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J Bacteriol. Jul 2012; 194(13): 3299–3306.
PMCID: PMC3434734

Environment-Mediated Accumulation of Diacyl Lipoproteins over Their Triacyl Counterparts in Staphylococcus aureus

Abstract

Bacterial lipoproteins are believed to exist in only one specific lipid-modified structure, such as the diacyl form or the triacyl form, in each bacterium. In the case of Staphylococcus aureus, recent extensive matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry analysis revealed that S. aureus lipoproteins exist in the α-aminoacylated triacyl form. Here, we discovered conditions that induce the accumulation of diacyl lipoproteins that lack α-aminoacylation in S. aureus. The accumulation of diacyl lipoproteins required a combination of conditions, including acidic pH and a post-logarithmic-growth phase. High temperatures and high salt concentrations additively accelerated the accumulation of the diacyl lipoprotein form. Following a post-logarithmic-growth phase where S. aureus MW2 cells were grown at pH 6, SitC lipoprotein was found almost exclusively in its diacyl structure rather than in its triacyl structure. This is the first report showing that the environment mediates lipid-modified structural alterations of bacterial lipoproteins.

INTRODUCTION

Bacterial lipoproteins are a class of membrane proteins that are encoded by 1% to 3% of genes within bacterial genomes (4). These lipoproteins are covalently modified with lipids at the conserved N-terminal cysteine residue and are located on the extracellular surface of the cell membrane or on the periplasmic surface of both the inner and outer cell membranes in monoderm and diderm bacteria, respectively. Bacterial lipoproteins are involved in a broad range of functions, including nutrient uptake, peptidoglycan synthesis and maintenance, outer membrane biogenesis, antibiotic resistance, and virulence of pathogenic bacteria (12, 15, 27, 30, 32, 33, 39). In addition, lipoproteins are among the proteins presenting pathogen-associated molecular patterns (PAMPs) that are recognized by host pattern recognition receptors (PRRs), such as Toll-like receptor-2 (TLR2) (2). Lipoproteins and lipopeptides are known to be recognized by mammalian TLR2 that is heterodimerized with TLR1 or TLR6, subsequently leading to inflammatory cytokine release and the establishment of adaptive immunity (5, 13, 34).

Thus far, two conventional lipid-modified structures, the diacyl form and the triacyl form, have been well recognized as bacterial lipoprotein structures, and three conserved enzymes are involved in the biosynthesis of the triacyl form (12, 29, 37). Specifically, after secretion of lipoprotein precursors (prepro-lipoproteins) across the cytoplasmic membrane, prepro-lipoprotein diacylglycerol transferase (Lgt) adds a phospholipid diacylglycerol moiety to the thiol group of the conserved cysteine residue within the lipobox domain, which is a characteristic domain inside the signal peptide of the prepro-lipoproteins, resulting in pro-lipoproteins (diacylated with signal sequence) (25). Next, pro-lipoprotein signal peptidase (Lsp) cleaves the signal peptide on the N-terminal side of the lipid-modified cysteine, resulting in the formation of diacyl lipoproteins (apolipoproteins). Diacyl lipoproteins are found in Listeria monocytogenes and some mycoplasmas, including Mycoplasma fermentans (18, 21, 22). Apolipoprotein N-acyltransferase (Lnt) further acylates the diacyl lipoproteins on their cysteine α-amino groups, resulting in the triacyl lipoprotein form (holo-lipoproteins) (6). The N-acylation step in Escherichia coli is required for sorting lipoproteins from the inner membrane to the outer membrane by the localization of lipoprotein (Lol) system (9, 28, 37, 41). The E. coli-type Lnt is conserved in most Gram-negative bacteria and in high-GC-content, Gram-positive bacteria, such as Mycobacterium tuberculosis and Streptomyces coelicolor, which were shown to contain triacyl lipoproteins (36, 38). However, in the absence of the E. coli-type Lnt homolog, some low-GC-content, Gram-positive bacteria and mycoplasmas, such as Staphylococcus aureus, Mycoplasma pneumoniae, and Acholeplasma laidlawii, contain the N-acylated triacyl lipoprotein form, too (3, 18, 31). Furthermore, we recently discovered new classes of N-acylated lipid-modified structures in low-GC-content Gram-positive bacteria, the lyso form and the N-acetyl form (18). The lyso form is named for the N-acyl-S-monoacyl-glyceryl cysteine-containing bacterial lipoprotein structure, where one of two acyl chains in the S-diacyl-glyceryl group does not exist. The lyso forms were found in resident bacteria in oral cavities or in the intestinal tract. The N-acetyl lipoproteins contain the N-acetyl-S-diacyl-glyceryl cysteine, which was found in five Bacillus-related bacteria residing in environmental soil or deep sea (18). These new discoveries of structural variants of bacterial lipoproteins expand new research topics in bacterial lipoprotein studies investigating how and why they are produced.

