The menaquinone composition of Micrococcus luteus strains grown in different media was examined. Cells of M. luteus IAM 1056 (type strain) grown in an ...
J. Gen. App!. Microbiol., 35, 311-318 (1989)
EFFECTS OF THE GROWTH MEDIUM COMPOSITION ON MENAQUINONE HOMOLOG FORMATION IN MICROCOCCUS LUTEUS AKIRA HIRAISHI* ANDKAZUO KOMAGATA Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Received July 24, 1989)
The menaquinone composition of Micrococcus luteus strains grown in different media was examined. Cells of M. luteus IAM 1056 (type strain) grown in an ordinary complex medium contained MK-8 as the predominant menaquinone. On the other hand, this bacterium produced both MK-8 and MK-8(H2) as major quinones when grown in a chemically defined medium containing glutamate and pyruvate as carbon sources. Unlike the type strain, M. luteus strains IAM 12009 and IAM 12144 had MK-8 (H2) as the major menaquinone, independent of cell growth media. In all strains the total amount of dihydrogenated homologs relative to the total menaquinone content increased more or less in cells grown in the chemically defined medium. Carbon sources and Mgt + concentrations in the growth medium had minor effects on the menaquinone composition of the M. luteus strains.
It is now recognized that the analysis of isoprenoid quinones involved in electron transport in the respiratory chain of bacteria provides invaluable criteria for their taxonomy (1, 2,11). Earlier work of Jeffries and associates (7,8) on the menaquinone composition of some aerobic gram-positive cocci was the first to demonstrate the value of respiratory quinones in microbial systematics. They reported that most strains of Micrococcus luteus contained dihydrogenated menaquinones with eight isoprene units (MK-8(H2)) as major isoprenoid quinones, while the strain now designated as the type strain of this species had MK-8 as the major type with significant proportions of MK-8(H2) and the next lower and higher homologs . Similar results were obtained by Yamada et al. (12). Later , Fujii et al. (3) found * Address reprint requests to: Dr . Akira Hiraishi, Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. Abbreviations used for quinones: MK-n, menaquinone with n isoprene units; MK-n(H x), MK-n saturated with x hydrogen atoms. 311
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that solanesyl pyrophosphate synthetase homologs of polyprenyl pyrophosphates suggested that might reflect
the relatively the variable
variable product
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from M. luteus ATCC 4698 formed different depending on Mgt + concentrations, and menaquinone composition of this bacterium specificity of its solanesyl pyrophosphate
synthetase. These findings raise the question whether the menaquinone of M. luteus is affected by Mgt + concentrations and other nutritional cell growth conditions, study, quinone
35
composition factors under
conditions. Until now, little has been known about the effects of cultural e.g., cell growth media, on microbial quinone systems. In the present
therefore, profiles
we report
the influence
of M, luteus
of the growth
medium
composition
on the
strains.
MATERIALS
AND
METHODS
Bacterial strains. Mainly Micrococcus luteus IAM 1056 ( = ATCC 4698, type strain) was studied. M. luteus strains TAM 12009 (= ATCC 7468) and IAM 12144 ( = ATCC 381) were also used for comparison. All test strains were obtained from the Culture Collection Center of the Institute of Applied Microbiology, The University of Tokyo, Tokyo, Japan. Media and cultivation. For culturing the organisms, complex and chemically defined media were used. The complex medium (designated as PBY) contained per liter of distilled water: 5.0 g of peptone (Difco Laboratories, Detroit, U.S.A.), 3.0 g of beef extract (Difco), and 1.0 g of yeast extract (Difco). The chemically defined medium (designated as GPS) had the following composition: mineral base RM2 (pH 6.8) (6), 1,000 ml; sodium L-glutamate, 3.6 g; sodium pyruvate, 2.2 g; vitamin-free Casamino Acids (Difco),1.0 g; inosine, 0.06 g; biotin, 0.1 mg; all organic compounds except Casamino Acids were filter-sterilized. In some cases, either glutamate, pyruvate, lactate, malate, or glucose was used as the sole carbon source, instead of glutamate plus pyruvate (see Table 3). The concentration of Mgt + (as MgClz) in GPS medium was usually 1.0 mM, but was changed between 0 (no addition) and 10.0 mM when its effects on quinone formation were examined. Preliminary experiments with M. luteus IAM 1056 established that biotin, inosine, and some amino acids (replaceable with Casamino Acids) were required as growth factors for maximum growth, as already reported elsewhere (4), and that glutamate and pyruvate served as the best carbon source among the organic compounds tested. In all strains, the doubling time for growth was slightly longer in GPS medium than in PBY medium, while the total cell yield was 2- to 3-fold higher in the former medium than in the latter. Strain IAM 1056 required an irregular lag period of 2 to 5 days, in some cases more than 1 week, for growth in the chemically defined medium. The reason for this is not yet known. For inoculation purposes, cells were grown aerobically for 2 to 3 days in 30-m1 test tubes containing 6 ml of PBY medium. For main cultures, the precultured cells were inoculated into 200 ml of the test medium (1% inoculum) and then incubated aerobically in Sakaguchi's flasks on a reciprocal shaker. To eliminate the possible
