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Journal of Bacteriology, September 2004, p. 5968-5971, Vol. 186, No. 17
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.17.5968-5971.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Overexpression of gnsA, a Multicopy Suppressor of the secG Null Mutation, Increases Acidic Phospholipid Contents by Inhibiting Phosphatidylethanolamine Synthesis at Low Temperatures

Rie Sugai, Hisayo Shimizu, Ken-ichi Nishiyama, and Hajime Tokuda*

Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japan

Received 22 April 2004/ Accepted 3 June 2004


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ABSTRACT
 
GnsA overproduction was previously found to suppress both the secG null mutation and the fabA6 mutation in Escherichia coli by increasing the unsaturated fatty acid contents. We report here that it also increased the acidic phospholipid contents at 20°C but not at 37°C. GnsA overproduction at 20°C specifically inhibited phosphatidylethanolamine synthesis and therefore caused the increase in the proportion of acidic phospholipids.


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TEXT
 
Escherichia coli contains three major phospholipids, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). The phospholipid composition is important for the activities of various membrane proteins. For example, acidic phospholipids PG and CL are essential for the function of SecA (3, 8, 23), which drives protein translocation across membranes. Nonbilayer lipid PE has been reported to be important for protein translocation (16) and development of the functional membrane topology of lactose permease (1). SecG is a membrane component of the protein translocation machinery and facilitates the SecA function (11). The secG null mutation significantly retards protein translocation (4, 22). All multicopy suppressors of the secG null mutation have been found to be involved in lipid synthesis (7, 19, 21-23). The gnsA gene is one of these suppressors and encodes a hydrophilic protein consisting of 57 amino acids (21). Overproduction of GnsA also corrects the defect in a temperature-sensitive unsaturated fatty acid auxotroph, the fabA6 mutant (17, 21), through an unknown mechanism.

Although GnsA overproduction was previously shown to increase the unsaturated fatty acid contents without affecting the phospholipid composition (21), detailed examinations described here revealed that GnsA overproduction affected the phospholipid composition at low temperatures (Table 1). E. coli strain K003 (24) harboring pKQ2 (ParaB) (10) or pSRA (ParaB gnsA) (21) was inoculated into Luria-Bertani medium containing 1% glycerol, 25 µg of ampicillin/ml, and 0.2% arabinose to give an optical density at 660 nm (OD660) of 0.1. Cells were cultivated in the presence of [32P]orthophosphoric acid (0.37 MBq/ml; NEN Life Science Products) at 37°C to an OD660 of 0.8 or at 20°C for 24 h. The OD of the culture reached ~1.0 after 24 h of incubation at 20°C. Phospholipids were extracted and analyzed by thin-layer chromatography (Silicagel 60; Merck) as described previously (21). The level of incorporation of radioactivity into phospholipids was determined at least twice throughout this study, and experimental error was within 5%. Although GnsA overproduction had no effect on the phospholipid composition at 37°C, as previously reported (21), it significantly decreased the PE content at 20°C, causing an increase in the acidic phospholipid content (PG plus CL) from ~19 to ~33%. To confirm that the effect of GnsA overproduction is specific to low temperatures, cells were grown at 37°C to an OD660 of 0.8 and then incubated at 20°C for 3 h. When the labeling was started at 37°C, the phospholipid composition remained normal irrespective of the presence or absence of GnsA overproduction, suggesting that incubation for 3 h at 20°C did not affect the overall phospholipid composition. In contrast, when phospholipids synthesized after the temperature downshift were labeled, GnsA overproduction decreased the PE content and increased the acidic phospholipid contents. The culture turbidity increased by ~10% during cultivation at 20°C. These results indicate that GnsA overproduction specifically increases the acidic phospholipid contents at low temperatures. Essentially the same effects of GnsA overproduction were observed with KN553 (K003 {Delta}secG::Kanr) (11), FS1576 (12, 20), and KN370 (FS1576 {Delta}secG::Kanr) (10). We therefore examined the growth and phospholipid synthesis of KN553 harboring pKQ2 or pSRA after the temperature downshift. GnsA overproduction had little stimulatory effect on growth (Fig. 1A). In contrast, labeling of phospholipids decreased to ~20% upon GnsA overproduction (Fig. 1B).


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  		TABLE 1. GnsA overproduction increases the acidic phospholipid contents at 20°C



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  		FIG. 1. GnsA overproduction decreases phospholipid synthesis. KN553 cells harboring pKQ2 or pSRA were grown at 37°C to an OD660 of 0.8, and the temperature was then shifted to 20°C. (A) The OD660 of the culture was monitored at specified times after the temperature downshift. (B) Labeling of the cells with [32P]orthophosphoric acid was started after the temperature downshift. Phospholipids were extracted from 100 µl of culture at the specified time points, and then radioactivity was determined.

