Dimannosyldiacylglycerol serves as a lipid anchor precursor in the assembly of the membrane-associated lipomannan in Micrococcus luteus

Leroy S. Pakkiri and Charles J. Waechter1

Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536


1 To whom correspondence should be addressed; e-mail: waechte{at}pop.uky.edu

Received on September 8, 2004; accepted on October 6, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Based on recent analytical and enzymological studies, a topological model for the role of {alpha}-D-mannosyl-(1->3)-{alpha}-D-mannosyl-(1->3)-diacylglycerol (Man2-DAG) as a lipid anchor precursor and mannosylphosphorylundecaprenol (Man-P-Und) as a mannosyl donor in the assembly of a membrane-associated lipomannan (LM) in Micrococcus luteus has been proposed. In this study, a [3H]mannose-suicide selection procedure has been used to identify temperature-sensitive (ts) mutants defective in LM assembly. Two micrococcal mutants with abnormal levels of Man2-DAG and LM at the nonpermissive temperature (37°C), mms1 and mms2, have been isolated and characterized. In vivo and in vitro biochemical assays indicate that mms1 cells have a defect in the mannosyltransferase catalyzing the conversion of Man-DAG to Man2-DAG, and mms2 has a temperature-sensitive defect in the synthesis of Man-P-Und. Because mms1 cells are depleted of endogenous Man2-DAG, membranes from this mutant efficiently converted purified, exogenous [3H]Man2-DAG to [3H]LM by a Man-P-Und-dependent process. An obligatory role for Man-P-Und as a mannosyl donor in the elongation process was also demonstrated by showing that the conversion of exogenous [3H]Man2-DAG to [3H]LM by membranes from mms1 cells in the presence of GDP-Man was inhibited by amphomycin. In addition, consistent with Man2-DAG serving as a lipid anchor precursor for LM assembly, endogenous, prelabeled [3H]Man2-DAG was converted to [3H]LM when membranes from mms2 cells were incubated with purified, exogenous Man-P-Und. These studies provide the first direct proof for the role of Man2-DAG as the lipid anchor precursor for LM, and suggest that Man2-DAG may be essential for the normal growth of M. luteus cells.

Key words: dimannosyldiacylglycerol / lipomannan / [3H]mannose-suicide / temperature-sensitive


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
It is well established that the cytoplasmic membrane of the Gram-positive bacterium Micrococcus luteus is enriched in Man2-DAG and a succinylated lipomannan (LM) apposed to the outer leaflet of the cytoplasmic membrane (Gilby et al., 1958Go; Lahav et al., 1969Go; Lennarz and Talamo, 1966Go; Pakkiri et al., 2004Go; Pless et al., 1975Go; Scher et al., 1968Go; Schmit et al., 1975Go). Cross-immunoelectrophoresis studies and staining of intact protoplasts with fluorescein isothiocyanate (FITC)-conjugated concanavalin A (Con A) localize the LM to the exterior face of the cytoplasmic membrane (Pakkiri et al., 2004Go; Powell et al., 1974Go). Recent topological studies indicate that the active sites of the mannosyltransferases catalyzing the synthesis of the mannolipid intermediates, Man-P-Und and Man1-2-DAG are oriented toward the inside of the cytoplasmic membrane, whereas the active site(s) of the lipid-mediated mannosyltransferases catalyzing the transfer of mannosyl units from Man-P-Und to LM are exposed on the exterior surface of the cytoplasmic membrane. Although earlier topological studies and the stoichiometry of the chemical composition of mannose (50), fatty acyl groups (2.1), and glycerol (1) (Pless et al., 1975Go; Powell et al., 1974Go) suggested that the Man2-DAG could serve as a lipid anchor precursor for LM bioassembly, there has not been any direct proof supporting this proposal.

To investigate the precise role(s) of the mannolipid intermediates, Man-P-Und and Man2-DAG, a [3H]mannose-suicide procedure was applied to isolate temperature-sensitive (ts) mutants that are defective for growth and LM synthesis by adapting the approach used for the selection of asparagine-linked glycosylation (alg) mutants in Saccharomyces cerevisiae by Huffaker and Robbins (1982Go, 1983)Go. The rationale is that the mutants survive the radiation damage sustained by the wild-type cells because ts defects in individual steps in the biosynthetic pathway block the accumulation of [3H]mannose in the membrane-anchored LM. Thus the defects prevent the mutant cells from sustaining lethal radiation damage.

Approximately 26 ts micrococcal mutants were identified after replica-plating the survivors on Luria Bertani (LB) agar by comparing the colonial patterns at the permissive (30°C) and nonpermissive temperatures (37°C). Cells that fail to form colonies at 37°C were isolated and screened for biochemical alterations in all of the steps shown in Figure 1. Two mutants with altered levels of Man2-DAG and LM were selected and characterized here. One mutant, designated mms1, is defective in Man2-DAG synthesis, and the second mutant (mms2) appears to have a ts defect in the mannosyltransferase synthesizing Man-P-Und. In vivo and in vitro studies with the mutant deficient in Man2-DAG (mms1) provide the first solid evidence that the mannolipid serves as the lipid anchor precursor for LM. The results of in vitro experiments with membrane fractions from mms2 confirm the obligatory role of Man-P-Und in the elongation of Man2-DAG. The potential of the [3H]mannose-suicide selection procedure for identifying genes encoding other key membrane proteins involved in LM assembly, including mannolipid flippases, and the possibility that Man2-DAG is required for normal growth of M. luteus cells are discussed.



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Fig. 1. Proposed model for the synthesis and transbilayer movement of mannolipid intermediates involved in LM assembly in M. luteus. Micrococcal mutants mms1 and mms2 have temperature-sensitive defects in protein(s) catalyzing steps 2 and 3, respectively. Other potential mutation(s) (1, 4–7) that would disrupt LM assembly are also shown.

