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Menaquinone Patterns of Amycolatopsis Species
Zbl. Bakt. 274, 465-470 (1991) © Gustav Fischer Verlag, StuttgartlNew York
Menaquinone Patterns of Amycolatopsis Species A. F. YASSIN h , K. P. SCHAAL 2 , H. BRZEZINKA3, M. GOODFELLOW4 **, and G. PULVERER 1 Institut ftir Medizinische Mikrobiologie und Hygiene, Universitat zu Koln, D-5000 Koln 41 Institut ftir Medizinische Mikrobiologie und Immunologie der Universitat Bonn, D-5300 Bonn 1 3 Institut ftir Rechtsmedizin der Universitat Bonn, D-5300 Bonn 1 4 Department of Microbiology, The Medical School, Newcastle upon Tyne, United Kingdom * Permanent address: Faculty of Science, University of Tanta, Tanta, Egypt. Fellow of the Alexander v. Humboldt-Foundation, D-5300 Bonn 1
2
Received March 2, 1990 . Accepted in revised form June 18, 1990
Summary The menaquinones of representative strains of the actinomycete genus Amycolatopsis were examined by reverse phase thin-layer chromatography and mass spectrometry. Representatives of all seven validly described species contained various combinations of di-, tetra- and hexahydrogenated menaquinones with nine isoprene units as predominant isoprenologues. It seems likely that the variation in the predominant menaquinones merely reflects the stages from the growth cycle from which biomass was taken. The detection of major proportions of hydrogenated menaquinones with nine isoprene units serves to distinguish Amycolatopsis strains from most other actinomycetes, notably those belonging to related genera such as Amycolata and Pseudonocardia.
Zusammenfassung Von reprasentativen Vertretern des Aktinomyzetengenus Amycolatopsis wurden ihre Menaquinone mit Hilfe der reversen Dtinnschichtchromatographie und der Massenspektrometrie bestimmt. Stamme aller 7 akzeptierten Spezies enthielten unterschiedliche Kombinationen von Di-, Tetra- und Hexahydrogen-menaquinonen mit 9 Isopreneinheiten als tiberwiegendes Isoprenolog. Die Unterschiede der vorherrschenden Menaquinone scheinen den verschiedenen Stufen des Wachstumszyklus zu entsprechen. Der Nachweis von hauptsachlichen Anteilen an hydrierten Menaquinonen mit 9 Isopreneinheiten kann zur Abgrenzung von Amycolatopsis-Stammen von den meisten tibrigen Aktinomyzeten dienen, speziell von Angehorigen der verwandten Genera Amycolata und Pseudonocardia.
* * Corresponding author
466
A. F. Yassin, K. P. Schaal, H. Brzezinka, M. Goodfellow, and G. Pulverer
Introduction The genus Amycolatopsis (14) was proposed for actinomycetes classified as Nocardia mediterranei, Nocardia orienta lis, Nocardia rugosa and Nocardia sulphurea; species cited as incertae sedis in the current edition of Bergey's Manual of Systematic Bacteriology (9). Members of the genus Amycolatopsis typically produce a substrate mycelium which tends to fragment into squarish elements, aerial hyphae, which when formed have the capacity to differentiate into squarish to oval fragments or spore-like structures, meso-diaminopimelic acid, arabinose and galactose in the wall peptidoglycan (wall chemotype IV; 12), major amounts of iso- and anteiso- fatty acids, a type II phospholipid pattern sensu Lechevalier et a1. (13), predominant amounts of di- and tetrahydrogenated menaquinones with nine isoprene units, and DNA rich in guanine plus cytosine. In contrast, to Nocardia strains organisms classified in the genus Amycolatopsis lack mycolic acids. Additional species, namely Amycolatopsis azurea (11); Amycolatopsis fastidiosa (11) and Amycolatopsis methanolica (3), have been described primarily by chemical and morphological properties. It can be important to differentiate Amycolatopsis strains from related actinomycetes given their commercial importance (6) and possible role as opportunistic pathogens in compromised hosts (17). Amongst lipids, menaquinones (2-methyl-3-polyprenyl-l,4-naphthoquinones) have been shown to be useful markers in actinomycete systematics (8). Various combinations of di-, tetra- and hexahydrogenated menaquinones with nine isoprene units (i.e., MK-9 [H2 , H 4 , H 6 ]) have been reported to be the predominant isoprenologues of A. azurea, A fastidiosa (2, 7,11), A. mediterranei (1, 2, 14), A. methanolica (3), A. orientalis (2,14,15), A. rugosa (14) and A. sulphurea (14) though not all of these investigators presented detailed menaquinone profiles of their test strains. It needs to be stressed that menaquinone data need to be interpreted with care as the proportions of isoprenologues can be dramatically influenced by the stage of the growth cycle at which biomass is harvested (16). In the present study representative strains of validly described species of the genus Amycolatopsis were examined to systematically determine the value of menaquinone composition in the classification and identification of the genus. Methods Strains and cultivation. Cultures (Table 1) were maintained on Brain Heart Infusion (BHI) agar (Difco) at 4°C. The organisms were grown in shake flasks of BHI broth (Difco) for 7 days at 36°C. After checking for purity at maximum growth, organisms were killed with 1% formalin, harvested by centrifugation, washed with distilled water and freezedried. Extraction, purification and analysis of isoprenoid quinones. Menaquinones were extracted from dried biomass (ca. 50 mg) and purified as described by Collins et a1. (4). The purified menaquinones were examined by reverse phase partition chromatography using Merck HPTLC RP-18F254 (Art. 13724) reverse thin-layer plates and a polar developing mixture of acetone-water (99: 1 v/v)j separated components were detected by ultraviolet light at 254 nm (5). The menaquinone composition of the test strains was confirmed by mass spectrometry: recordings were made on a Finnigan MAT 212 instrument using a direct insertion probe, an ionizing voltage of 80 eV and a temperature range of 200-300 0C.
Menaquinone Patterns of Amycolatopsis Species
467
Table 1. Description and source of Amycolatopsis strains A. azurea DSM 43854*
A. fastidiosa DSM 43855"·
A. mediterranei ATCC 13685"
DSM 40501 * A. methanolica NCIB 11946* A. orientalis ATCC 19795"
DSM 43387"· DSM 43388 N875 N876 N878 N879
A. Henssen, Fachbereich Biologie der Phillips-Universitiit, Marburg! Lahn, Federal Republic of Germany (Pseudonocardia azurea); NRRL 11412; S Omura, AM 3696, soil, Japan; produces azureomycin A and B A.Henssen (Pseudonocardia fastidiosa); ATCC 31181; Chas. Pfizer Inc., FD 25028, soil, Egypt; produces A macrobicyclic peptide antibiotic Lepetit Laboratories, ME83/973 (Streptomyces mediterranei) , soil; produces rifamycin Lepetit Laboratories, ME83/973
J. G. Kuenen,
Laboratory of Microbiology, Technical University of Delft, The Netherlands, LMD 80.32 (Nocardia sp.) soil, New Guinea
E. B. Shirling, Ohio Wesleyan University, Delaware, Ohio, USA, ISP 5040 (Streptomyces orientalis); R.Pittinger, M43-05865, soil, Asia; produces vancomycin E. B. Shirling, ISP 5040 M. Goodfellow, N858; R. E. Gordon, IMRU 1390; J. Scharfen, W113/66 R. E. Gordon, Rutgers University, New Jersey, USA; IMRU 1311 (Nocardia-Streptomyces group); M. P. Lechevalier, FT 19; clinical speCimen R. E. Gordon, IMRU 1409 (Nocardia brasiliensis); J. Scharfen, AK 4348; clinical specimen R. E. Gordon, IMRU 1592 (Micropolyspora sp.); L. K. Georg, W2101; soil, Sudan R. E. Gordon, IMRU 1601 (Micropolyspora sp.); L. K. Georg, W2156; soil, Sudan
A.orientalis subsp. lurida DSM 43134* L.Ettlinger, LBG A 3091 (Nocardia lurida); R.Hiitter, ETH 24315; W.E.Grundy, NA 3-TE-19; produces ristocetin A and B A. rugosa ATCC 43014" A. sulphurea ATCC 27624"
M. P. Lechevalier, Rurgers University, New Brunswick, New Jersey, USA, IMRU 3760; R. E. Gordon; C. di Marco; rumen of cow Abbott Laboratories, M-319 (Nocardia sulphurea); garden soil; produces chelocardin
" Type strain ATCC, American Type Culture Collection, Rockville, Maryland, USA DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Federal Republic of Germany
468
A. F. Yassin, K. P. Schaal, H. Brzezinka, M. Goodfellow, and G. Pulverer
