Laboratory of Developmental Biochemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 3-1 7-Chome, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Correspondence
Kenji Kurokawa
kurokawa{at}mol.f.u-tokyo.ac.jp
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ABSTRACT |
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INTRODUCTION |
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LPG is involved in the regulatory mechanisms of the initiation of DNA replication in S. aureus (Ichihashi et al., 2003). The initiation activity of DnaA protein, the initiator of bacterial DNA replication, is regulated by its adenine-nucleotide binding (Sekimizu et al., 1987
; Katayama et al., 1998
; Kurokawa et al., 1999
; Nishida et al., 2002
). The affinity of DnaA protein for adenine nucleotides is affected by acidic phospholipids (Sekimizu & Kornberg, 1988
; Castuma et al., 1993
; Xia & Dowhan, 1995
; Mizushima et al., 1996
; Kitchen et al., 1999
). LPG inhibits the interaction between DnaA protein and acidic phospholipids, PG and CL, and the S. aureus mprF mutant exhibits an increased amount of replication origins (Ichihashi et al., 2003
). These findings suggest that LPG is also involved in cell cycle regulation.
LPG synthetase activity in S. aureus exists in the cell membrane fraction and catalyses the transfer of L-lysine from lysyl-tRNA to PG (Lennarz et al., 1967; Nesbitt & Lennarz, 1968
), but the gene encoding the enzyme has not yet been identified. Because the mprF deletion mutant did not synthesize LPG, the mprF gene was suggested to encode the LPG synthetase (Peschel et al., 2001
). In the present study, we trans-expressed the S. aureus MprF protein in Escherichia coli cells and obtained biochemical evidence that the mprF gene product encodes LPG synthetase.
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METHODS |
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Bacterial culture conditions.
S. aureus and E. coli cells were grown at 37 °C in LuriaBertani (LB) medium [1 % tryptone (Difco), 0·5 % yeast extract (Difco) and 1 % NaCl] containing, where appropriate, 50 µg thymine ml-1, 50 µg ampicillin ml-1, 5 µg tetracycline ml-1 or 12·5 µg chloramphenicol ml-1. When bacterial cells were metabolically labelled with L-[14C]lysine (300 mCi mmol-1, Amersham Pharmacia Biotech) or [32P]orthophosphate (10 mCi ml-1, Amersham Pharmacia Biotech), LB medium containing 0·5 µCi (18·5 kBq) [14C]lysine ml-1 or 10 µCi (0·37 MBq) [32P]orthophosphate ml-1 was used. For preparation of the membrane fraction from S. aureus RN4220 or CK1001 cells, cells were grown at 37 °C in 5 l LB medium to an OD600 value of 0·9 and then harvested. For preparation of the membrane fraction from E. coli JM109 cells harbouring either pBADmprF or pBAD24, cells were grown at 37 °C in 1 l LB medium to an OD600 value of 0·5, then up to 1 % L-(+)arabinose (Tokyo Kasei) was added; the cells were incubated further for 2 h, and then harvested.
Analysis of lipids of bacteria.
For analysis of the E. coli membrane lipid composition, total lipid was extracted from 1 ml of each bacterial cell culture using a modified BlighDyer method as described previously (Sekimizu & Kornberg, 1988). Extracted lipids were separated by TLC developed with chloroform/methanol/acetic acid [65 : 25 : 10 (by vol.)] using a silica gel plate (Silicagel 60, type PK6F, Whatman). To detect lipids, TLC plates were sprayed with 100 mg CuSO4 ml-1 containing 8 % phosphoric acid and heated. To detect [14C]lysine- or [32P]orthophosphate-labelled lipids, TLC plates were fluorographed using an image analyser (Fuji Film Bas2000).
Preparation of membrane fraction.
Membrane fractions from S. aureus or E. coli were prepared as described previously (Lennarz et al., 1966) with slight modifications. S. aureus cells (wet weight 11·2 g), harvested as described above, were suspended in 2·24 ml 20 mM Tris/HCl (pH 6·8), and incubated with 200 µg lysostaphin (Wako) ml-1 at 4 °C for 30 min. The resulting lysate was sonicated for 30 s at output 2·0 using a Branson Sonifier 450. Sonicated lysate was centrifuged at 15 000 g for 15 min at 4 °C, followed by centrifugation at 100 000 g for 60 min at 4 °C. The resultant pellet was suspended in 250 µl buffer containing 20 mM Tris/HCl and 1 mM 2-mercaptoethanol. The suspension was recentrifuged at 100 000 g for 60 min at 4 °C; the pellet was suspended in 250 µl buffer containing 20 mM Tris/HCl (pH 6·8) and 1 mM 2-mercaptoethanol, and used as the membrane fraction. To prepare E. coli cell membrane fractions, harvested cells (wet weight 2·8 g) were suspended in 560 µl 20 mM Tris/HCl (pH 6·8), and lysed with 200 µg lysozyme ml-1 at 4 °C for 30 min, followed by the addition of spermidine hydrochloride to 20 mM. Sonication and centrifugation of samples from E. coli were performed as described above. Protein concentration was determined by the Bradford method (Bradford, 1976
) using BSA as a standard.
Preparation of [3H]lysyl-tRNA.