1989
Menaquinones
of Micrococcus
luteus
313
effects of nutrients carried over from the preculture, cells grown in GPS medium were further transferred into 200 ml of the "fresh" medium (1 % inoculum) and incubated with shaking as noted above. All media were incubated at 30°C. Analysis of quinones. Unless otherwise specified, fresh wet cells harvested from cultures at the stationary phase of growth were used for quinone analysis. Qunnones were extracted with a chloroform-methanol mixture (2: 1, v/v), purified by thin-layer chromatography, and analyzed spectrophotometrically to verify the purity and determine the concentration. Menaquinone components were separated and identified by high-performance liquid chromatography (HPLC) with internal and external standards, and the relative peak area of the separated components was calculated with a microcomputer. Detailed information on the analytical procedures has been given in the previous papers (5,10). RESULTS
Menaquinone profiles of cells grown in complex and chemically defined media Experiments with cells of Micrococcus luteus TAM 1056 grown in PBY medium confirmed that MK-8 predominated (63 to 78% of the total menaquinones) in this strain at all growth stages. This is in agreement with the previous reports (3, 7,12). On the other hand, when grown in GPS medium, the bacterium was found to produce both MK-8 and MK-8(H2) as major menaquinones, with relative amounts of 32 to 48 and 24 to 50%, respectively. Typical HPLC profiles of menaquinones from strain IAM 1056 grown in the two media are shown in Fig. 1. The cells grown in GPS medium after the second transfer had much the same quinone composition as those from the first batch culture. Therefore, only data obtained with the first batch culture are reported below. The above findings on menaquinone profiles of strain IAM 1056 raised the question whether the shift of the menaquinone composition from cells grown in PBY medium to those in GPS medium also occurs in other M. luteus strains. Table 1 shows comparative data on the distribution of menaquinone homologs in JAM 1056 and other M. luteus strains grown in the complex and the chemically defined media. Unlike the type strain, strains IAM 12009 and IAM 12144 formed MK-8(H2) as the predominant menaquinone with a considerable proportion of MK-7(H2), independent of cell growth media. Although there were some differences in the menaquinone composition between the type strain and the other two, the hydrogenation of menaquinones in those strains seemed to take place in a similar fashion depending on the cell growth media. The results shown in Table 2 indicate that, in all strains, the total content of dihydrogenated menaquinones did not vary so significantly as that of the unsaturated types in response to changes in cell growth media, and the total amount of the hydrogenated homologs relative to the total menaquinone content increased more or less in cells grown in the chemically defined medium.
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KOMAGATA
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Fig, 1. HPLC elution profiles of menaquinones from Micrococcus luteus IAM 1056 cells grown in the complex (A) and the chemically defined (B) media. Analytical conditions: pump, Shimadzu LC-5A; column, Zorbax ODS (4.6 i.d. x 250 mm) with a short guard column; mobile phase, methanol-isopropyl ether (3:1, v/v); column temperature, 23°C; flow rate, 1.0 ml/min; samples were monitored in a spectrophotometric detector at 270 nm. Table 1. Menaquinone composition of Micrococcus luteus strains grown in complex (PBY) and chemically defined (GPS) media.°
35
1989
Menaquinones
Table
2.
Contents
in Micrococcus
Table
3. Menaquinone in the chemically
of Micrococcus
of dihydrogenated luteus
and
luteus
unsaturated
strains grown in complex defined media.°
and
315
menaquinones chemically
composition of Micrococcus luteus IAM 1056 grown defined medium with different carbon sources.
Effects of carbon sources When M. luteus IAM 1056 was grown in the chemically defined medium containing either glutamate, pyruvate, lactate, malate, or glucose as the carbon source, it produced MK-8 as a major component with comparable or significant amounts of MK-8(H2) in all cases, as shown in Table 3. The proportion of dihydrogenated menaquinones in glucose-grown cells was considerably lower than it was in cells cultivated with other carbon sources. Since the strain showed only poor growth in the glucose-containing medium, the growth might not result from the consumption of glucose but from the utilization of organic nutrients carried over from the preculture. In fact, the second transfer of the "glucose-grown" culture
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HIRAIsHI and KOMAGATA
316
Table
4.
Effects of Mgt + concentration on the menaquinone of Micrococcus luteus JAM 1056.Q
composition
to the same medium resulted in no growth within at least 10 days This may explain why the cells cultivated in the glucose-containing more
similar
to those
grown
in the complex
profiles. The above-noted results suggest that as a factor affecting the menaquinone menaquinone cells grown
medium
35
with respect
of incubation. medium were to the quinone
the carbon source is of little significance composition of M. luteus, although
homolog formation in strain IAM 1056 differed in the complex and the chemically defined media.