Results and Discussion Components that co-chromatographed with vitamin K were the only isoprenoid quinones detected in the lipid extracts of the test strains. The most intense peaks in the mass spectra of the menaquinones occurred at mle 187 and 225 and were derived from the naphthoquinone nucleus. The mass spectra in the high mass region contained strong peaks corresponding to molecular ions (M+) with smaller peaks at M+ -15 corresponding to the loss of a methyl group from the molecular ions. The results of the mass spectral analyses are shown in Table 2. These results are in good agreement with those from reverse phase partition thin-layer chromatography. However, the isoprenologues MK-8 (H6) and MK-9 (H4 ) as well as MK-8 (~), MK-9 and MK-9 (Wg ), MK-10 (H6) were only separated by mass spectrometry as they migrated as a single band on reverse phase d.c. The detection of various combinations of di-, tetra- and hexahydrogenated menaquinones with nine isoprene units as the predominant isoprenologues of Amycolatopsis strains is in line with earlier investigations (1, 2, 3, 4, 7, 11, 14, 15). Strains of A. orientalis from a numerically circumscribed taxospecies (10) can be divided into three subgroups on the basis of the predominant isoprenologue. Thus, some strains contained major proportions of MK-9 (~) and others either MK-9 (H2) or MK-9 (H6). It was especially interesting that the type strain of A. orientalis obtained from the ATCC contained MK-9 (H 2 ) as the predominant component whereas the corresponding organism from the DSM was characterised by major proportions of MK-9 (H6). These results are not inconsistent with earlier findings as the type strain of A. orientalis has been reported to contain either predominant amounts of MK-9 (H6) (2) or major proportions of MK-9 (H2' H 4 ) (14). Similarly, discordent results were obtained with the two A. mediterranei strains; the type strain of this organism had previously been reported to contain MK-9 (~) (7, 15) or MK-9 (H2, H 4 ) (11), one strain contain MK-9 (H2 ) the other MK-9 (H6). However, the detection of major proportions of MK-9 (H2) in the type strains of A. rugosa and A. sulphurea, and MK-9 (~) in A. methanolica, is in good agreement with previous findings (3, 14). The results of this and earlier studies suggest that the menaquinone composition of Amycolatopsis strains is sensitive to the stage in the growth cycle from which biomass is taken. It has already been shown (16) that the menaquinone composition of a representative strain of Streptomyces cyaneus is markedly influenced by culture age. In contrast, Ruan et al. (15) found that the major and minor menaquinone composition of Actinoplanes aurantiacus, Amycolatopsis (Pseudonocardia) azurea and Streptomyces griseus were neither media nor age dependent though they did note some changes in the relative proportions of isoprenologues in biomass taken at different stages of the growth cycle. It is clear from this and earlier studies that the menaquinone profiles of Amycolatopsis strains need to be critically examined if a distinction is to be drawn between pattern and vigour differences. It is also evident that additional studies are needed to determine the relationship between menaquinone composition and the growth cycle in organisms like Amycolatopsis and Streptomyces. Nevertheless, the detection of major proportions of hydrogenated menaquinones with nine isoprene units serves to distinguish Amycolatopsis strains from many other actinomycetes, notably those belonging to related genera such as Amycolata and Pseudonocardia (8).
w
N
+
A. sulphurea ATCC 27624
+ +
+
+
+
+
+
(784)
H6 (786)
H2
+++
+++
+++ +++
+
+++
++
+++
+
+
+++
+ + ++ +++ +++ +++ +++
+++
+ +
++
++
(788)
H4
MK-9
+++
+
+++ +++
+
+++
+++
+++
(790)
H6
+ +
+
(792)
H4
+ +
+ +
+
(858)
H6
MK-10
(856)
H4
" Type strain +++, the main component of each series; ++, components greater than 50% of the main peak; +, other significant components
+
A. rugosa ATCC 43014*
+
++
+
+ + ++
+
+
A.orientalis subsp. lurida DSM 43134*
A.orientalis subsp. orientalis ATCC 19795" DSM 43387" DSM 43388 N875 N876 N878 N879
A. methanolica NCIB 11946'
A. mediterranel ATCC 13685" DSM 40501 "-
+
(722)
H6
+
(720)
H4
MK-8
+
(718)
H2
A. fastidiosa DSM 43855~'
(650)
H2
MK-7
A.aurea DSM 43854*
Menaquinone isoprenologue Degree of hydrogenation Microorganisms m/e M+
Table 2. Peaks corresponding to molecular ions (M+) in the mass spectra of menaquinones from Amycolatopsis strains
"'c'"
\0
~
C1\
~
'"D.