To analyze the synthesis of individual phospholipids with or without GnsA overproduction, K003 cells harboring pKQ2 or pSRA were grown in the presence of 0.2% arabinose at 37°C to an OD660 of 0.8 and then transferred to 20°C. At the indicated time points, aliquots of the culture were pulse-labeled with [32P]orthophosphoric acid for 2 min and then radioactivities incorporated into individual phospholipids were determined (Fig. 2). GnsA overproduction decreased the incorporation of 32P into the three phospholipids even at 37°C, although the decrease was less significant than that at 20°C. Incorporation of 32P into PG was greater than that into PE at 37°C whether GnsA was overproduced or not (Fig. 2A and B). It is known that the rate of PG synthesis is higher than that of PE synthesis, while the PG content is lower than the PE content due to the unstable property of PG (13, 14). The profile of 32P incorporation was significantly different at 20°C, at which the labeling of phospholipids drastically decreased. A higher level of radioactivity was incorporated into PE than PG without GnsA overproduction (Fig. 2A). In contrast, the level of radioactivity incorporated into PE, but not PG, was significantly decreased by GnsA overproduction (Fig. 2B). The level of radioactivity incorporated into CL remained low irrespective of the presence or absence of GnsA overproduction (Fig. 2A and B). The proportion of each phospholipid pulse-labeled for 2 min was determined at different time points (Fig. 2C and D). GnsA overproduction did not affect the proportions of the three phospholipids at 37°C. In marked contrast, the proportion of newly synthesized PE increased to ~60% at 20°C without GnsA overproduction (Fig. 2C) whereas it decreased to less than 20% upon GnsA overproduction (Fig. 2D). Essentially the same results were obtained with KN553 ({Delta}secG) cells (data not shown), indicating that SecG has no effect on the specificity of phospholipids synthesized at 20°C. Taken together, these results indicate that GnsA overproduction at 20°C specifically reduces the synthesis of PE, thereby causing increases in the acidic phospholipid contents.



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  		FIG. 2. GnsA overproduction specifically inhibits PE synthesis at 20°C. K003 cells harboring pKQ2 (A and C) or pSRA (B and D) were grown at 37°C to an OD660 of 0.8, and the temperature was then shifted to 20°C (0 time). An aliquot (667 µl) of the culture was withdrawn at the indicated times before and after the temperature downshift, followed by labeling with [32P]orthophosphoric acid for 2 min at 37°C (gray area) or 20°C. (A and B) The phospholipids were extracted and analyzed by thin-layer chromatography. The levels of radioactivity incorporated into PE, PG, and CL were determined. (C and D) The proportions of PE, PG, and CL at different time points were calculated from the results in A and B.

The effects of GnsA overproduction on cell morphology were examined at 37 and 20°C. Although GnsA overproduction had no apparent effect on the cell morphology at 37°C (data not shown), more than 30% of both K003 and KN553 cells became filamentous upon the overproduction of GnsA at 20°C (Fig. 3). Some cells overproducing GnsA were longer than 20 µm, while the length of cells was ~3 µm without GnsA overproduction. It has been reported that PE is important for FtsZ ring formation, which is required for cell division (9). Indeed, a decrease in the level of PE causes the appearance of filamentous cells (2, 5, 15). Therefore, a decrease in the level of PE due to GnsA overproduction at 20°C is responsible for the appearance of elongated cells.



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  		FIG. 3. GnsA overproduction makes E. coli cells filamentous at low temperatures. K003 and KN553 cells harboring pKQ2 or pSRA were grown at 20°C for 24 h.

Although the increases in the unsaturated fatty acid contents were independent of temperature (17, 21), acidic phospholipid contents were increased by GnsA overproduction only at low temperatures. The increases in acidic phospholipid contents most likely resulted from inhibition of PE synthesis and not stimulation of PG and CL synthesis. However, it is not clear at present why the overproduction of GnsA, a small cytosolic protein possessing a helix-turn-helix motif (21), has such drastic and pleiotropic effects on the compositions of both fatty acids and phospholipids.

PE synthesis is dependent on phosphatidylserine (PS) synthase (18), which catalyzes the formation of PS from CDP-diacylglycerol and L-serine. PS is then rapidly converted to PE by PS decarboxylase. The synthesis of phospholipids and the maintenance of their compositions in membranes are regulated in complex manners and are not fully understood (18). GnsA overproduction may affect transcription, translation, and even enzyme activity. It has been reported that the inhibition of phospholipid synthesis rapidly blocks fatty acid synthesis (6). However, the relationship between the increases in the unsaturated fatty acid contents and the inhibition of PE, both of which are induced by GnsA overproduction, remains to be clarified.

Overproduction of GnsB, a homologue of GnsA, also caused increases in acidic phospholipid contents at low temperatures (data not shown). However, it had a lesser effect than GnsA overproduction. The increases in unsaturated fatty acid contents were also more significant with GnsA than with GnsB (21). Disruption of both gnsA and gnsB affected neither the phospholipid composition nor growth (data not shown). The levels of GnsA and GnsB are under the detection limits in wild-type cells (17, 21). More analyses are therefore necessary to reveal the physiological function of GnsA.


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ACKNOWLEDGMENTS
 
We thank Akihiko Okuno for his efforts in the initial stage of this study and Rika Ishihara for technical assistance and secretarial support.

This work was supported by grants to H.T. from the Ministry of Education, Science, Sports and Culture of Japan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Phone: 81-3-5841-7830. Fax: 81-3-5841-8464. E-mail: htokuda{at}iam.u-tokyo.ac.jp. Back


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Journal of Bacteriology, September 2004, p. 5968-5971, Vol. 186, No. 17
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.17.5968-5971.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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