 

    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Experimental strategy for [3H]mannose-suicide selection in M. luteus
The principle of radiological suicide selection has been applied to the isolation of cells with mutations in protein synthesis (Miller, 1972Go), phospholipid biosynthesis (Cronan et al., 1970Go), hexose transport (O'Rear et al., 1999Go), glycoprotein fucosylation (Hirschberg et al., 1981Go) and in yeast mannolipid and glycoprotein biosynthesis (Huffaker and Robbins, 1982Go, 1983Go) using targeted isotopic precursors. The approach used to isolate alg mutants in yeast by Robbins and co-workers (Huffaker and Robbins, 1982Go) was adapted to select micrococcal mutants with defects in specific steps in the assembly of LM (Figure 1) that would block the accumulation of [3H]mannose in the membrane-associated LM and thereby protect the mutant cells from radiation damage.

Micrococcal mutants mms1 and mms2 are temperature-sensitive for growth
Based on the assumption that ts defects in Man1–2-DAG and LM biosynthesis could affect cell viability, ethyl methanesulfonate (EMS)-treated cells were subjected to two rounds of [3H]mannose-suicide selection, replica plated (Miller, 1972Go) on LB agar, and grown at 30°C (permissive temperature) and 37°C (nonpermissive temperature). Two ts mutants, mms1 and mms2, were isolated by this procedure, and the enzymatic mutations have been characterized.

After 30 h of incubation at 30°C, all strains showed normal growth on LB agar (Figure 2A). In contrast to the wild-type cells, mms1 and mms2 cells failed to produce visible colonies at 37°C (Figure 2B). To determine if incubation at the nonpermissive temperature would lead to lethality, mms1 and mms2 cells were incubated at 37°C for 30 h in LB agar (no visible colonies formed), and then shifted to 30°C for an additional 30 h. Under these conditions, both mutants resumed normal growth (Figure 2C), indicating that the temperature-sensitive growth defects are reversible.



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Fig. 2. Mms1 and mms2 cells have growth defects at the nonpermissive temperature (37°C), but resume normal growth after shifting back to permissive temperature (30°C). Cells plated on LB agar were incubated for 30 h at 30°C (A) and 37°C (B). After this time, the plate shown at B was shifted back to 30°C (C) for an additional 30 h.

 
Man1-2-DAG, phospholipid, and LM content in wild-type and mutant cells
To characterize the ts mutants further, the mannolipid, phospholipid, and LM composition was compared at the nonpermissive temperature. After exponential growth for 16 h at 30°C, the cell cultures were shifted to 37°C and incubated for an additional 6 h. The crude lipid extracts were analyzed for mannolipid and phospholipid content. From the qualitative analysis as illustrated in Figure 3 it can be seen that Man2-DAG is the major mannolipid in wild-type cells, and the precursor, Man-DAG, is barely detectable. However, mms1 cells contained significantly lower amounts of Man2-DAG but accumulated large amounts of the precursor Man-DAG. No difference was seen in the intensity of the zones corresponding to the phospholipids, phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidylinositol (PI) in wild type and the two mutant strains by this chromatographic analysis.



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Fig. 3. Thin-layer chromatographic analysis of Man1–2-DAG and phospholipid composition of wild-type, mms1, and mms2 cells. Crude lipids extracted from membranes of wild-type, mms1, and mms2 cells that were grown exponentially at 30°C and shifted to 37°C for 6 h were partially purified by chromatography on silicic acid as described in Materials and methods. Fractions containing Man1–2-DAG and phospholipids were analyzed by TLC on silica gel plates after developing with CHCl3/CH3OH/H2O (65:25:4) and staining with orcinol/sulfuric acid solution and incubation for 5 min at 100°C (Churms and Zweig, 1982Go) for mannolipids or by spraying with phospholipid reagent (Dittmer and Lester, 1964Go).

 
The results of a quantitative compositional analysis (Table I) are consistent with the differences seen when the lipid extracts were analyzed chromatographically. Chemical analysis of mms1 cells revealed that this mutant accumulated a substantial amount of Man-DAG, and the level of Man2-DAG was reduced 84% compared to wild-type cells (Table I). Similarly, the mannose content of the LM fraction was reduced 83% relative to wild-type cells, offering a plausible explanation for the survival of the mutant cells during [3H]mannose-suicide treatment.


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Table I. Phospholipid, mannolipid, and LM composition of wild-type and mutant cells

 
In contrast to mms1 cells, mms2 cells showed a fivefold higher level of Man2-DAG compared to wild-type cells and contained 73% less LM based on the mannose content. No changes were seen in the content of the phospholipids in either mms1 or mms2 cells at the nonpermissive temperature, indicating that the mutations in these cells do not affect the phospholipid biosynthetic pathways.

Microscopy of NAO or FITC-Con A-stained protoplasts
CL-specific 10-N-nonyl-acridine orange (NAO) staining has been widely utilized to evaluate the level of CL in cell membranes (Mileykovskaya and Dowhan, 2000Go). Previously, it was shown that FITC-conjugated Con A binds to LM present on the outer surface of intact protoplasts of M. luteus (Pakkiri et al., 2004Go). To corroborate that CL levels are apparently normal but the LM levels are reduced in the two ts mutants, protoplasts from wild-type, mms1, and mms2 cells were exposed to NAO (Figure 4a–f) and FITC-conjugated Con A (Figure 4g–l). When exposed to NAO, protoplasts from all strains exhibited the same fluorescence intensity (Figure 4d–f). In contrast to wild-type protoplasts, the fluorescence intensity from mms1 and mms2 was severely reduced when exposed to FITC-conjugated Con A (Figure 4j–l), indicative of lower amounts of LM present on the outer surface of the micrococcal protoplasts. These results confirm that the phospholipid levels are unaffected, but the LM level is drastically reduced in mms1 and mms2 cells at the nonpermissive temperature.



