Masakazu KIKUCHI and Yoshio NAKAO

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1 Agr. Biol. Client., 37 (3), 515 `519, 1973 Relation between Cellular Phospholipids and the Excretion of L-Glutamic Acid by a Glycerol Auxotroph of Corynebacterium alkanolyticum õ Masakazu KIKUCHI and Yoshio NAKAO Microbiological Research Laboratories, Central Research Division. Takeda Chemical Industries, Ltd., Osaka, Japan Received July 12, 1972 Relation between cellular phospholipids and L-glutamic acid excretion was investigated using Corynebacterium alkanolyticum GL-21 (a glycerol auxotroph). When strain GL-21 was cultured in glycerol-limited medium which contained n-hexade cane, acetic acid or fructose as carbon source, there occurred the limitation of cellular phos pholipid content and the over-accumulation of L-glutamic acid in the broth. Two-dimensional thin-layer chromatograms provided evidence that both the parent and the mutant strains contained the same phospholipids such as cardiolipin, phosphatidylethanolamine, phosphadityl glycerol, phosphatidylinositol and phosphatidic acid. Limited supply of glycerol to the mutant did not greatly alter the proportions of the individual phospholipids. Using a glycerol auxotroph of Corynebac terium alkanolyticum, the authors suggested that the over-excretion of L-glutamic acid would be initiated by the regulation of cellular pho spholipids rather than of cellular fatty acids.1 `3) Moreover, evidence was presented that phos pholipids were required for the transport of some kind of substances.4 `6) These findings suggest that phospholipids are involved in the mechanism of the membrane permeability. The present investigation was undertaken in order to clarify the relation between cellu lar phospholipids and L-glutamic acid excre tion. METHODS AND MATERIALS Organisms and culture conditions. C. alkanoly ticum No. 314 and C. alkanolyticum GL-21 (a glycerol auxotroph)1) were used in all experiments. Cultiva tion was carried out according to the method described õ Microbial Production of L-Glutamic Acid by Glycerol Auxotrophs. Part VI. See reference 3). Part of this work was presented at the 4th International Fermentation Symposium, held in Kyoto, March 19, Following abbreviations were used in this report: CL, cardiolipin; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PA, phosphatidic acid; PI, phosphatidylinositol. previously.7) Strain GL-21 grown on media con taining 0.02% and 0.1% of glycerol is designated as GL-21 (0.02) and GL-21 (0.1), respectively. Assay of L-glutamic acid. L-Glutamic acid was determined by the microbiological method described previously.8) Extraction of phospholipids. Phospholipids were extracted by the method described earlier.2) Thin-layer chromatography of phospholipids. Com mercial thin-layer plates of 0.25mm thickness (TLCplates Silica Gel F254; Merck) were used for the separa tion of phospholipids. Phospholipids were chromato graphed with the following solvent systems: Sol vent 1, chloroform-methanol-water (65:25:4, v/v/v), solvent 2, chloroform-methanol-7n ammonia (46:18:3, v/v/v); solvent 3, diisobutyl ketone-glacial acetic acid water (40:20:3, v/v/v); solvent 4, chloroform-metha nol-diisobutyl ketone-glacial acetic acid-water (45:15: 30:20:4, v/v/v/v/v). Detection of phospholipids. Phospholipids on TLC plates were detected with the following spray re agents9): (1) total lipid with iodine, (2) amino nitrogen with 0.2% solution of ninhydrin in acetone, (3) organic phosphorus with Zinzadze reagent, (4) choline with Dragendorff reagent and (5) sugar with periodate benzidine. Determination of phospholipids. Spots of phospho lipids on TLC plates were made visible by exposure to iodine vapour and scraped. The phospholipids were