'"0
Vl
]. en
0
...0;-
n 0
'<
>3
'"'"'
0
'"
'""'en
;::;
"'
'"<:J
(1)
'"
S c
.D
2:::: (1)
470
A. F. Yassin, K. P. Schaal, H. Brzezinka, M. Goodfellow, and G. Pulverer
References
1. Alderson, G., M. Goodfellow, E. M. H. Wellington, S. T. Williams, S. M. Minnikin, and D. E. Minnikin: Chemical and numerical taxonomy of Nocardia mediterranei Zbl. Bakt. Suppl. 11 (1981) 39-46 2. Alderson, G., M. Goodfellow, and D. E. Minnikin: Menaquinone composition in the classification of Streptomyces and other sporoactinomycetes. J. gen. Microbiol. (1985) 1671-1679 3. De Boer, L., L. Dijkhuizen, G. Grobben, M. Goodfellow, E. Stackebrandt,J. H. Parlett, D. Whitehead, and D. Witt: Amycolatopsis methano/ica sp. nov., a facultatively methylotrophic actinomycete. Int. J. system. Bact. 4. Collins, M. D., T. Pirouz, M. Goodfellow, and D. E. Minnikin: Distribution of menaquinones in actinomycetes and corynebacteria. J. gen. Microbiol. 100 (1977) 221-230 5. Collins, M. D., H. N. Shah, and D. E. Minnikin: A note on the separation of natural mixtures of bacterial menaquinones using reverse phase thin-layer chromatography. J. appl. Bact. 48 (1980) 277-282 6. Embley, T. M.: The family Pseudonocardiaceae. In: The Prokaryotes, second edition, eds. A. Balows, H. G. Truper, M. Dworkin, W. Harder, and K. H. Schleifer. SpringerVerlag, New York (in press) 7. Embley, T. M., A. G. O'Donnell,]. Rostron, and M. Goodfellow: Chemotaxonomy of wall chemotype IV actinomycetes which lack mycolic acids. J. gen. Microbiol. 134 (1988) 953-960 8. Goodfellow, M.: Suprageneric classification of actinomycetes. In: Bergey's Manual of Systematic Bacteriology, Volume 4, pp. 2333-2339, eds. S. T. Williams, M. E. Sharpe, and]. G. Holt. Williams & Wilkins, Baltimore (1989) 9. Goodfellow, M. and M. P. Lechevalier: Genus Nocardia Trevisan 1889, 9 AL • In: Bergey's Manual of Systematic Bacteriology, Volume 4, pp. 2350-2361, eds. S. T. Williams, M. E. Sharpe. and]. G. Holt. Williams & Wilkins, Baltimore (1989) 10. Goodfellow, M., ]. Lacey, M. Athalye, T. M. Embley, and T. Bowen: Saccharopolyspora gregorii and Saccharopolyspora hordei: Two new actinomycete species from fodder. J. gen. Microbiol. 135 (1989) 2125-2139 11. Henssen, A., H. W. Kothe, and R. M. Kroppenstedt: Transfer of Pseudonocardia azurea and "Pseudonocardia fastidiosa" to the genus Amycolatopsis, with emended species descriptions. Int. J. system. Bact. 37 (1987) 292-295 12. Lechevalier, M. P. and H. Lechevalier: Chemical composition as a criterion in the classification of aerobic actinomycetes. Int. J. system. Bact. 20 (1970) 435-443 13. Lechevalier, M. P., C. De Bievre, and H. A. Lechevalier: Chemotaxonomy of aerobic actinomycetes: phospholipid composition. Biochem. Syst. Ecol. 5 (1977) 249-260 14. Lechevalier, M. P., H. Prauser, D. P. Labeda, and ].-S. Ruan: Two new genera of nocardioform actinomycetes: Amycolata gen. nov. and Amycolatopsis gen. nov. Int. J. system. Bact. 36 (1986) 29-37 15. Ruan,]. S., L. Xiaotao, Z. Yamei, and S. Yanlin: Numerical classification and chemotaxonomy of actinoplanetes and nocardiae. In: Biology of Actinomycetes '88, pp. 221-226, eds. Y. Okami. T. Beppu, and H. Ogawara. Japan Scientific Societies Press, Tokyo (1988) 16. Saddler, G. S., M. Goodfellow, D. E. Minnikin, and A. G. O'Donnell: Influence of the growth cycle on the fatty acid and menaquinone composition of Streptomyces cyaneus NCIB 9616. J. appl. Bact. 60 (1986) 51-56 17. Schaal, K. P. and B. L. Beaman: Clinical significance of actinomycetes. In: The Biology of the Actinomycetes, pp. 389-424, Eds. M. Goodfellow, M. Mordarski, and S. T. Williams. Academic Press, London (1984) Dr. M. Goodfellow, Dept. of Microbiology, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K.