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Fig. 4. Protoplasts from mms1 and mms2 cells contain normal levels of CL but reduced amounts of LM on the outer surface of the cytoplasmic membrane. Intact protoplasts of wild-type, mms1, and mms2 cells were visualized by bright field differential interference contrast (DIC) or by fluorescence microscopy at 1000x magnification after exposure to NAO (af). A different set of protoplasts from these cells were exposed to FITC-conjugated Con A (gl).

 
In vivo synthesis of [3H]Man1-2-DAG and [3H]LM in wild type and mutant cells
To determine the enzymatic defect in the LM assembly process, in vivo metabolic labeling experiments with [3H]mannose were conducted. Some potential mutation(s) in mannolipid biosynthesis or utilization are illustrated in Figure 1 (steps 1–7). To determine if there was a temperature-sensitive defect in any of these biosynthetic steps, cells were grown at 30°C, shifted to 37°C for 1 h, and metabolically labeled with [3H]mannose to assess the rates of synthesis of mannolipids and LM. Under these conditions, large amounts of [3H]mannose were incorporated into [3H]Man2-DAG and LM in wild-type cells (Figure 5A). However, the rate of incorporation of [3H]mannose into [3H]Man2-DAG and [3H]LM was significantly reduced in mms1, whereas the isotopic precursor accumulated in [3H]Man-DAG (Figure 5B), suggesting a ts defect in the addition of the second mannosyl unit to Man2-DAG.



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Fig. 5. Mms1 cells accumulate [3H]Man-DAG and mms2 cells accumulate [3H]Man2-DAG when metabolically labeled in vivo with [3H]mannose. Wild-type, mms2, and mms1 cells that were exponentially grown on LB media (5 ml) at 30°C were sedimented by centrifugation (3000 x g, 10 min) and resuspended in fresh LB media (0.2 ml) containing 25 µCi of [3H]mannose. Incubation was carried out at 37°C, and at the end of each indicated time point, lysozyme (1.5 mg) and DNase 1 (0.2 mg) were added for 10 s, prior to termination of reaction by the addition of 0.3 ml CH3OH and 5 ml (final vol) CHCl3/CH3OH (2:1). The tubes were placed on ice for 5–10 min and radioactivity incorporated into Man-DAG (squares), Man2-DAG (circles), and LM (triangles) of wild-type (A), mms1 (B), and mms2 cells (C) was determined as described in Materials and methods.

 
In mms2 cells, [3H]mannose accumulated in [3H]Man2-DAG, and the rate of labeling of LM was significantly reduced (Figure 5C), suggesting a possible defect in the use of the mannolipid for LM assembly or possibly in the synthesis of the obligatory mannosyl donor, Man-P-Und. The large amounts of [3H]mannose incorporated into the two neutral mannolipids in mms1 and mms2 cells indicate that there are no defects in the entry of mannose into these cells or its utilization for the formation of GDP-Man.

Assay of micrococcal mannosyltransferases in vitro in crude homogenates from wild-type and mutant cells
To examine the biochemical defects in mms1 and mms2 in more detail, the rates of synthesis of the mannolipids and LM were compared in vitro with homogenates from wild-type and mutant cells at 37°C. In contrast to homogenates from wild type cells, [3H]mannose accumulated in [3H]Man-DAG when homogenates from mms1 cells were incubated with GDP-[3H]Man (Table II). The synthesis of [3H]Man2-DAG and [3H]LM was markedly lower in mms1 cells, indicating that the mannosyltransferase catalyzing the transfer of the second mannosyl unit to Man2-DAG is defective at the nonpermissive temperature. The rates of [3H]Man-P-Und synthesis in wild-type and mms1 cell homogenates were virtually identical. However, the synthesis of [3H]Man-P-Und by homogenates from mms2 cells was drastically reduced (Table II). Consequently, the synthesis of [3H]LM was also reduced, and the transfer of [3H]mannosyl units from GDP-[3H]Man was diverted to the synthesis of [3H]Man1–2DAG.


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Table II. In vitro incorporation of mannose from GDP-[3H]Man into [3H]mannolipids and [3H]LM by crude cell homogenates

 
Enzymatic conversion of exogenous [3H]Man-DAG to [3H]Man2-DAG by homogenates from wild-type and mms1 cells
To corroborate that mms1 cells contained a ts defect in the mannosyltransferase activity adding the second mannosyl unit to Man2-DAG, the direct conversion of purified, exogenous [3H]Man-DAG to [3H]Man2-DAG was assayed. From the results in Table III it can be seen that homogenates from wild-type cells readily converted [3H]Man-DAG to [3H]Man2-DAG in the presence of GDP-Man. In contrast to this result, the rate of Man2-DAG formation catalyzed by homogenates from mms1 cells was reduced 84% compared to wild-type cells. These results are consistent with the compositional studies and the in vivo and in vitro assays indicating that the mannosyltransferase converting Man-DAG to Man2-DAG is impaired at the nonpermissive temperature in mms1 cells.


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Table III. Conversion of exogenous [3H]Man-DAG to [3H]Man2-DAG in cell homogenates

 
To determine if the decrease in Man2-DAG synthesis was due to a ts mutation in the mannosyltransferase or the presence of an inhibitory factor produced at 37°C, homogenates from wild-type and mms1 cells were premixed for 10 min, and the rate of synthesis of Man2-DAG was assayed separately or in mixed homogenates. The data in Table IV clearly show that the rate of conversion of Man-DAG to Man2-DAG is reduced in mms1 cells and that the predicted average value is seen with mixed homogenates from both cells. Thus the lower level of Man2-DAG in the temperature-sensitive mms1 mutant appears to be due to a defect in the mannosyltransferase adding the second mannosyl unit to Man-DAG, and not due to the presence of an inhibitory factor. The inability to synthesize Man2-DAG, the putative lipid anchor precursor, also provides a mechanistic explanation for the lower levels of LM present in mms1 cells (Figure 1).