2 516 M. KIKUCHI and Y. NAKAO eluted twice with 3ml of chloroform-methanol (2:1, v/v), and then with 2ml of methanol-acetic acid-water (95:1:5, v/v/v). The residual silica gel was removed and the solvent was evaporated in a boiling water bath. Phospholipids were determined by estimating phosphorus in phospholipids by the method of Bart lett.10) The amount of phospholipids in other ex periments were shown as the dry weight as described previously.2) The amount of each phospholipids is shown as per cent by weight of the total phospholipids. Phospholipid standards. Cardiolipin and phospha tidylserine were purchased from Nutritional Biochemi cals Corporation, and phosphatidylethanolamine and lecithin from Calbiochem. Phosphatidic acid was prepared from lecithin according to the method of Davidson and Long.11) RESULTS L-Glutamic acid accumulation based on the regulation of cellular phospholipid content It has been reported that the regulation of phospholipid synthesis was responsible for the over-accumulation of L-glutamic acid in the culture broth.1 `3) To further investigate this phenomenon, n-hexadecane (C16), acetic acid or fructose was employed as a typical carbon source for the production of L-glutamic acid. As will be seen from Table I, strain GL-21 (a glycerol auxotroph) grown under limited supply of glycerol produced L-glutamic acid from each carbon source. L-Glutamic acid production was always accompanied by a decrease in cellular phospholipid content. Phospholipid content of L-glutamic acid accumulating cells was one-half to one-third that of L-glutamic acid non-accumulating cells. The cells at the logarithmic phase (24hr culture) generally maintained a high level of phospholipid content, compared with those at the stationary phase (64hr culture). Characterization of phospholipids of C. alka nolyticum Figure 1 shows two-dimensional thin-layer chromatograms of cellular phospholipids ex tracted from C. alkanolyticum No. 314 and GL-21 grown on n-hexadecane. These seven phosphorus-containing lipids were detected with the Zinzadze reagent spray. The parent and the mutant strains were composed of the same phospholipids (A `X2). As is evident from Fig. 1, this chromatographic method permitted almost complete separation of the major phospholipids. A spot A was revealed with ninhydrin reaction, and its chromato graphic mobilities were identical with those of PE in various solvent systems described in the Methods. The chromatographic mobi lities of spots B and C were identical with TABLE I. RELATION BETWEEN PHOSPHOLIPID CONTENT AND L-GLUTAMIC ACID PRODUCTION

3 Cellular Phospholipids and the Excretion of L-Glutamic Acid 517 Moreover, a spot X2 might be a sugar-contain ing phospholipid from the low mobilities on chromatographic plates in various organic solvent systems. But a spot X1 was uniden tified. No phospholipid was detectable with Dragendorff reagent. The same phospholipid patterns were obtained from the cells grown on fructose and acetic acid. On the basis of the data presented, the kind of cellular phospholipid does not seem to be influenced by the carbon sources employed. FIG. 1. Thin-layer Chromatogram of Phospho lipids of C. alkanolyticum. Phospholipids were applied to silica gel plates (Merck) and chromatographed in the first dimension with Solvent I of chloroform-methanol-water (65:25:4, v/v/v). The chromatogram was then rotated 90 and developed in the second dimension with Solvent II of chloroform-methanol-7n ammonia (46:18:3, v/v/v). A, PE; B: CL; C, PA; D, PG; E, PI; X1 and X2, unidentified phospholipids. those of authentic CL and PA, respectively. However, identification of spots D, E, X1 and X2 was limited by the lack of certain standards so it was based on a color reaction and the comparison of their chromatographic mobilities in various solvent systems with those described in the other reports.12 `15) Spots D, E and X2 were detectable with periodate benzidine spray. In addition, from the chromatographic mobilities, spots D and E seemed identical with PG and PI, respectively. Relation between L-glutamic acid accumulation and phospholipid composition To represent a key to solve the mechanism of L-glutamic acid excretion, further study on phospholipids was carried out. Phospholipid composition of the parent and the mutant strains grown on n-hexadecane is illustrated in Fig. 2. CL was the main phospholipid of these strains at every growth phase. Phospholipid patterns of three cul tures were almost the same at the logarithmic growth phase. PA could not be detected in the cultures at this growth phase. At the stationary phase, a small change in the phos pholipid pattern was observed. Proportions of PE and X1, especially PE, in L-glutamic acid-accumulating cells (GL-21 (0.02)) were higher than those in L-glutamic acid non-ac cumulating cells (No. 314 and GL-21 (0.1)). However, the reverse relation was observed in the proportions of PA and X2. FIG. 2. Phospholipid Composition of C. alkanolyticum Grown on n-hexadecane.,, Phospholipids from 24hr and 64hr cultured cells, respectively. GL-21 (0.02), GL-21 (0.1): Strain GL-21 was grown in the presence of 0.02 or 0.1% glycerol. * Recorded as percentage of each phospholipid to total phospholipids.