Currently, our knowledge of lipid-modified lipoprotein structures is still not perfect, due in part to the difficulty of native lipoprotein purification and the subsequent structural analysis. Here, we report alterations of the N-acylation status of bacterial lipoproteins induced by changes in environmental and growth-phase conditions in S. aureus. The obtained results were unexpected, because, except for M. fermentans, only one lipid-modified structure was previously identified in each bacterium. The M. fermentans lipoproteins exist in one of two lipid-modified structures, the conventional diacyl form or the peptidyl form, the latter having two amino acids prior to the lipidated cysteine residue at the N terminus (18). Taking these data together, our report represents the first description of structural alterations of bacterial lipoproteins induced by environmental changes.

MATERIALS AND METHODS

Bacterial strains.

The methicillin-resistant S. aureus MW2 strain, methicillin-sensitive S. aureus MSSA476 strain, and S. aureus USA300 JE2 strain, which is a plasmid-cured derivative of the community-associated methicillin-resistant USA300 LAC strain (14), were obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (Chantilly, VA). The S. aureus RN4220 strain is a laboratory stock strain. Two-component-system gene mutants derived from the S. aureus MW2 strain were described previously (20). These S. aureus strains were grown in Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) at 37°C unless otherwise indicated. Ampicillin (100 μg · ml−1), chloramphenicol (12.5 μg · ml−1), or tetracycline (3 μg · ml−1) was added when necessary.

Lipoprotein preparation by Triton X-114 phase separation.

Lipoproteins were isolated from S. aureus cells by Triton X-114 (TX-114) phase separation as described previously (3) with some modifications. Briefly, bacterial cultures at an optical density at 600 nm (OD600) of 0.05 were diluted into 125 ml of medium in 500-ml flasks, grown with vigorous shaking, and harvested at an OD600 of 0.6 to 0.7 (logarithmic-growth phase) or after an 18-h incubation period (post-logarithmic-growth phase). Harvested cells were disrupted with 12 g of glass beads at 2,000 oscillations per min (MSK cell homogenizer; B. Braun Biotech International) for a total of 7 min in 20 ml of 1 M NaCl. The disrupted cells were centrifuged at 2,000 × g for 10 min to remove the glass beads and unbroken cells, and the supernatant was centrifuged at 13,000 × g for 10 min. The resulting supernatant was collected as the cell lysate and supplemented with TX-114 solution to reach a final concentration of 2% and then incubated at 4°C for 1 h. Next, the mixture was incubated at 37°C for 10 min for phase separation. After centrifugation at 10,000 × g for 10 min at 25°C, the upper phase was removed and replaced with the same volume of TBSE solution (20 mM Tris-HCl [pH 8], 130 mM NaCl, 5 mM EDTA). This procedure was repeated twice. Components of the final TX-114-phase solution were precipitated with 2.5 volumes of ethanol and loaded onto a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins separated by SDS-PAGE were subjected to in-gel digestion with trypsin, and the resulting digests (10 to 20 μl) were acidified with 1 μl of 70% formic acid and then mixed with the same volume of a chloroform/methanol mixture (2/1 [vol/vol]) to extract lipid-modified peptides. Finally, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) was performed on the lipopeptides in the lower organic phase as previously described (3).

MALDI-TOF MS and MS/MS.