markedly
between
Effects of Mgt + concentration The effects of Mgt + concentrations on the menaquinone composition of strain IAM 1056 grown in the chemically defined medium are shown in Table 4. There was no significant difference in menaquinone profiles among cells grown on different Mgt + concentrations. Both MK-8 and MK-8(H2) were detected as the major quinones in all cases. The menaquinone composition of the other M. luteus strains was also unaffected by the Mgt + concentration in the growth medium (data not shown). This suggests that menaquinone homolog formation in M. luteus is stringently regulated under physiological conditions, irrespective of Mgt + strength. DISCUSSIC)N
The value of isoprenoid quinones in microbial systematics is based on the wide range of their structural variations among different taxa and their relative uniformity and stability within strains of the same taxon (1, 2,11). However, there has been little information about environmental factors affecting microbial quinone composition. The results of this study demonstrate that the menaquinone composition of the type strain of M. luteus varies depending on cell growth media,
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Menaquinones
of Micrococcus
luteus
317
although it is not affected by Mgt + concentration. In concurrent studies it has been found that significant variations in isoprenoid quinone profiles occur in some other species of gram-positive bacteria in response to the composition of growth medium and the period of cultivation (Hiraishi and Komagata, unpublished data). We are reluctant to consider that these observations discount the taxonomic importance of isoprenoid quinones. Nevertheless, we are decidedly of the opinion that cell growth media and other cultural conditions for gram-positive bacteria used for quinone analysis should be strictly defined. The results obtained with cells grown in an ordinary complex medium indicate differences in menaquinone profiles between the type strain and the other strains of M. luteus. However, it is worth noting that, like the other strains, the type strain had MK-8(H2) as a major menaquinone when grown in chemically defined media, and the absolute amount of dihydrogenated menaquinones in this bacterium was relatively constant, independent of cell growth media. These findings may lead to the idea that the hydrogenated menaquinones are more important than the unsaturated ones for the physiology of strain IAM 1056, as well as for that of the other M. luteus. Therefore, the difference in menaquinone systems between the type strain and the other strains of M. luteus may be of little taxonomic significance. While the physiological significance of variations in the length and the hydrogenation of the side chain of respiratory quinones is far from being well understood, there is evidence for the existence of a specific binding between quinones and protein in mitochondrial and bacterial electron transport systems (13). Thus, it can be assumed that the primary function of the side chain of quinones is to participate in this specific binding and to stabilize the free form of quinones in the cytoplasmic membrane. It has been shown that a specific quinone homolog together with phospholipids is required for maximum activity of the re-constituted NADH oxidase system in certain gram-negative bacteria (9). Further study in this field would provide more comprehensive insight into evaluating the taxonomic significance of structural variations in the side chain of isoprenoid quinones. REFERENCES
1) Collins, M. D., Analysis of isoprenoid quinones. In Methods in Microbiology, Vol. 18, ed. by Gottschalk, G., Academic Press, London (1985), p. 329-366. 2) Collins, M. D. and Jones, D., Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. Microbiol. Rev., 45, 316-354 (1981). 3) Fujii, H., Sagami, H., Koyama, T., Ogura, K., and Seto, S., Variable product specificity of solanesyl pyrophosphate synthase. Biochem. Biophys. Res. Commun., 96, 1648-1653 (1980). 4) Grula, E. A., Luk, S.-K., and Chu, Y.-C., Chemically defined medium for growth of Micrococcus lysodeikticus. Can. J. Microbiol., 7, 27-32 (1961). 5) Hiraishi, A., Hoshino, Y., and Kitamura, H., Isoprenoid quinone composition in the classification of Rhodospirillaceae. J. Gen. Appl. Microbiol., 30, 197-210 (1984). 6) Hiraishi, A. and Kitamura, H., Distribution of phototrophic purple nonsulfur bacteria in activated sludge systems and other aquatic environments. Bull. Jpn. Soc. Sci. Fish., 50, 1929-1937 (1984). 7) Jeffries, M. A., Cawthorne, M. A., Harris, M., Cook, B., and Diplock, A. T., Menaquinone
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determination in the taxonomy of Micrococcaceae. J. Gen. Microbiol., 54, 365-380 (1969). Jeffries, M. A., Cawthorne, M. A., Harris, M., Diplock, A. T., Green, J., and Price, S. A., Distribution of menaquinones in aerobic Micrococcaceae. Nature, 215, 257-259 (1967). King, M. T. and Drews, G., The function and localization of ubiquinone in the NADH and succinate oxidase systems of Rhodopseudomonas palustris. Biochim. Biophys. Acta, 305, 230-248 (1973). Tamaoka, J., Katayama-Fujimura, Y., and Kuraishi, H., Analysis of bacterial menaquinone mixtures by high performance liquid chromatography. J. App!. Bacteriol., 54, 31-36 (1983). Yamada, Y., Kokyusa ni kanyosuru kinonrui no bunshishu ni motozuku biseibutsu no bunrui (Classification of microorganisms on the basis of respiratory quinone systems) (in Japanese). Hakko to Kogyo, 37, 940-954 (1979). Yamada, Y., Inouye, G., Tahara, Y., and Kondo, K., The menaquinone system in the classification of aerobic gram-positive cocci in the genera Micrococcus, Staphylococcus, Planococcus, and Sporosarcina. J. Gen. App!. Microbiol., 22, 227-236 (1976). Yu, C.-A. and Yu, L., Ubiquinone-binding proteins. Biochim. Biophys. Acta, 639,99-128(1981).