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Table IV. Effect of mixing cell homogenates from wild-type and mms1 cells on Man1-2-DAG biosynthesis

 
Enzymatic conversion of exogenous [3H]Man2-DAG to [3H]LM by membranes from mms1 cells
Previous studies with membranes from wild-type cells have failed to demonstrate the direct conversion of purified, exogenous [3H]Man2-DAG into LM, as proposed in Figure 1, presumably due to isotopic dilution of the exogenous substrate by the large amount of endogenous Man2-DAG. Because mms1 cells contain greatly reduced levels of endogenous Man2-DAG (Table I), membranes from this mutant were tested for their ability to elongate exogenous [3H]Man2-DAG to [3H]LM enzymatically. The results in Table V show that a substantial amount of exogenous [3H]Man2-DAG was converted to [3H]LM in the presence of GDP-Man when incubated with membranes from mms1 cells. The dependence of the conversion of Man2-DAG to [3H]LM on the formation of Man-P-Und is supported by the observation that the elongation system was blocked by the addition of amphomycin, which inhibits Man-P-Und formation by forming a complex with Und-P (Pakkiri et al., 2004Go).


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Table V. The conversion of exogenous [3H]Man2-DAG to [3H]LM by membranes from mms1 cells is inhibited by amphomycin

 
Similarly, the conversion of exogenous unlabeled Man2-DAG to [3H]LM, in the presence of GDP-[3H]Man asthe isotopic precursor, was also inhibited by the lipopeptide inhibitor amphomycin (Table VI). These results providethe first direct proof that the polymannose chain of the LM is formed by elongation of Man2-DAG with Man-P-Und serving as mannosyl donor for a yet to be determined number of lipid-mediated mannosyltransferases, as depicted in Figure 1.


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Table VI. Effect of amphomycin on the GDP-[3H]Man-dependent conversion of nonradioactive, exogenous Man2-DAG to LM by membranes from mms1 cells

 
Characterization of [3H]LM formed from exogenous [3H]Man2-DAG by membranes from mms1 cells
To characterize and estimate the size of the product synthesized from purified, exogenous [3H]Man2-DAG in vitro by mms1 membranes, the size of the [3H]LM fraction deacylated by mild alkaline methanolysis was estimated by comparative gel filtration with the deacylated [3H]LM synthesized by membranes from wild-type cells using GDP-[3H]Man as the isotopic precursor. From the elution profiles illustrated in Figure 6, it can be seen that the major products from wild type (A) and mms1 cells (B) eluted near the exclusion volume with virtually identical Ve/Vo ratios. When both deacylated LM products were digested with {alpha}-mannosidase, free [3H]mannose was released (Figure 6A and B).



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Fig. 6. Gel-filtration analysis of deacylated [3H]LM from wild-type, mms1, and mms2 cells before (open circles) and after (closed circles) digestion with {alpha}-mannosidase. [3H]LM (20,000 cpm) synthesized from GDP-[3H]Man by wild-type membranes and deacylated, and a second aliquot treated with {alpha}-mannosidase (A) were applied to a Bio-Gel P-10 column (1.8 x 20 cm) and eluted with 0.1 N acetic acid as described in Materials and methods. Similarly, an aliquot of [3H]LM (20,000 cpm) synthesized from purified, exogenous [3H]Man2-DAG and GDP-Man by mms1 membranes and deacylated, and a second aliquot treated with {alpha}-mannosidase (B) were applied to the column. In C, [3H]LM (20,000 cpm) was synthesized from the conversion of endogenous, prelabeled [3H]Man2-DAG to [3H]LM due to exogenous addition of Man-P-Und by mms2 membranes and deacylated, and a second aliquot treated with {alpha}-mannosidase were applied to the column. Fractions (1.4 ml) were collected, dried, and analyzed for radioactivity by liquid scintillation spectrometry. The elution positions of the calibration markers, blue dextran (Vo), Glc3Man9GlcNAc2 (G14), Man2-glycerol (M2G), and mannose (M) are indicated by arrows.

 
These results provide convincing evidence that the large-molecular-weight product formed by sequential Man-P-Und-dependent mannosylation of [3H]Man2-DAG in membrane preparations from mms1 cells is similar in size to the LM formed in vitro with membranes from wild-type cells, ranging from 40–50 mannose residues.

Man-P-Und-mediated transfer of mannosyl units to LM by membranes from mms2 cells
To characterize the enzymatic defect in mms2 cells further, exogenous Man-P-Und was added to membranes from mms2 containing endogenous, prelabeled [3H]Man2-DAG. The putative mannolipid anchor precursor was converted to [3H]LM (Figure 7) in a concentration-dependent manner, and the [3H]LM formed under these conditions also appeared to be chromatographically identical to glycerol-(Man)48–50 following deacylation (Figure 6C). These results confirm that mms2 cells are defective for the synthesis of the lipophilic mannosyl donor, Man-P-Und, and that the elongation of the lipid anchor precursor, Man2-DAG, requires Man-P-Und as the mannosyl donor.