MALDI-TOF MS was conducted using an Ultraflex mass spectrometer (Bruker Daltonics) in positive reflectron mode as previously described (3). The saturated α-cyano-4-hydroxycinnamic acid (CHCA) solution in a chloroform/methanol (2/1 [vol/vol]) solvent was used as a matrix. A thin layer of CHCA matrix was prepared, and the samples were deposited on the matrix. Tandem MS (MS/MS) spectra were acquired using a MALDI-TOF/TOF instrument (Ultraflex; Bruker Daltonics) and CHCA as the matrix. Fragments of tryptic lipopeptides observed in MALDI MS/MS spectra are mainly C-terminal-containing sequence ions (y-type), because the positive charge tends to localize at C-terminal lysine or arginine residues of the peptides. We confirmed that both diacyl and triacyl synthetic peptides [Pam2 (dipalmitoyl)-CSK4 and Pam3-CSK4] could be analyzed with respect to organic solvent extraction and ionization (3). The efficiencies of ionization of the synthetic diacyl and triacyl synthetic peptides are slightly different: the triacyl peptide was slightly more easily ionized than its diacyl counterpart in our MALDI-TOF MS analysis. Generally, major compounds are more effectively ionized and detected than minor compounds in the analysis. Therefore, the ratio of the diacyl form to the total amount, which was calculated from the MS analysis in this study, might be not fully accurate but a rough indication.

Expression and detection of a truncated form of SitC lipoprotein.

A truncated form of the SitC protein was expressed under the control of the S. aureus HU promoter by the use of the pKE515pHU plasmid (17). The SitC N-terminal region containing a signal peptide sequence was amplified using the following primers: pSsitC-5PKpnI (5′-ACGAGGAAGGGTACCATGAAAAAATTA-3′) and pSsitCdelD-His-3PSacI (5′-GGCGAGCTCAAACCAACCGTTACCAGTCT-3′). The amplified regions were then digested with KpnI and SacI and cloned into the pET52b vector at the KpnI and SacI sites (Novagen). After confirming the inserted DNA sequence in the resulting plasmid, the SitCdelD-His region was amplified using the following primers: pSsitC-5PKpnI and T7terminator-SalI (5′-CCGTTTAGTCGACCCAAGGGGTTATGCTAGTTA-3′). The amplified region was cloned into the pKE515pHU vector at the KpnI and SalI sites. The resulting pSsitCdelD-His plasmid was introduced into S. aureus RN4220 and transferred to the S. aureus JE2 strain via phage transduction. A preculture of the resulting strain, JE2 harboring pSsitCdelD-His, was diluted to an OD600 of 0.02 and grown in LB medium buffered at pH 5.5, 6.5, or 7.5 until the post-logarithmic-growth phase was reached (24 h) at 37°C with vigorous shaking. Harvested cells were lysed with lysostaphin, and the lipoprotein fraction was prepared by TX-114 phase separation, separated by 16% SDS-PAGE (20- by 20-cm gel size glass plate), and visualized by Western blotting using an anti-His-horseradish peroxidase (anti-His-HRP)-conjugated antibody (Qiagen).

RESULTS

Diacyl lipoproteins accumulated in amounts exceeding those of their triacyl counterparts under the combined conditions of acidic pH values and a post-logarithmic-growth phase in S. aureus.

Previously, we reported that lipoproteins of exponentially growing S. aureus cells in LB or brain heart infusion (BHI) medium were found only in the triacyl form (3). In contrast, for the first time, we describe the environmental conditions that cause the accumulation of the diacyl form of S. aureus lipoproteins. To determine the N-terminal structure of lipoproteins, lipoprotein-enriched TX-114 fractions were separated by SDS-PAGE, and the SitC lipoprotein band was subjected to in-gel digestion with trypsin and analyzed using MALDI-TOF MS. SitC is the one of the predominant lipoproteins in S. aureus (3, 23). Consistent with our previous results, N-terminal SitC lipopeptides obtained from S. aureus RN4220 cells grown in LB medium showed a series of 14-Da-interval ions between m/z 1,324.9 and 1,381.0 (Fig. 1A). The presence of these ions is explained by an increasing number of methylene (CH2) groups in the lipopeptide fatty acids, and they correspond to triacyl N-terminal SitC lipopeptides harboring 49:0 (49 and 0 refer to the numbers of total carbons and double bonds in acyl chains, respectively) to 53:0 fatty acids in total. In contrast, when 0.2% glucose was added to the LB medium, a series of 14-Da-interval ions between m/z 1,072.7 and 1,114.8 were observed. These interval ions correspond to the diacyl form of SitC (32:0 to 35:0 in total), which was found to be more prevalent than the triacyl form (Fig. 1B). To prove the absence of α-aminoacylation in the diacyl structure, the N-terminal sequence of the diacyl form was confirmed by Edman degradation, enabling us to obtain the correct amino acid sequence following the lipid-modified cysteine (data not shown). In addition, MS/MS analysis of diacyl ions was performed as described below. Because glucose decreases the medium pH due to an increase in bacterial production of carboxylic acids (Fig. 1C, squares), we attempted to prevent the pH drop by buffering the medium with 100 mM HEPES at pH 7.5. This buffering prevented the glucose-mediated pH drop (Fig. 1C, triangles) and allowed the cells to continue synthesizing triacyl SitC (Fig. 1D, triangles), suggesting that the apparent effect of the presence of glucose on the accumulation of the diacyl form was due to the pH decrease. Taken together, these results suggest an acidic pH-mediated accumulation of diacyl SitC lipoprotein in S. aureus.