4 518 M. KIKUCHI and Y. NAKAO The same results were obtained from the analysis of phospholipids of bacteria producing L-glutamic acid from carbohydrates, such as Breuibacterium lactofermentum, Microbacterium ammoniaphilum and Corynebacterium gluta micum.12) However, the main phospholipid of bacteria seems to differ from species to species: PE is generally the main phospho lipid in Escherichia coli5,16,17) and Micrococcus FIG. 3. Phospholipid Composition of C. alkanoly ticum Grown on Fructose. Phospholipids were extracted from 72hr cultured cells. * Recorded as percentage of each phospholipid to total phospholipids. GL-21 (0.02) is the same as in Fig. 2. Phospholipid composition of the parent and the mutant strains grown on fructose is also presented in Fig. 3. CL was the main component in both strains, while PA was seldom detectable. A high proportion of PE in L-glutamic acid-accumulating cells (GL-21 (0.02)) was similarly found. The proportions of CL and PI in strain GL-21 (0.02) were a little lower than those in strain No The relation between L-glutamic acid ac cumulation and the amount of individual phospholipids remains to be solved. DISCUSSION It is interesting and important to clarify the regulating factor in L-glutamic acid excretion. This elucidation is expected to present a possibility to control the membrane permeability artificially. Our previous reports using a glycerol auxotroph of C. alkanolyticum indicated that the regulation of phospholipid synthesis was necessary for the microbial production of L-glutamic acid from n-paraffins.1 `3) This mutant GL-21 also produced a large amount of L-glutamic acid in the culture broth from various carbon sources,3,7) only when the cellular content of phospholipids was limited (Table I). From the analysis of phospholipid composi tion, CL was the main phospholipid in C. alkanolyticum, as shown in Fig. 2 and Fig. 3. cerificans,18) and PG in Bacillus subtilis.13,19) During the growth under a limited supply of glycerol, maintenance of high proportion of PE was common to L-glutamic acid-accumulat ing cells grown on n-hexadecane and fructose (Fig. 2 and Fig. 3), whereas change in the proportions of other components was found to be small and irregular. During glycerol starvation, no marked difference was found in phospholipid composition of E. coli glycerol auxotroph,5) while the proportion of phos pholipids of B. subtilis19) and Staphylococcus aureus20) glycerol auxotrophs changed. As the experimental conditions in these reports were different from ours, exact comparison is difficult. But, PE appears not to be a controlling factor in L-glutamic acid excretion, because PE was reported to be absent in other L-glutamic acid producing bacteria.12) Shibukawa et al.21) reported that the relation between the amount of CL and PA had a profound influence on the excretion of L-glutamic acid. However, such a phenomenon was not observed in our experiments. Moreover, turnover of CL and PG in phospholipid metabolism was pro ved22 `25) and PA was sometimes not detected in our experiments, so that these phospholipids may not control the excretion of L-glutamic acid. Takinami et al.12) pointed out phospha tidyl inositol mannoside (PIM) as a controlling factor in L-glutamic acid excretion. The spot X2 appears to be PIM in our experiments. However, in view of the fact that much im provement remains in the method of extrac tion or assay of phospholipids, it might be too early to discuss the relation between such a minor phospholipid and L-glutamic acid excretion.