Menaquinone Patterns of Amycolatopsis Species A. F. YASSIN h , K. P. SCHAAL 2 , H. BRZEZINKA3, M. GOODFELLOW4 **, and G. PULVERER 1 Institut ftir Medizinische Mikrobiologie und Hygiene, Universitat zu Koln, D-5000 Koln 41 Institut ftir Medizinische Mikrobiologie und Immunologie der Universitat Bonn, D-5300 Bonn 1 3 Institut ftir Rechtsmedizin der Universitat Bonn, D-5300 Bonn 1 4 Department of Microbiology, The Medical School, Newcastle upon Tyne, United Kingdom * Permanent address: Faculty of Science, University of Tanta, Tanta, Egypt. Fellow of the Alexander v. Humboldt-Foundation, D-5300 Bonn 1
2
Received March 2, 1990 . Accepted in revised form June 18, 1990
Summary The menaquinones of representative strains of the actinomycete genus Amycolatopsis were examined by reverse phase thin-layer chromatography and mass spectrometry. Representatives of all seven validly described species contained various combinations of di-, tetra- and hexahydrogenated menaquinones with nine isoprene units as predominant isoprenologues. It seems likely that the variation in the predominant menaquinones merely reflects the stages from the growth cycle from which biomass was taken. The detection of major proportions of hydrogenated menaquinones with nine isoprene units serves to distinguish Amycolatopsis strains from most other actinomycetes, notably those belonging to related genera such as Amycolata and Pseudonocardia.
Zusammenfassung Von reprasentativen Vertretern des Aktinomyzetengenus Amycolatopsis wurden ihre Menaquinone mit Hilfe der reversen Dtinnschichtchromatographie und der Massenspektrometrie bestimmt. Stamme aller 7 akzeptierten Spezies enthielten unterschiedliche Kombinationen von Di-, Tetra- und Hexahydrogen-menaquinonen mit 9 Isopreneinheiten als tiberwiegendes Isoprenolog. Die Unterschiede der vorherrschenden Menaquinone scheinen den verschiedenen Stufen des Wachstumszyklus zu entsprechen. Der Nachweis von hauptsachlichen Anteilen an hydrierten Menaquinonen mit 9 Isopreneinheiten kann zur Abgrenzung von Amycolatopsis-Stammen von den meisten tibrigen Aktinomyzeten dienen, speziell von Angehorigen der verwandten Genera Amycolata und Pseudonocardia.
* * Corresponding author
466
A. F. Yassin, K. P. Schaal, H. Brzezinka, M. Goodfellow, and G. Pulverer
Introduction The genus Amycolatopsis (14) was proposed for actinomycetes classified as Nocardia mediterranei, Nocardia orienta lis, Nocardia rugosa and Nocardia sulphurea; species cited as incertae sedis in the current edition of Bergey's Manual of Systematic Bacteriology (9). Members of the genus Amycolatopsis typically produce a substrate mycelium which tends to fragment into squarish elements, aerial hyphae, which when formed have the capacity to differentiate into squarish to oval fragments or spore-like structures, meso-diaminopimelic acid, arabinose and galactose in the wall peptidoglycan (wall chemotype IV; 12), major amounts of iso- and anteiso- fatty acids, a type II phospholipid pattern sensu Lechevalier et a1. (13), predominant amounts of di- and tetrahydrogenated menaquinones with nine isoprene units, and DNA rich in guanine plus cytosine. In contrast, to Nocardia strains organisms classified in the genus Amycolatopsis lack mycolic acids. Additional species, namely Amycolatopsis azurea (11); Amycolatopsis fastidiosa (11) and Amycolatopsis methanolica (3), have been described primarily by chemical and morphological properties. It can be important to differentiate Amycolatopsis strains from related actinomycetes given their commercial importance (6) and possible role as opportunistic pathogens in compromised hosts (17). Amongst lipids, menaquinones (2-methyl-3-polyprenyl-l,4-naphthoquinones) have been shown to be useful markers in actinomycete systematics (8). Various combinations of di-, tetra- and hexahydrogenated menaquinones with nine isoprene units (i.e., MK-9 [H2 , H 4 , H 6 ]) have been reported to be the predominant isoprenologues of A. azurea, A fastidiosa (2, 7,11), A. mediterranei (1, 2, 14), A. methanolica (3), A. orientalis (2,14,15), A. rugosa (14) and A. sulphurea (14) though not all of these investigators presented detailed menaquinone profiles of their test strains. It needs to be stressed that menaquinone data need to be interpreted with care as the proportions of isoprenologues can be dramatically influenced by the stage of the growth cycle at which biomass is harvested (16). In the present study representative strains of validly described species of the genus Amycolatopsis were examined to systematically determine the value of menaquinone composition in the classification and identification of the genus. Methods Strains and cultivation. Cultures (Table 1) were maintained on Brain Heart Infusion (BHI) agar (Difco) at 4°C. The organisms were grown in shake flasks of BHI broth (Difco) for 7 days at 36°C. After checking for purity at maximum growth, organisms were killed with 1% formalin, harvested by centrifugation, washed with distilled water and freezedried. Extraction, purification and analysis of isoprenoid quinones. Menaquinones were extracted from dried biomass (ca. 50 mg) and purified as described by Collins et a1. (4). The purified menaquinones were examined by reverse phase partition chromatography using Merck HPTLC RP-18F254 (Art. 13724) reverse thin-layer plates and a polar developing mixture of acetone-water (99: 1 v/v)j separated components were detected by ultraviolet light at 254 nm (5). The menaquinone composition of the test strains was confirmed by mass spectrometry: recordings were made on a Finnigan MAT 212 instrument using a direct insertion probe, an ionizing voltage of 80 eV and a temperature range of 200-300 0C.