Fig 1
Accumulation of the diacyl form of S. aureus SitC lipoprotein under acidic conditions. (A and B) MALDI-TOF MS spectrum of a fraction containing in-gel-digested SitC lipoprotein of S. aureus RN4220 cells grown at 37°C for 12 h in LB medium (A) ...

Next, we examined whether acidic conditions accumulate diacyl lipoproteins by the use of LB medium buffered at various pH values (6.0, 6.5, and 7.5) with 100 mM MES (morpholineethanesulfonic acid), 100 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)], and 100 mM HEPES (Fig. 2). At an acidic pH of 6.0 or 6.5, S. aureus MW2 cells produced diacyl SitC in the post-logarithmic-growth phase. The percentage of diacyl SitC at pH 6.0 was higher than the percentage at pH 6.5. In contrast, LB medium buffered at pH 7.5 caused S. aureus MW2 cells to continue to synthesize triacyl SitC in the post-logarithmic-growth phase. Interestingly, diacyl SitC did not accumulate in cells during the logarithmic-growth phase, even at pH 6.0. Similar to the MW2 strain, the S. aureus MSSA476 strain exhibited an accumulation of diacyl SitC in the post-logarithmic-growth phase and under acidic conditions (pH 6.0 or pH 6.5); however, in the logarithmic-growth phase or under neutral conditions (pH 7.5), differences in diacyl lipoprotein percentages among S. aureus strains were observed (data not shown). These results provide evidence for the acidic pH- and growth-phase-dependent accumulation of diacyl SitC in S. aureus cells.

Fig 2
Accumulation of the diacyl lipoprotein in amounts exceeding those of their triacyl counterparts under combined conditions of acidic pH and a post-logarithmic-growth phase. S. aureus MW2 cells were cultured at 37°C until an OD600 of 0.5 was reached ...

Next, we monitored the structural differences between the diacyl and triacyl forms by the use of SDS-PAGE, under conditions in which an N-acylation-induced increase in lipoprotein molecular size has been previously detected (9). Because the molecular mass of native SitC is approximately 33 kDa, which is too large to allow us to distinguish between the two forms with respect to size, a plasmid expressing an N-terminal SitC fragment harboring a His tag at the C terminus, named SitCdelD-His, was constructed. S. aureus USA300 JE2 cells harboring the plasmid expressing SitCdelD-His under the control of the histone-like protein HU promoter were grown to the post-logarithmic-growth phase in LB medium buffered at pH 5.5, 6.5, or 7.5, and lipoprotein-enriched TX-114 fractions were separated by SDS-PAGE and visualized by Western blotting using an anti-His tag antibody. The SitCdelD-His produced in cells in the post-logarithmic-growth phase under acidic conditions (pH 5.5) was smaller than the SitC fragment produced under neutral conditions (pH 7.5) (Fig. 3). Taken together, these results suggest that the diacyl form of SitC accumulates under the combined conditions of acidic pH values and post-logarithmic-growth phase in S. aureus.

Fig 3
The mobility differences between the diacyl and triacyl forms of lipoproteins, as shown by SDS-PAGE. S. aureus USA300 JE2 cells harboring a plasmid expressing SitCdelD-His, the N-terminal SitC fragment containing a His tag at the C terminus, were grown ...