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Fig. 7. Conversion of endogenous, prelabeled [3H]Man2-DAG to [3H]LM by membranes from mms2 cells is dependent on the addition of exogenous Man-P-Und. Enzymatic reaction mixtures contained membrane proteins (1.0 mg) with endogenous [3H]Man2-DAG prelabeled by incubating with GDP-[3H]Man as described in Materials and methods, 50 mM Tris–HCl (pH 7.6), 10 mM MnCl2, and purified exogenous Man-P-Und (0–130 mM) dispersed by ultrasonication in 1% Triton X-100 (final conc. 0.1%, w/v) in a total volume of 0.1 ml. Following incubation at 37°C for 30 min, the reactions were terminated by the addition of 20 vols of CHCl3/CH3OH (2:1), and the amount of endogenous [3H]Man2-DAG converted to [3H]LM was determined as described in Materials and methods.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In a previous report (Pakkiri et al., 2004Go), a topological model was proposed in which Man2-DAG was formed on the inner leaflet of the cytoplasmic membrane and served as a lipid anchor precursor for LM assembly after flip-flopping to the outer monolayer in M. luteus (Figure 1). To obtain more support for this hypothesis, a [3H]mannose-suicide selection procedure was devised to isolate ts mutants that are blocked in specific enzymatic steps in the biosynthetic pathway for LM. Approximately 35,000 surviving colonies were screened at permissive (30°C) temperature and nonpermissive temperature (37°C), and 26 colonies were identified that are temperature-sensitive for growth and defects in LM synthesis. Two of these mutants, mms1 and mms2, exhibited dramatic changes in Man2-DAG and LM content and were characterized. This article describes studies with mms1 and mms2 cells that provide experimental support for specific stages of the proposed model illustrated in Figure 1.

In vivo and in vitro biochemical studies establish that mms1 cells have a ts defect in the mannosyltransferase(s) catalyzing the conversion of Man-DAG to Man2-DAG (Figure 1, step 2), and consequently accumulate the precursor intermediate, Man-DAG. The ts growth defect in this mutant strongly suggests that Man2-DAG synthesis is required for the normal growth of these micrococcal cells.

In a key direct experiment, membranes from mms1 cells are able to efficiently convert exogenous [3H]Man2-DAG to [3H]LM by a process dependent on Man-P-Und formation due to the deficiency of endogenous Man2-DAG. The radiolabeled LM formed from either exogenous [3H]Man2-DAG and unlabeled GDP-Man or from exogenous Man2-DAG and GDP-[3H]Man by membranes from mms1 cells appear to be virtually identical in size (48–50 residues) to LM formed by membranes from wild-type cells as assessed by gel filtration analysis of the deacylated products onBio-Gel P-10. The role of Man-P-Und as the mannosyl donor for the elongation process is confirmed by the inhibitory effect of amphomycin.

The second mutant characterized in this study, mms2, has a temperature-sensitive defect in the synthesis of Man-P-Und (Figure 1, step 3), as determined by in vitro studies. The inability to synthesize Man-P-Und, the obligatory mannosyl donor, blocked the elongation of the membrane-anchored precursor, Man2-DAG, in mms2 cells on the outer surface of the cytoplasmic membrane (Pakkiri et al., 2004Go), resulting in the accumulation of Man2-DAG. In accord with the proposed pathway in Figure 1, Man2-DAG could be readily converted to LM when exogenous Man-P-Und was added in vitro.

The data from these studies clearly indicate that the synthesis of the lipid intermediates, Man2-DAG and Man-P-Und, is essential for LM formation and normal cell growth as indicated by growth defects at the restrictive temperature (37°C). The mutant cells are also partially defective in these biosynthetic steps under normal growth condition (data not shown), suggesting that residual enzyme activities may be sufficient for the viability and propagation of these mutant cells. It has been reported that some ts mutants of S. cerevisiae showed similar phenotypes and are also partially defective at the nonrestrictive temperature (Huffaker and Robbins, 1983Go; Runge and Robbins, 1986Go).

These studies establish that the [3H]mannose-suicide selection procedure is an effective approach to isolating specific micrococcal mutant variants with defects in the enzymes catalyzing various steps in LM assembly. Based on the potential biochemical defect(s) in the LM assembly steps, the mutants can be categorized into three classes that would ostensibly allow the mutant cells to survive [3H]mannose-suicide. Class I micrococcal mutants are those that are blocked in mannose transport/entry or the biosynthesis of GDP-Man. Several class 1 mutants were identified after metabolic labeling with [3H]mannose in vivo (data not shown). The in vivo results in this study firmly establish that mms1 and mms2 cells do not belong to this category because [3H]mannose is incorporated into [3H]Man1-2DAG (Figure 5). Micrococcal mutants that are defective in the synthesis of mannolipid intermediates, Man1-2-DAG and Man-P-Und from GDP-Man or the exoplasmic Man-P-Und-mediated transfer of mannosyl units to LM (Figure 1, steps 6–7), are classified as class II.

Finally, class III mutants, which are potentially the most interesting, are defined as mutants that are impaired in the transverse diffusion of Man-P-Und or Man2-DAG (Figure 1, steps 4–5) from the inner leaflet to outer monolayer of the cytoplasmic membrane. Prospective class III mutants with defects in membrane proteins mediating the transbilayer movement of Man-P-Und and Man2-DAG will be based on assaying the loss of transport of the chemoenzymatically synthesized, water-soluble analogs, Man-P-nerol (Man-P-Und) and sn-1,2-diC4-DAG-[3H]Man2 (Man2-DAG) into intact protoplasts or sealed micrococcal membrane vesicles. This approach has successfully implicated endoplasmic reticulum proteins in the transbilayer movement of Man-P-dolichol (Rush and Waechter, 1995Go, 2004Go) and Glc-P-dolichol (Rush and Waechter, 1998Go) in liver and brain microsomes, and Fuc4NAc-ManNAcA-GlcNAc-P-P-Und (Lipid III), the trisaccharyl donor in the biosynthesis of enterobacterial common antigen in Escherichia coli (Rick et al., 2003Go).