Menaquinone Patterns of Amycolatopsis Species
467
Table 1. Description and source of Amycolatopsis strains A. azurea DSM 43854*
A. fastidiosa DSM 43855"·
A. mediterranei ATCC 13685"
DSM 40501 * A. methanolica NCIB 11946* A. orientalis ATCC 19795"
DSM 43387"· DSM 43388 N875 N876 N878 N879
A. Henssen, Fachbereich Biologie der Phillips-Universitiit, Marburg! Lahn, Federal Republic of Germany (Pseudonocardia azurea); NRRL 11412; S Omura, AM 3696, soil, Japan; produces azureomycin A and B A.Henssen (Pseudonocardia fastidiosa); ATCC 31181; Chas. Pfizer Inc., FD 25028, soil, Egypt; produces A macrobicyclic peptide antibiotic Lepetit Laboratories, ME83/973 (Streptomyces mediterranei) , soil; produces rifamycin Lepetit Laboratories, ME83/973
J. G. Kuenen,
Laboratory of Microbiology, Technical University of Delft, The Netherlands, LMD 80.32 (Nocardia sp.) soil, New Guinea
E. B. Shirling, Ohio Wesleyan University, Delaware, Ohio, USA, ISP 5040 (Streptomyces orientalis); R.Pittinger, M43-05865, soil, Asia; produces vancomycin E. B. Shirling, ISP 5040 M. Goodfellow, N858; R. E. Gordon, IMRU 1390; J. Scharfen, W113/66 R. E. Gordon, Rutgers University, New Jersey, USA; IMRU 1311 (Nocardia-Streptomyces group); M. P. Lechevalier, FT 19; clinical speCimen R. E. Gordon, IMRU 1409 (Nocardia brasiliensis); J. Scharfen, AK 4348; clinical specimen R. E. Gordon, IMRU 1592 (Micropolyspora sp.); L. K. Georg, W2101; soil, Sudan R. E. Gordon, IMRU 1601 (Micropolyspora sp.); L. K. Georg, W2156; soil, Sudan
A.orientalis subsp. lurida DSM 43134* L.Ettlinger, LBG A 3091 (Nocardia lurida); R.Hiitter, ETH 24315; W.E.Grundy, NA 3-TE-19; produces ristocetin A and B A. rugosa ATCC 43014" A. sulphurea ATCC 27624"
M. P. Lechevalier, Rurgers University, New Brunswick, New Jersey, USA, IMRU 3760; R. E. Gordon; C. di Marco; rumen of cow Abbott Laboratories, M-319 (Nocardia sulphurea); garden soil; produces chelocardin
" Type strain ATCC, American Type Culture Collection, Rockville, Maryland, USA DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Federal Republic of Germany
468
A. F. Yassin, K. P. Schaal, H. Brzezinka, M. Goodfellow, and G. Pulverer
Results and Discussion Components that co-chromatographed with vitamin K were the only isoprenoid quinones detected in the lipid extracts of the test strains. The most intense peaks in the mass spectra of the menaquinones occurred at mle 187 and 225 and were derived from the naphthoquinone nucleus. The mass spectra in the high mass region contained strong peaks corresponding to molecular ions (M+) with smaller peaks at M+ -15 corresponding to the loss of a methyl group from the molecular ions. The results of the mass spectral analyses are shown in Table 2. These results are in good agreement with those from reverse phase partition thin-layer chromatography. However, the isoprenologues MK-8 (H6) and MK-9 (H4 ) as well as MK-8 (~), MK-9 and MK-9 (Wg ), MK-10 (H6) were only separated by mass spectrometry as they migrated as a single band on reverse phase d.c. The detection of various combinations of di-, tetra- and hexahydrogenated menaquinones with nine isoprene units as the predominant isoprenologues of Amycolatopsis strains is in line with earlier investigations (1, 2, 3, 4, 7, 11, 14, 15). Strains of A. orientalis from a numerically circumscribed taxospecies (10) can be divided into three subgroups on the basis of the predominant isoprenologue. Thus, some strains contained major proportions of MK-9 (~) and others either MK-9 (H2) or MK-9 (H6). It was especially interesting that the type strain of A. orientalis obtained from the ATCC contained MK-9 (H 2 ) as the predominant component whereas the corresponding organism from the DSM was characterised by major proportions of MK-9 (H6). These results are not inconsistent with earlier findings as the type strain of A. orientalis has been reported to contain either predominant amounts of MK-9 (H6) (2) or major proportions of MK-9 (H2' H 4 ) (14). Similarly, discordent results were obtained with the two A. mediterranei strains; the type strain of this organism had previously been reported to contain MK-9 (~) (7, 15) or MK-9 (H2, H 4 ) (11), one strain contain MK-9 (H2 ) the other MK-9 (H6). However, the detection of major proportions of MK-9 (H2) in the type strains of A. rugosa and A. sulphurea, and MK-9 (~) in A. methanolica, is in good agreement with previous findings (3, 14). The results of this and earlier studies suggest that the menaquinone composition of Amycolatopsis strains is sensitive to the stage in the growth cycle from which biomass is taken. It has already been shown (16) that the menaquinone composition of a representative strain of Streptomyces cyaneus is markedly influenced by culture age. In contrast, Ruan et al. (15) found that the major and minor menaquinone composition of Actinoplanes aurantiacus, Amycolatopsis (Pseudonocardia) azurea and Streptomyces griseus were neither media nor age dependent though they did note some changes in the relative proportions of isoprenologues in biomass taken at different stages of the growth cycle. It is clear from this and earlier studies that the menaquinone profiles of Amycolatopsis strains need to be critically examined if a distinction is to be drawn between pattern and vigour differences. It is also evident that additional studies are needed to determine the relationship between menaquinone composition and the growth cycle in organisms like Amycolatopsis and Streptomyces. Nevertheless, the detection of major proportions of hydrogenated menaquinones with nine isoprene units serves to distinguish Amycolatopsis strains from many other actinomycetes, notably those belonging to related genera such as Amycolata and Pseudonocardia (8).
w
N
+
A. sulphurea ATCC 27624
+ +
+
+
+
+
+
(784)
H6 (786)
H2
+++
+++
+++ +++
+
+++
++
+++
+
+
+++
+ + ++ +++ +++ +++ +++
+++
+ +
++
++
(788)
H4
MK-9
+++
+
+++ +++
+
+++
+++
+++
(790)
H6
+ +
+
(792)
H4
+ +
+ +
+
(858)
H6
MK-10
(856)
H4
" Type strain +++, the main component of each series; ++, components greater than 50% of the main peak; +, other significant components
+
A. rugosa ATCC 43014*
+
++
+
+ + ++
+
+
A.orientalis subsp. lurida DSM 43134*
A.orientalis subsp. orientalis ATCC 19795" DSM 43387" DSM 43388 N875 N876 N878 N879
A. methanolica NCIB 11946'
A. mediterranel ATCC 13685" DSM 40501 "-
+
(722)
H6
+
(720)
H4
MK-8
+
(718)
H2
A. fastidiosa DSM 43855~'
(650)
H2
MK-7
A.aurea DSM 43854*
Menaquinone isoprenologue Degree of hydrogenation Microorganisms m/e M+
Table 2. Peaks corresponding to molecular ions (M+) in the mass spectra of menaquinones from Amycolatopsis strains
"'c'"
\0
~
C1\
~
'"D.