MS/MS analysis revealed α-aminoacylation-free structure of diacyl SitC lipoprotein.

To obtain clear evidence of the α-aminoacylation-free structure of the diacyl lipoprotein, we performed MALDI-TOF MS/MS analysis using the diacylated N-terminal SitC lipopeptide ions. The SitC lipopeptides were prepared from S. aureus MW2 cells grown for 18 h in medium at pH 6.0. Ions with 14 mass differences between m/z 1,072.8 and 1,114.9 that corresponded to diacyl SitC lipopeptides (between 32:0 and 35:0 in total) were more prevalent than the triacyl lipopeptides between m/z 1,311.0 and 1,395.1 (between 48:0 and 54:0 in total) (Fig. 4A), as shown in Fig. 2. MS/MS analysis was performed for each major diacyl ion. Each MS/MS spectrum represented y-series ions that confirmed the GTGGK peptide sequence of N-terminal SitC lipoprotein. Importantly, they represented the characteristic fragment ion at m/z 488.2 that corresponded to the α-amino group-free dehydroalanyl GTGGK peptide generated by the neutral loss of diacylthioglycerol (Fig. 4B to toE).E). These results suggest that S. aureus MW2 cells accumulate the α-aminoacylation-free, conventional diacyl form of SitC lipoproteins. Interestingly, as shown in Fig. 4, each MS/MS spectrum represented a product ion that had lost a 15:0 fatty acid at m/z 830.5 (panel B), 844.5 (C), 858.5 (D), or 872.5 (E) from each precursor ion and ions that had lost two fatty acids at m/z 560.2 (panels B to E). In general, modification of the sn-1 position of the phospholipid glyceryl group is more stable than the sn-2 modification (24). Because our results showed that only the 17:0- to 20:0-containing ions, and not the 15:0-containing ion, were represented in the MS/MS spectrum (Fig. 4B to toE),E), the sn-1 and sn-2 positions of the S-glyceryl group of S. aureus SitC lipoprotein could be modified by the 17:0-to-20:0 fatty acids and 15:0 fatty acid, respectively (Fig. 4F). The fatty acid content of the diacylglyceryl group of SitC is consistent with that of membrane phospholipids of S. aureus, where approximately 40% of fatty acids are 15:0 fatty acids (8, 40). It is unclear how the composition of phospholipid fatty acids changes upon pH shock; we speculate that acidic pH may not significantly change the composition of phospholipid fatty acids, because triacylated SitC lipopeptide ions obtained from log-phase cells grown at pH 6.0, pH 6.5, and pH 7.5 (Fig. 2) showed similar distribution patterns on MALDI-TOF MS spectrums with the total fatty acid length of 48:0 to 54:0. Figure 4 represents updated data on the accurate fatty acid-modified structure of an S. aureus lipoprotein.

Fig 4
MS/MS analysis reveals α-aminoacylation-free diacyl SitC lipoprotein in post-exponential-growth-phase S. aureus cells under acidic conditions. (A) A MALDI-TOF MS spectrum of an organic phase fraction containing in-gel-digested SitC lipoprotein ...

Other lipoproteins also accumulated in the diacyl form under acidic conditions.