5 Cellular Phospholipids and the Excretion of L-Glutamic Acid 519 Tarlov and Kennedy4) indicated that phos pholipids would participate in the function of the membrane such as permeability. More over, PG was found to be required for the sugar transport system6) and net synthesis of phospholipids was necessary for lactose transport.5) Our previous reports showed that regulation of phospholipid synthesis was effective for the excretion of L-glutamic acid1 `3) and possibly of ƒ -ketoglutaric acid.7) From a consideration of these facts, it is an important approach to find out the sort of phospholipid operative in the excretion of L-glutamic acid. Although the relation be tween the excretion of L-glutamic acid and the sort of phospholipid was not clear from our study, the limitation of cellular phospholipid content was at least necessary for the excretion of L-glutamic acid (Table I). Until now, the structure of the membrane has not fully been clarified, however, there remain some possibilities to answer the ques tion on the mechanism of L-glutamic acid excretion as described in the previous report8): mutant requiring each constituent of phospho lipids such as ethanolamine, inositol, choline, serine and mannose will give some clues to this problem. Acknowledgement. The authors wish to thank Drs. R. Takeda and H. Fukuda, Microbiological Research Laboratories, Takeda Chemical Industries, Ltd. for their continuing interest and encouragement during this study. REFERENCES 1) Y. Nakao, M. Kikuchi, M. SUZUKI and M. Doi, Agr. Biol. Chem., 34, 1875 (1970). 2) M. Kikuchi and Y. Nakao, ibid., 36, 1135 (1972). 3) M. Kikuchi and Y. Nakao, ibid., 37, 507 (1973). 4) A. R. Tarlov and E. P. Kennedy, J. Biol. Chem., 240, 49 (1965). 5) C. C. Hsu and C. F. Fox, J. Bacteriol., 103, 410 (1970). 6) W. Kundig and S. Roseman, J. Biol. Chem., 246, 1407 (1971). 7) M. Kikuchi, M. Doi, M. Suzuki and Y. Nakao, Agr. Biol. Chem., 36, 1141 (1972). 8) Y. Nakao, M. Kikuchi, M. Suzuki and M. Doi, ibid., 36, 490 (1972). 9) "Data for Biochemical Research," ed. by R. M. C. Dawson et al., Oxford University Press, Great Britain, 1969, p ) G. R. Bartlett, J. Biol. Chem., 234, 466 (1959). 11) F. M. Davidson and C. Long, Biochem. J., 69, 458 (1958). 12) K. Takinami, Y. Yamada and T. Shiro, Proceed ings of Japanese Conference on the Biochemistry of Lipids, (Sapporo), No. 11, 49 (1969). 13) J. L. Beebe, J. Bacteriol., 107, 704 (1971). 14) A. J. De Siervo and M. R. J. Salton, Biochim. Biophys. Acta, 239, 280 (1971). 15) T. G. Tornabene and J. E. Ogg, ibid., 239, 133 (1971). 16) J. E. Cronan, Jr., J. Bacterial., 95, 2054 (1968). 17) A. J. De Siervo, ibid., 100, 1342 (1969). 18) R. A. Makula and W. R. Finnerty, ibid., 103, 348 (1970). 19) T. T. Lillich and D. C. White, ibid., 107, 790 (1971). 20) P. H. Ray and D. C. White, ibid., 109, 668 (1972). 21) M. Shibukawa, M. Kurima and S. Ohuchi, Agr. Biol. Chem., 34, 1136 (1970). 22) J. Kanfer and E. P. Kennedy, J. Biol. Chem., 238, 2919 (1963). 23) Y. Kanemasa, Y. Akamatsu, and S. Nojima, Biochim. Biophys. Acta, 144, 382 (1967). 24) G. F. Ames, J. Bacteriol., 95, 833 (1968). 25) D. C. White and A. N. Tucker, ibid., 97, 199 (1969).

Phospholipase D Activity of Gram-Negative Bacteria

JOURNAL OF BACTERIOLOGY, Dec. 1975, p. 1148-1152 Copyright 1975 American Society for Microbiology Vol. 124, No. 3 Printed in U.S.A. Phospholipase D Activity of Gram-Negative Bacteria R. COLE AND P. PROULX*