In summary, the in vitro and in vivo results with the two ts mutants strongly suggest that mms1 cells have a defect in the mannosyltransferase forming Man2-DAG and mms2 cells have a defect in the enzyme synthesizing Man-P-Und. The results also suggest that Man2-DAG may be essential for normal growth of M. luteus cells. To obtain definitive evidence for these conclusions, it will be necessary to identify the genes encoding the mannosyltransferases by complementing the ts defects. Work is in progress to identify the micrococcal genes encoding the mutations in mms1 and mms2 by complementing the ts phenotype with a wild-type genomic DNA library, similar to the cloning of the alg genes (Aebi et al., 1996Go; Couto et al., 1984Go; Jackson et al., 1993Go) and other genes related to the dolichol pathway (Shridas et al., 2003Go). In any case, the deficiencies in Man2-DAG and LM in mms1 cells have provided an excellent model system for establishing for the first time that Man2-DAG functions as the lipid anchor precursor for LM in M. luteus cells. Another future goal in the micrococcal system will be to elucidate the mechanism and topology of the formation of the succinyl ester linkages on the LM chains as well as their physiological function(s). M. luteus and the related micrococcal species, Micrococcus flavus and Micrococcus sodonensis, contain succinylated mannosyl residues in LM. The micrococcal LM is proposed to bind Mg2+ (Powell et al., 1975Go) essential for functional DD-carboxypeptidases, transpeptidases, and autolytic enzymes involved in peptidoglycan remodeling during cell growth and division (Salton, 1980Go). It remains to be established if the loss of the succinylated LM and consequently the inability to bind Mg2+ is related to the growth sensitivity of the LM-deficient mms1 and mms2 cells at the restrictive temperature.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
M. luteus (ATCC 4698) was obtained from American Type Culture Collection (Rockville, MD). {alpha}-Mannosidase from Canavalia ensiformis (M-7257), NAO (A-7847), chicken egg white lysozyme (L-6876), DEAE cellulose (D-8507), DNase 1 (DN-25), EMS (M-0880), FITC-conjugated Con A (C-7642), guanosine diphosphomannose, sodium salt (G-5131), rhodamine 6G (R-4127), RNase (R-7003), sodium taurocholate (T-4009), silicic acid, 100–200 mesh (Sil 350), and Triton X-100 (T-9284) were purchased from Sigma Chemical (St. Louis, MO). Orcinol (O-244) was obtained from Fisher Scientific (Fairlawn, NJ), and blue dextran 2000 was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Sodium dodecyl sulfate (811034) was purchased from ICN Biomed (Aurora, OH). Silica gel plates, Baker Si250 (7000-00) were purchased from J. T. Baker (Phillipsburg, NJ), and Bio-Gel P-10, 100–200 mesh was from Bio-Rad (Richmond, CA). Econosafe liquid scintillation counting cocktail was purchased from Research Products International (Mount Prospect, IL). [2-3H]mannose (50 Ci/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO), and Und-P was purchased from Dr. Taduesz Chojnacki (Polish Academy of Sciences, Warsaw). GDP-[3H]mannose (150–200 cpm/pmol) was enzymatically also synthesized as described (Rush et al., 1993Go), and amphomycin (calcium salt) was a gift from Dr. M. Bodanszky (Case Western ReserveUniversity, Cleveland, OH). All other chemicals are reagent grade and purchased from standard commercial sources.

Preparation of cell homogenates and membranes
Cells were grown by shaking liquid cultures at 30°C on LB media consisting of 1% bactopeptone, 1% NaCl, and 0.5% yeast extract. During exponential growth (A600 1.2), the cells were shifted to 37°C for 6 h. These were then sedimented by centrifugation (3000 x g, 10 min), and washed twice with distilled water and once with 50 mM Tris–HCl (pH 7.6) containing 5 mM EDTA (buffer A) before resuspension in the same buffer (20 ml). Lysozyme (150 µg/ml), DNase 1 (10 µg/ml), and RNase (5 µg/ml) were added to a stirred cell suspension for 45 min at 4°C, and the cells were disrupted by passage through a French pressure cell (18,000 psi). This procedure was repeated three times at 4°C before placing the crude cell lysate on ice for an additional 15 min until a decrease in viscosity was observed. Unbroken cells were sedimented by centrifugation (3000 x g, 10 min) and discarded. The resulting lysate was homogenized by 8–10 strokes with a Dounce homogenizer using pestle B and stored at –20°C at a final protein concentration of 10–15 mg/ml. Crude membranes were sedimented by centrifugation of cell homogenates at 4°C (50,000 x g, 1 h). The supernatant fluid was removed, and the membrane pellet washed four times with buffer A. The membrane pellet was resuspended at a final protein concentration of 8–10 mg/ml and stored at –20°C until further use.

Preparation of intact protoplasts
Cells harvested during exponential growth were sedimented by centrifugation (3000 x g, 10 min), washed with distilled water as described, and resuspended in 50 mM Tris–HCl (pH 7.6) containing 0.8 M sucrose, 150 mM NaCl, and 10 mM MgCl2 (buffer B). Following incubation for 20 min at 22°C with lysozyme (150 µg/ml) and DNase 1 (1 µg/ml), protoplasts were sedimented by centrifugation (20,000 x g, 20 min). The protoplasts were washed once with buffer B, resuspended at a final protein concentration of 10 mg/ml, and stored at –20°C. The intactness of the protoplasts was assessed by incubating with GDP-[3H]Man as described (Pakkiri et al., 2004Go).