To obtain evidence that not only SitC but also other lipoproteins exist in their diacyl forms under acidic conditions, we performed MALDI-TOF MS analysis on a fraction containing in-gel-digested SA2079 (ferrichrome ABC transporter substrate-binding protein homolog) and SA1659 (peptidyl-prolyl cis/trans isomerase homolog) lipoproteins obtained from MW2 cells grown to the post-exponential-growth phase at pH 5.5. Four different series of 14-Da-interval peaks were detected (Fig. 5A). The first series of ions at m/z 1,285.9, 1,299.9, 1,313.9, and 1,327.9 (highlighted by green letters) corresponded to diacyl ions of the N-terminal SA2079 lipopeptide. The second series of ions at m/z 1,375.9, 1,389.9, 1,403.9, 1,417.9, and 1,432.0 (highlighted by orange letters) corresponded to diacyl ions of the N-terminal SA1659 lipopeptide. The third series (green) and fourth series (orange) of ions corresponded to triacyl ions of the N-terminal SA2079 and SA1659 lipopeptides, respectively. As expected, the first and second series of peaks were not detectable in similar samples obtained from cells grown at pH 7.5. To obtain evidence of the α-aminoacylation-free structure of the diacyl SA1659 lipopeptide, we performed MS/MS analysis on the ion at m/z 1,403.9 that had a 33:0 modification in total. The C-terminal-containing y-series ions confirmed the GASATDSK peptide sequence of the N-terminal SA1659 lipopeptide (Fig. 5B). Importantly, a characteristic fragment ion corresponding to the α-amino group-free dehydroalanyl peptide generated by the neutral loss of diacylthioglycerol was observed at m/z 805.2 (Fig. 5B). In addition, a product ion that had lost a 15:0 fatty acid was observed at m/z 1,161.6, suggesting that the sn-1 and sn-2 positions of the S-glyceryl group of the 33:0-modified SA1659 lipopeptide are occupied by the 18:0 and 15:0 fatty acids, respectively (Fig. 5C). These results demonstrate that other lipoproteins in addition to SitC, such as SA1659 and SA2079, accumulated as α-aminoacylation-free, conventional diacyl structures in post-exponential-growth-phase cells under acidic conditions.

Fig 5
Determination of α-aminoacylation-free diacyl SA1659 lipoprotein in S. aureus cells grown under acidic conditions. (A) MALDI-TOF MS spectrum of an organic-phase fraction containing in-gel-digested SA1659 and SA2079 lipoproteins derived from post-exponential-growth-phase ...

Control of gene expression may determine the N-acylation states of lipoproteins.

To examine how α-aminoacylation of lipoproteins is controlled, we performed a pH shift assay in the presence or absence of the protein synthesis inhibitor chloramphenicol (Fig. 6). When MW2 cells grown to the post-logarithmic-growth phase at pH 7.5 (Fig. 6A, column 1) were diluted 10-fold with pH 6.0 medium and cultured for 6 h in the absence of chloramphenicol, we observed an increase in the amount of the diacyl form (Fig. 6A, column 2), thereby indicating a decrease in the amount of the triacyl form. Conversely, in the presence of chloramphenicol, a similar pH downshift did not produce the diacyl form and kept the amount of the triacyl form constant (Fig. 6A, column 3). Therefore, a pH downshift is not sufficient for accumulation of the diacyl form, and new protein synthesis is required for the accumulation. These results also indicate that the diacyl form was not produced from the triacyl form via hydrolysis of α-aminoacyl group of the triacyl lipoproteins, at least in the presence of the protein synthesis inhibitor. The interpretation that acidic pH is not the sole factor required for the diacyl form accumulation is consistent with the facts that logarithmic-growth-phase cells at pH 6.0 continued to synthesize the triacyl form (Fig. 2) and that the combined conditions of acidic pH and a post-logarithmic-growth phase were required for diacyl form accumulation (Fig. 2). Next, we performed a pH upshift experiment in which we shifted post-logarithmic-growth-phase MW2 cells from pH 6.0 to pH 7.5 by adding a 10-fold volume of pH 7.5 medium. This pH upshift recovered N-acylation in the absence of chloramphenicol (Fig. 6B, column 2) but not in the presence of chloramphenicol (Fig. 6B, column 3), suggesting that new protein synthesis is required for the N-acylation of diacyl lipoproteins during the pH upshift. Taken together, these results suggest that α-aminoacylation of lipoproteins might be controlled by gene expression levels; however, a pH-mediated enzyme activity change corresponding to an unidentified Lnt cannot be ruled out.

Fig 6
Requirement of new protein synthesis for pH shift-mediated changes in N-acyl states of S. aureus SitC lipoprotein. (A) MW2 cells grown for 18 h in LB medium buffered with 100 mM MES at pH 7.5 (column 1 [Pre]) were diluted 10-fold with LB medium buffered ...

High-temperature and high-salt conditions accelerate the accumulation of the diacyl lipoprotein form in S. aureus.