[3H]Lysyl-tRNA was prepared as described by von Ehrenstein (1967) with slight modification. To prepare the tRNA synthetase enzyme fraction, E. coli W3110 cells (wet weight 5·4 g) were suspended in 10·4 ml buffer A [1 mM Tris/HCl (pH 7·2) and 10 mM MgCl2] and incubated with 200 µg lysozyme ml-1, followed by the addition of spermidine hydrochloride up to 20 mM. The supernatant was centrifuged at 105 000 g for 1 h. The resultant supernatant was passed through a DE52 (Whatman) column equilibrated with buffer A to remove endogenous tRNAs, and flowthrough fractions were pooled as the tRNA synthetase enzyme fraction. The reaction mixture (0·5 ml) for [3H]lysyl-tRNA synthesis contained 100 mM Tris/HCl (pH 7·2), 10 mM MgCl2, 10 mM KCl, 1 mM ATP, 0·4 mM [3H]lysine (100 mCi mmol-1, Amersham Pharmacia Biotech), 200 µg E. coli tRNA ml-1 (Sigma), and 50 µg tRNA synthetase enzyme fraction. Synthesized [3H]lysyl-tRNA was purified by salt-ethanol precipitation with two washes. To make sure that free [3H]lysine was removed in the [3H]lysyl-tRNA fraction, we simultaneously performed experiments omitting the tRNA synthetase fraction and washed the fractions until the background radioactivities disappeared.
In vitro LPG synthetase activity assay.
This assay was performed as described by Lennarz et al. (1967) with minor modifications. The reaction mixture (50 µl) contained 17·4 mM Tris/maleate (pH 7·0), 94 mM KCl, 7 mM MgCl2, 0·24 mM L-lysine, 1·55 mM phosphatidylglycerol (Sigma), 30 µM [3H]lysyl-tRNA prepared above, and 040 µg protein from the membrane fraction. The reaction was performed at 30 °C for 10 min and terminated by the addition of 550 µl chloroform/methanol [2 : 1 (v/v)]. The mixture was vigorously vortexed, incubated at 55 °C for 3 min to denature proteins, vortexed again, incubated at 55 °C for 7 min, and filtered through a paper filter (no. 2, Toyo Roshi). The filter was washed with 100 µl chloroform/methanol [2 : 1 (v/v)] and 250 µl 0·9 % NaCl to get reproducible recovery of LPG in the organic phase. The filtrate was mixed, vigorously vortexed for 1 min, placed on ice for 5 min, and centrifuged at 3000 r.p.m. for 5 min at 4 °C. The chloroform layer was washed once with 0·9 % NaCl to extract [3H]lysyl-tRNA that distributed in the chloroform layer independently of enzyme reaction. The amount of radioactivity incorporated into the chloroform layer was measured with a liquid scintillation counter (LS3801, Beckman).
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RESULTS AND DISCUSSION |
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When LPG was synthesized via MprF protein expression in E. coli cells, the relative amount of CL increased, and that of phosphatidylethanolamine (PE) decreased (Fig. 2a). One interpretation of the result is that a homeostatic control of the net charge might exist on the cell membrane. The present E. coli strain, in which negatively charged LPG can be induced, will be a useful tool to elucidate novel regulatory mechanisms for phospholipid biosynthesis in bacterial membranes.
Identification of MprF protein and LPG synthetase activity in the membrane fraction of E. coli cells expressing MprF protein
MprF protein is proposed to be a transmembrane protein containing 13 putative transmembrane domains (Peschel et al., 2001). Membrane fractions were prepared from E. coli cells transformed with pBADmprF or pBAD24 vector, and proteins were separated by SDS-polyacrylamide (7·5 %) gel electrophoresis. An approximately 93 kDa protein, whose molecular size was consistent with the predicted mass of the MprF protein, was induced by the addition of arabinose with dependence on plasmid pBADmprF (Fig. 4
a). Repeated washing of the membrane fractions increased the content of the 93 kDa protein (data not shown). The results support the prediction that MprF protein is a 93 kDa membrane protein.
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Conclusion
The present study examined the LPG synthetase activity of the S. aureus mprF gene product. The membrane fraction prepared from the S. aureus mprF deletion mutant cells had lost LPG synthetase activity (Fig. 1). E. coli cells that expressed the S. aureus MprF protein synthesized LPG (Figs 2 and 3
). Co-induction of a 93 kDa protein whose molecular size corresponds to that of MprF protein and LPG synthetase activity was demonstrated in the membrane fraction prepared from the E. coli cells in which MprF protein was expressed (Fig. 4
). The enzyme reaction mediated by this E. coli cell membrane fraction was dependent on both PG and lysyl-tRNA, consistent with that from S. aureus cells. Km values obtained from the E. coli membrane fraction for PG and lysyl-tRNA were consistent with the values of the LPG synthetase activity from S. aureus cells. These results suggest that MprF protein functions as the LPG synthetase in the S. aureus cell membrane.
Although this study strongly suggests that MprF protein is LPG synthetase, we cannot deny the possibility that there is a polypeptide(s) other than the MprF protein that is essential for the enzyme activity. To exclude this possibility, purification and characterization of the LPG synthetase must be performed. LPG is suggested to be involved in resistance against cationic antimicrobial peptides (Peschel et al., 2001; Kristian et al., 2003
), cationic antimicrobial peptide-like bacteriocins (Staubitz & Peschel, 2002
), or antibiotics (Ruzin et al., 2003
), and to have a role in cell cycle regulation (Ichihashi et al., 2003
). The cellular content of LPG varies depending on the growth phase and culture conditions (Houtsmuller & van Deenen, 1965
; Kenward et al., 1979
; Ichihashi et al., 2003
). To understand the roles of LPG in various aspects of cell physiology, it is critical to elucidate the mechanism that controls the membrane LPG content. LPG localization in cells is an important factor in the regulation of LPG function. The E. coli strain expressing MprF protein constructed in the present study will serve as a useful tool for further research on this basic phospholipid.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 August 2003;
revised 6 October 2003;
accepted 10 October 2003.