Staining of protoplasts with NAO and FITC-Con A conjugate
Protoplasts (15 mg) were washed twice with a buffer containing 25 mM HEPES (pH 7.4), 0.8 M sucrose, 150 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, and 0.5 mM MgCl2 (buffer C). The washed protoplasts were resuspended and incubated with buffer C (0.2 ml) containing 2% bovine serum albumin (BSA) at 30°C. After 1 h, the protoplasts were sedimented by centrifugation (20,000 x g, 20 min) to remove the BSA and resuspended in buffer C containing 20 µg of either NAO or FITC-conjugated Con A in a final volume of 0.2 ml. After 1 h at 30°C, the incubation mixture was centrifuged to remove unbound fluorescent conjugates, washed three times, and resuspended in 0.05 ml of buffer C. Samples (1 µl) were placed on a microscope slide and visualized by light microscopy (Nikon Eclipse E600) under bright field or fluorescence to assess the surface exposure of CL and LM. Images were recorded with a SPOT camera (Diagnostic Instruments) and manipulated with Adobe Photoshop 7.0.

Isolation of ts mutants
A [3H]mannose-suicide selection was performed essentially by the procedure of Huffaker and Robbins (1982)Go with minor modifications. Cells were grown exponentially in 10 ml LB media at 30°C and sedimented by centrifugation (3000 x g, 10 min). The cell pellet was resuspended in 0.5 ml of 100 mM sodium phosphate (pH 7.0) containing 3% EMS. After 2 h at 30°C, the mutagenic reaction was terminated by diluting with 40 vols of 5% sodium thiosulfate, and the cells were sedimented by centrifugation (3000 x g, 10 min) and washed three times with ice-cold distilled water (autoclaved). The survival rate of the EMS-treated cells was ~40% as determined by plating on LB agar at 30°C. The mutagenized cells were grown in 5 ml of LB media by shaking at 30°C for 16 h (A600 1.2), shifted to 37°C for 30 min, and sedimented and resuspended in 0.3 ml LB media containing 0.5 mCi of [2-3H]mannose. In control experiments, nonradioactive mannose was used. After 1 h at 37°C, the cells were sedimented by centrifugation (3000 x g, 10 min), washed three times with sterile ice-cold distilled water, and resuspended in 2.0 ml of 25% glycerol and 50% LB. Aliquots of the suspensions were stored at –80°C.

Growth of the radiation-exposed mutants was monitored by periodically thawing an aliquot and scoring for growth on LB agar at 30°C. After 35 days of storage at –80°C, cell viability was reduced to less than 1% of the unexposed cell population. The surviving cells were subjected to a second round of [3H]mannose-suicide selection and were stored at –80°C. After 35 days, the cells were serially diluted, plated on LB agar (150–200 colonies/plate) and incubated at 30°C for 30 h. This master plate was replica-plated onto LB agar and incubated at either 30°C (permissive) or 37°C (nonpermissive). After 30 h, the growth of the colonies on each plate was compared with the master plate. Micrococcal strains that were able to grow at 30°C, but not at 37°C were isolated and analyzed further for ts defects in mannolipid and LM biosynthesis.

Extraction and purification of DAG and Man1–2DAG
Cells were grown exponentially at 30°C, shifted to 37°C for 6 h, and sedimented by centrifugation (3000 x g, 10 min). Crude lipids (0.5 g) were extracted from membranes with 20 vols of CHCl3/CH3OH (2:1). The lipid extracts were then washed sequentially with 1/5 vol of 0.9% NaCl and CHCl3/CH3OH/H2O (3:48:47). The washed lower (organic) phases were concentrated by rotary evaporation under reduced pressure at 30°C (Buchi Rotavapor). The lipid extracts were redissolved in CHCl3 (0.3 ml) and applied to a silicic acid column (2.5 x 15 cm) preequilibrated with CHCl3. The column was sequentially eluted with 5 column vols of CHCl3 to recover free fatty acids and DAG, 20 column vols of acetone to elute neutral mannolipids (Man1–2-DAG), and 5 column vols of CHCl3/CH3OH (1:1) to elute phospholipids. The partially purified fractions were concentrated to dryness by rotary evaporation and resuspended in 100–150 µl of CHCl3/CH3OH (2:1). Mannolipids were analyzed by thin-layer chromatography on silica gel plates by developing with CHCl3/CH3OH/H2O (65:25:4) and detected by spraying with an orcinol/sulfuric acid solution (Churms and Zweig, 1982Go). Phospholipids are detected by spraying with a phosphate-specific reagent (Dittmer and Lester, 1964Go). For further purification, the lipid fractions were redissolved in CHCl3 (0.3 ml) and applied to a DEAE-cellulose column (2.5 x 10 cm) preequilibrated with CHCl3. Neutral lipids were eluted with 8 column vols of CHCl3, Man-DAG was eluted with 8 column vols of CHCl3/CH3OH (98:2), and Man2-DAG was eluted with 10 column vols of CHCl3/CH3OH (90:10) (Kates, 1972Go). Highly purified samples were obtained by preparative thin-layer chromatography on silica plates developed with petroleum ether/ethyl ether/acetic acid (80:20:1) for DAGs or CHCl3/CH3OH/H2O (65:25:4) for Man1–2-DAG.

Radiolabeled Man1–2-DAG were synthesized by metabolically labeling wild-type cells grown to A600 1.2 on 50 ml LB media, and sedimented by centrifugation (3000 x g, 10 min). The cells were resuspended in fresh LB media (1 ml) containing 50–100 µCi of [2-3H]mannose and incubated for 3 h at 22°C. The metabolically labeled [3H]Man1–2-DAGs were extracted and purified by chromatography on DEAE-cellulose and silica plates as described. Man-P-Und was enzymatically synthesized and purified from partially purified preparations of Man-P-Und synthase (Rush et al., 1993Go).