In addition to pH and growth phase, we assumed that other environmental conditions might also enhance diacyl form accumulation in S. aureus. After our continued search, we found that high temperature also accelerated diacyl lipoprotein accumulation. As shown in Fig. 7A, when S. aureus MW2 cells were grown in LB medium at 37°C, 41°C, or 42°C, the percentage of diacyl SitC increased at higher temperatures in both logarithmic- and post-logarithmic-growth-phase cells. The absence of α-aminoacylation in the diacyl SitC under these conditions was confirmed by MS/MS analysis (data not shown).

Fig 7
High-temperature and high-salt conditions accelerate the accumulation of the diacyl lipoprotein in S. aureus. (A) The percentages of the diacyl form of SitC lipoprotein were determined from S. aureus MW2 cells grown in LB medium until the logarithmic ...

In addition to high temperature, a high NaCl concentration also enhanced accumulation of the diacyl form. As shown in Fig. 7B, the percentage of diacyl SitC lipoprotein in LB medium containing 7.5% NaCl was higher than that in LB medium containing 1% NaCl in S. aureus MW2 cells grown at 41°C. This NaCl effect was more obvious at 41°C or 42°C than at 37°C (data not shown). The high-salt-mediated accumulation of the diacyl form was also observed under acidic conditions (e.g., at pH 5.5 or pH 6.0) but not under neutral or alkaline conditions (e.g., at pH 7.5 or pH 8.5) in S. aureus RN4220 cells (Fig. 7C). Therefore, it seems that a high NaCl concentration is the stimulatory factor for high-temperature-mediated or acidic-pH-mediated accumulation of diacyl lipoproteins.

In bacteria, two-component systems are known to sense and respond to changes in environmental conditions (for a recent review, see reference 10). Specifically, among the two-component systems in S. aureus, the saeRS genes are reported to respond to acidic pH and high salt concentrations (1, 11, 19). Therefore, we examined the role of two-component systems in the regulation of N-acylation. All 15 viable two-component system mutants in S. aureus (20), including agr and saeRS mutants, were tested for their N-acylated states; however, all mutants accumulated the N-acylated triacyl form at pH 7.5 and accumulated the diacyl form in the post-exponential-growth phase at pH 6.0 (data not shown). Therefore, these two-component systems do not seem to be principal regulators of Lnt expression. Mutations in the alternative sigma factor sigB, which is involved in gene regulation under various conditions of pH, heat, NaCl, and growth-phase shifts (7, 16, 26), also had little effect on N-acyl states. Further studies are necessary to address the issues behind the molecular basis of N-acyl state regulation of S. aureus lipoproteins.

DISCUSSION

Until now, the structure of bacterial lipoproteins has been considered to be constant in each bacterium. For example, Gram-negative E. coli contains only the triacyl lipoprotein form. In addition, we recently demonstrated that lipoproteins in each Gram-positive bacterium exist in only one lipid-modified structure, such as the triacyl, diacyl, lyso, or N-acetyl form (18). However, here we show that each lipoprotein in S. aureus can exist in two lipidated forms, the diacyl form and the triacyl form, as determined by pH and the bacterial growth phase. In addition to the pH and growth phase, other environmental factors, such as salt concentration and temperature, were involved in regulating the N-acyl states of S. aureus lipoproteins. Because we also recently discovered two different N-terminal structures of lipoproteins in M. fermentans (18), the features distinguishing S. aureus and M. fermentans should be noted. In M. fermentans, we revealed that two lipoproteins exist in the conventional diacyl form, while two other lipoproteins exist in the peptidyl form, an unusual diacyl form containing two amino acids in front of the lipid-modified cysteine. In other words, lipoproteins in M. fermentans exist in only one of two structures, probably based on their lipobox sequence. In contrast, each lipoprotein in S. aureus can exist in two different structures, depending on both environmental conditions and growth phase. This finding supports the novel hypothesis that lipid-modified structures of bacterial lipoproteins are modulated by environmental conditions.

Regulation of the N-acyl state of S. aureus lipoproteins seems to be modulated by controlling Lnt expression. Requirements of new protein synthesis for the pH shift-mediated alteration of lipid-modified structures support this interpretation (Fig. 5). Therefore, although the lnt gene in S. aureus is currently unidentified, Lnt expression seems to be regulated by environmental conditions and the bacterial growth phase in S. aureus, while other explanations, such as Lnt activity regulation mediated by environmental conditions and conversion of triacyl forms to diacyl counterparts by an acid- and stationary-phase-inducible deacylase, should be considered.