'"0
Vl
]. en
0
...0;-
n 0
'<
>3
'"'"'
0
'"
'""'en
;::;
"'
'"<:J
(1)
'"
S c
.D
2:::: (1)
470
A. F. Yassin, K. P. Schaal, H. Brzezinka, M. Goodfellow, and G. Pulverer
References
1. Alderson, G., M. Goodfellow, E. M. H. Wellington, S. T. Williams, S. M. Minnikin, and D. E. Minnikin: Chemical and numerical taxonomy of Nocardia mediterranei Zbl. Bakt. Suppl. 11 (1981) 39-46 2. Alderson, G., M. Goodfellow, and D. E. Minnikin: Menaquinone composition in the classification of Streptomyces and other sporoactinomycetes. J. gen. Microbiol. (1985) 1671-1679 3. De Boer, L., L. Dijkhuizen, G. Grobben, M. Goodfellow, E. Stackebrandt,J. H. Parlett, D. Whitehead, and D. Witt: Amycolatopsis methano/ica sp. nov., a facultatively methylotrophic actinomycete. Int. J. system. Bact. 4. Collins, M. D., T. Pirouz, M. Goodfellow, and D. E. Minnikin: Distribution of menaquinones in actinomycetes and corynebacteria. J. gen. Microbiol. 100 (1977) 221-230 5. Collins, M. D., H. N. Shah, and D. E. Minnikin: A note on the separation of natural mixtures of bacterial menaquinones using reverse phase thin-layer chromatography. J. appl. Bact. 48 (1980) 277-282 6. Embley, T. M.: The family Pseudonocardiaceae. In: The Prokaryotes, second edition, eds. A. Balows, H. G. Truper, M. Dworkin, W. Harder, and K. H. Schleifer. SpringerVerlag, New York (in press) 7. Embley, T. M., A. G. O'Donnell,]. Rostron, and M. Goodfellow: Chemotaxonomy of wall chemotype IV actinomycetes which lack mycolic acids. J. gen. Microbiol. 134 (1988) 953-960 8. Goodfellow, M.: Suprageneric classification of actinomycetes. In: Bergey's Manual of Systematic Bacteriology, Volume 4, pp. 2333-2339, eds. S. T. Williams, M. E. Sharpe, and]. G. Holt. Williams & Wilkins, Baltimore (1989) 9. Goodfellow, M. and M. P. Lechevalier: Genus Nocardia Trevisan 1889, 9 AL • In: Bergey's Manual of Systematic Bacteriology, Volume 4, pp. 2350-2361, eds. S. T. Williams, M. E. Sharpe. and]. G. Holt. Williams & Wilkins, Baltimore (1989) 10. Goodfellow, M., ]. Lacey, M. Athalye, T. M. Embley, and T. Bowen: Saccharopolyspora gregorii and Saccharopolyspora hordei: Two new actinomycete species from fodder. J. gen. Microbiol. 135 (1989) 2125-2139 11. Henssen, A., H. W. Kothe, and R. M. Kroppenstedt: Transfer of Pseudonocardia azurea and "Pseudonocardia fastidiosa" to the genus Amycolatopsis, with emended species descriptions. Int. J. system. Bact. 37 (1987) 292-295 12. Lechevalier, M. P. and H. Lechevalier: Chemical composition as a criterion in the classification of aerobic actinomycetes. Int. J. system. Bact. 20 (1970) 435-443 13. Lechevalier, M. P., C. De Bievre, and H. A. Lechevalier: Chemotaxonomy of aerobic actinomycetes: phospholipid composition. Biochem. Syst. Ecol. 5 (1977) 249-260 14. Lechevalier, M. P., H. Prauser, D. P. Labeda, and ].-S. Ruan: Two new genera of nocardioform actinomycetes: Amycolata gen. nov. and Amycolatopsis gen. nov. Int. J. system. Bact. 36 (1986) 29-37 15. Ruan,]. S., L. Xiaotao, Z. Yamei, and S. Yanlin: Numerical classification and chemotaxonomy of actinoplanetes and nocardiae. In: Biology of Actinomycetes '88, pp. 221-226, eds. Y. Okami. T. Beppu, and H. Ogawara. Japan Scientific Societies Press, Tokyo (1988) 16. Saddler, G. S., M. Goodfellow, D. E. Minnikin, and A. G. O'Donnell: Influence of the growth cycle on the fatty acid and menaquinone composition of Streptomyces cyaneus NCIB 9616. J. appl. Bact. 60 (1986) 51-56 17. Schaal, K. P. and B. L. Beaman: Clinical significance of actinomycetes. In: The Biology of the Actinomycetes, pp. 389-424, Eds. M. Goodfellow, M. Mordarski, and S. T. Williams. Academic Press, London (1984) Dr. M. Goodfellow, Dept. of Microbiology, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K.