The stimulation of 4-hydroxylase activity by reduced pyridine nucleotide, as shown here, is consistent with Syr2p being a member of this enzyme family. Molecular oxygen has been shown to be the main source of oxygen added to dihydroceramide (46), but little else is known of the mechanism of this reaction. Knowledge about Syr2p will also reveal mechanisms about mammalian sphingolipid biosynthesis. Phytoceramide is produced by certain mammalian tissues (47) as well as by fungi and plants, and these tissues are predicted to contain a Syr2p homolog. The primary mammalian sphingolipids based on ceramide contain the long chain base sphingosine, which has a C-4,5 double bond rather than the 4-hydroxyl group. Recent reports concerning the enzyme in rats that catalyzes this reaction, dihydroceramide desaturase, suggest it also has properties similar to diiron-containing lipid desaturases and hydroxylases (43, 48).

How sphingolipids, and more specifically sphingolipid 4-hydroxylation, allow susceptibility to syringomycin E can only be speculated. One possibility is that 4-hydroxylated sphingolipids directly bind this antifungal compound at the cell surface. The 4-hydroxyl group is expected to influence the degree of sphingolipid exposure on the membrane surface, but it will also affect lipid and protein nearest neighbor interactions in the plane of the membrane. Another possibility is that 4-hydroxylated sphingolipids indirectly influence syringomycin E-cell interaction by modulating sterol or glycerophospholipid compositions or both. Syringomycin E action is influenced by sterols (8, 10), phospholipid bilayers facilitate ion channel formation by syringomycin E molecules (49), and syr2 mutants have lowered cellular glycerophospholipid levels (11). Despite evidence for cross-regulation of the biosynthetic pathways of these various lipid classes in yeast (50), it is difficult to predict precisely how an alteration in the hydroxylation state of sphingolipids could influence cellular sterol and phospholipid composition. Furthermore, 4-hydroxylation could influence the insertion and assembly of lipids as well as proteins into the plasma membrane. Finally, the requirement for sphingolipid 4-hydroxylation may reflect the involvement of phytoceramide-mediated growth inhibition processes in syringomycin E action. Phytoceramide and ceramide, but not dihydroceramide, are reported to mediate cell death in animal cells and growth inhibition in yeast (18, 51, 52), although the phenomenon is not always observed (53). Exposure of yeast cells to syringomycin E may cause increased cellular levels of phytoceramide (perhaps by activating sphingolipid turnover), which in turn may activate specific protein kinases and phosphatases (18, 51, 52), leading to growth arrest. Without the ability to hydroxylate dihydroceramide to phytoceramide and the consequent substitution of dihydroceramide into mature sphingolipids, syr2 mutants would be incapable of undergoing this process.

The observation that SYR2 encodes a nonessential function raises questions about the cellular roles of 4-hydroxylated sphingolipids in yeast growth and survival. Normal SYR2 strains produce sphingolipids that are based almost exclusively on phytoceramide (this study and Ref. 54), but syr2 mutants grow well with dihydroceramide-based sphingolipids. Sphingolipids are indicated to be required for maintaining proton permeability barriers across the membrane or for proton extrusion (55) and for maturation of glycosylphosphatidylinositol-anchored proteins (56). Preliminary observations, however, show that syr2 mutants display wild type growth phenotypes under conditions where proper functioning of these processes may be essential, namely at acidic pHs (4.1), high temperatures (39 °C), and high salt concentrations (0.75 M NaCl).3 Also, Calcofluor staining of chitin, a probe of cell wall structure, was unperturbed in the syr2 mutant, although growth of the syr2 mutant was slightly retarded by Calcofluor.4 The two phenotypes previously associated with syr2/sur2 mutations, resistance to syringomycin E, and suppression of rvs161 mutations can now be said to be associated with a loss of 4-hydroxylation of the long chain base moiety of sphingolipids. The only apparent commonality of these two phenotypes is growth restoration under conditions that inhibit growth of wild type cells. The mechanism(s) responsible for these effects await elucidation, as does a clear definition of the role of 4-hydroxy-sphingolipids in yeast biology and syringomycin E action.

    ACKNOWLEDGEMENTS

We thank Gerald Wells and Elizabeth Nagiec for expert technical assistance.

    FOOTNOTES

* This work was supported by grants from Eli Lilly, the Utah Agricultural Experiment Station Project 607, and the National Science Foundation Grant 9003398 (to J. Y. T.) and National Institutes of Health Grant GM41302 (to R. C. D. and R. L. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This is Utah Agricultural Experiment Station Journal Paper 6022.

To whom correspondence should be addressed: Dept. of Biology, Utah State University, Logan, UT 84322-5305. Tel.: 435-797-1909; Fax: 435-797-1575; E-mail: takemoto{at}cc.usu.edu.

1 The abbreviations used are: PHS, phytosphingosine; DHS, dihydrosphingosine; HPLC, high performance liquid chromatography; IPC, inositolphosphoryl ceramide; MIPC, mannosylinositolphosphoryl ceramide; M(IP)2C, mannosyl-di(inositolphosphoryl) ceramide; PI, phosphatidylinositol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

2 L. Parks, S. Kelly, and M. Bard, personal communications.

3 J. Y. Takemoto, unpublished results.

4 M. M. Grilley and J. Y. Takemoto, unpublished results.

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