We have reported here that the sn-1 and sn-2 positions of the S-glyceryl group of SitC lipoprotein from S. aureus MW2 strain can be modified by the 17:0-to-20:0 fatty acids and 15:0 fatty acid, respectively, which seems to be consistent with the fatty acid contents of S. aureus phospholipids (8, 40) corresponding to the substrate of Lgt for the S-glyceryl group modification. The asymmetric distribution of fatty acids at the sn-1 and sn-2 positions of SitC lipoprotein was also confirmed in other S. aureus strains, including strains RN4220 and SA113, and in other lipoproteins, including lipoproteins SA1659 and SA2202 (data not shown). In contrast to our results, Tawaratsumida et al. reported a single diacyl structure of an SA2202 lipoprotein modified with a 32:0 fatty acid, in which both the sn-1 and sn-2 positions are modified by the 16:0 fatty acid, from S. aureus SA113 cells grown in BHI medium for 6 h at 37°C (35). Apparently, their α-aminoacylation-free diacyl structure can be explained by their culturing conditions for the bacteria, such as the post-logarithmic-growth phase under acidic conditions as revealed in this study. However, there are marked discrepancies between their study and ours. In fact, they detected only the diacyl form but not the triacyl form (35). Also, they isolated only one S-dipalmitoylglyceryl form rather than a series of diacyl and triacyl lipopeptide forms with different length fatty acids in the sn-1 and a 15:0 fatty acid in the sn-2 positions. Moreover, we previously detected a series of unsaturated fatty acid-containing lipopeptides in considerable amounts in cultures of the S. aureus SA113 strain that was provided by Hashimoto's group (3) in both LB medium and BHI medium, while they detected only the single, saturated fatty acid-containing lipopeptide. As discussed before (3), lipoproteins were recovered by Hashimoto's group at levels that were several-hundred-fold lower than ours, suggesting that we might analyze unbiased structures and conversely that they might analyze minor components. It is possible that the S-dipalmitoylgylceryl form exists; if so, however, it must be not a major component but a very tiny fraction in S. aureus cells, because asymmetric distribution of fatty acids at the sn-1 and sn-2 positions is now evident. Despite this, it was not mentioned in their paper that there are any reasonable methods that allow us to purify specifically the S-dipalmitoylglyceryl lipopeptide from a series of diacyl and triacyl lipopeptides containing mainly the S-diacyl(sn-1:17:0–20:0, sn-2:15:0)glyceryl group (35). Except for analyses performed using synthetic lipopeptide, we assume that it would be almost impossible to obtain MS and MS/MS spectra of a single S-dipalmitoylgylceryl form of native S. aureus lipoproteins.

In S. aureus, N-acylation might play different roles, in addition to simply anchoring lipoproteins on the plasma membrane; this is because two acyl chains of the S-glyceryl group can achieve lipoprotein anchoring. While N-acylation in E. coli is required for sorting lipoproteins from the inner membrane to the outer membrane by the Lol system (9, 28), S. aureus bacteria do not have an outer membrane. One of the possible roles of N-acylation may be comparable to that of N-myristoylation in eukaryotic proteins, functioning in protein localization and protein-protein interactions (10). Because N-acylation of lipoproteins in S. aureus occurred in exponentially growing cells at all tested pH values and in post-exponential-growth-phase cells at neutral and alkaline pH values (Fig. 2), it is possible that N-acylation plays roles specifically under these conditions. Conversely, N-acylation might act as an obstacle under acidic conditions in post-exponential-growth phases. The alteration of lipid-modified structures may work to maintain homeostasis of bacterial cell membrane integrity in response to different environmental conditions. The discoveries of environmental factors mediating structural alterations of bacterial lipoproteins in S. aureus provide a new field of study on bacterial lipoproteins and accelerate further research to identify Lnt in Gram-positive bacteria.

ACKNOWLEDGMENTS

We are grateful to M. Sugai for S. aureus two-component system mutants. We also thank C. Wolz and R. P. Novick for S. aureus sae mutants.

This work was supported by grants from a Bio-Program (2008-2004086) and the BK21 program of the National Research Foundation of Korea to B.L.L.

Footnotes

Published ahead of print 30 March 2012

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