In vitro assay for mannosyltransferase activities
The micrococcal mannosyltransferases were assayed in vitro by procedures described previously (Pakkiri et al., 2004Go). Typical reaction mixtures contained cell homogenates (1.9 mg protein), 50–80 mM Tris–HCl (pH 7.6–8.0),20 mM MgCl2, and 15 µM GDP-[3H]Man (150 cpm/pmol) in a total volume of 0.15 ml. After incubation at 37°C, the enzymatic reactions were terminated by the addition of 20 vols of CHCl3/CH3OH (2:1). The lipid extracts containing the [3H]mannolipids were saved, and the delipidated membrane residues were extracted twice with 1 ml CHCl3/CH3OH (2:1). The lipid extracts (5 ml) were pooled and washed with 1/5 volume of 0.9% NaCl to remove unreacted GDP-[3H]Man. The organic (lower) phase was washed twice with CHCl3/CH3OH/H2O (3:48:47), and the organic solvent was evaporated to dryness under N2. An aliquot was taken to determine the total amount of [3H]mannose incorporated into the [3H]mannolipids, and another aliquot was used to analyze the reaction products by chromatography on thin-layer silica gel plates developed with CHCl3/CH3OH/H2O (65:25:4). The plates were dried at room temperature, and the radiolabeled products were located with an AR-2000 Imaging Scanner (BioScan, Washington, DC) to determine the distribution of [3H]Man-DAG, [3H]Man2-DAG, and [3H]Man-P-Und. The rate of synthesis of each mannolipid was determined by multiplying the percentage of each radiolabeled mannolipid by the total amount of [3H]mannolipid formed. The incorporation of [3H]mannose into the membrane-associated LM was determined by washing the delipidated membrane residue with 3 ml CH3OH-0.9% NaCl (1:1) and then twice with 3 ml CH3OH/H2O (1:1) to remove residual unreacted GDP-[3H]Man. The delipidated membrane residue was solubilized in 0.5 ml of 1% Sodium dodecyl sulfate (100°C, 5 min) and mixed with 4 ml scintillation cocktail. The amount of [3H]LM formed was determined by liquid scintillation spectrometry.

Deacylation of [3H]LM from wild-type and mms1 cells by mild alkali treatment
[3H]LM from wild-type and mms1 cells was synthesized in vitro as described earlier by incubating membranes (1 mg protein) with 74 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 1 mM GDP-Man, and purified exogenous [3H]Man2-DAG (50,000 cpm) dispersed in 0.5% sodium taurocholate (final conc. 0.05%, w/v) by ultrasonication, in a total volume of 0.15 ml for 1 h at 30°C. The enzymatically radiolabeled preparations were deacylated by mild alkali treatment (0.2 ml 0.1 N KOH in CH3OH/toluene, 3:1 for 1 h at 4°C) (Waechter and Lester, 1973Go). The reactions were neutralized by adding 0.2 ml 0.1 N acetic acid followed by 20 volumes of CHCl3/CH3OH (2:1) and 3 vols of 0.9% NaCl. The binary mixtures were mixed vigorously and placed on ice for 5 min before centrifugation (2000 x g, 1 min). The deacylated [3H]LM from wild-type and mms1 membranes were recovered from the upper (aqueous) phase and carefully separated from the lower (organic) phase. The molecular size of the deacylated LM was estimated by gel filtration chromatography on a Bio-Gel P-10 column (1.8 x 20 cm), equilibrated, and eluted with 0.1 N acetic acid. Fractions (1.4 ml) were collected using a Gilson (Middleton, WI) FC-80K microfractionator, and the amount of radioactivity in each fraction was determined by liquid scintillation spectrometry.

{alpha}-Mannosidase treatment of deacylated [3H]LM from wild-type and mms1 cells
[3H]LM from wild-type and mms1 cells was deacylated as described and treated with jack bean {alpha}-mannosidase (5 U) in 0.1 M sodium acetate (pH 6.0) in a total volume of 0.2 ml for 4 h at 30°C. The release of free [3H]mannose was assessed by gel filtration chromatography on Bio-Gel P-10.

Quantitative compositional analysis of phospholipids, mannolipids, and LM
Lipids extracted from membranes of wild-type, mms1, and mms2 cells grown exponentially at 30°C and shifted to 37°C for 6 h were sequentially purified by chromatography on silicic acid and DEAE-cellulose as described earlier. The phosphorus content in phospholipids (CL, PG, and PI) was determined by the method of Bartlett (1959)Go and the mannose content in Man1–2DAG and deacylated LM was determined by phenol-sulfuric method (Dubois et al., 1956Go).

General analytical methods
Protein concentrations were estimated with the Pierce (Rockford, IL) BCA Protein Assay Reagent using BSA as standard. Und-P and Man-P-Und were detected by staining the thin-layer chromatography plates with phospholipid reagent (Dittmer and Lester, 1964Go), and Man1–2-DAG were detected by spraying with water, Rhodamine 6G (Kates, 1972Go) or orcinol-sulfuric acid (Churms and Zweig, 1982Go). The amount of radioactivity in the various samples was determined by scintillation counting in a Packard Tri-Card 2100TR liquid scintillation spectrometer (Packard Instrument, Meriden, CT) after the addition of Econosafe Liquid Scintillation Counting Cocktail.


    Acknowledgements
 
We thank Drs. Jeffrey S. Rush and Preetha Shridas for carefully editing the manuscript and Susan Wang for her technical assistance. The authors also thank Wally Whiteheart with the fluorescence microscopy studies. This research was supported by NIH grant GM36065 to C.J.W.


    Abbreviations
 
BSA, bovine serum albumin; CL,; Con A, concanavalin A; EMS, ethyl methanesulfonate; FITC, fluorescein isothiocyanate; LB, Luria Bertani; LM, lipomannan; NAO, 10-N-nonyl-acridine orange


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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