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Chemotaxonomy of aerobic Actinomycetes: Phospholipid composition
Biochemical Systematics and Ecology, 1977, Vol. 5, pp. 249 to 260. PergamonPress.Printedin England.
Chemotaxonomy of Aerobic Actinomycetes: Phospholipid Composition MARY P. LECHEVALIER, CLAUDE DE BIEVRE* and HUBERT LECHEVALIER Waksman Institute of Microbiology, Rutgers, The State University, P.O. Box 759, Piscataway, NJ 08854, USA Key Word Index--Actinoplanaceae; Micromonosporaceae; Mycobacteriaceae; Nocardiaceae; cetaceae; Thermomonosporaceae; Actinomycetales; phospholipids; fatty acids; chemotaxonomy.
Streptomy-
A b s t r a c t - - A survey of the phospholipid composition of 97 strains representing 20 genera of the Actinomycetales showed that five groups could be distinguished on the basis of the presence or absence of certain nitrogenous phospholipids. Phospholipid type PI (no nitrogenous phospholipids) is characteristic of the genera Actinomadura (madurae, pelletieri), Corynebacterium, Microtetraspora and Nocardioides. Actinomycetes of Type PII contain only one nitrogenous phospholipid, phosphatidyl ethanolamine. These include members of the genera Actinoplanes,
Chainia, Dactylosporangium, Microellobosporia, Micromonospora, Micropolyspora (brevicatena), Mycobacterium, Nocardia (all species examined but autotrophica), Streptomyces and Streptoverticillium. Phospholipid pattern type PIII (characteristic phospholipid, phosphatidyl choline) was found in Actinomadura (dassonvillei), Micropolyspora (faeni), Nocardia (autotrophica), and Pseudonocardia. Actinomycetes having a type P IV pattern contain an unknown, previously undescribed phospholipid containing glucosamine (GluNU) which was found to be characteristic of members of the genera Intrasporangium, Microbispora and Streptosporangium. Actinomycetes of type PV contain phosphatidyl glycerol in addition to GluNU and include members of the genera Promicromonospora and Oerskovia. Other phospholipids found variably in all groups included phosphatidyl inositol, phosphatidyl inositol mannosides, phosphatidyl methylethanolamine, acyl phosphatidyl glycerol (APG) and diphosphatidyl glycerol (DPG). The fatty acids present in DPG (or APG when DPG was absent) may be species-specific. The chemical heterogeneity of the genera Actinomadura, Corynebacterium, Micropolyspora and Nocardia is discussed.
Introduction The chemistry of cell constituents of actinomycetes has played an increasingly important role in the taxonomy of these economically important, gram-positive branching bacteria [1]. Cell wall composition [2, 3], whole cell amino acids [4], whole cell sugars r5] and lipids such as mycolic acids r6-8] have enabled taxonomists to clarify relationships among different groups of actinomycetes and has led to the proposal of at least five new genera [9-13]. The phospholipid composition of actinomycetes has been the subject of a number of reports [14-48], but no systematic attempt has been made to apply this criterion to the taxonomy of broad groups of actinomycetes. In this paper we wish to report on the phospholipid composition of 97 strains representing species of 20 genera of actinomycetes. Results
More than ninety strains of actinomycetes representing twenty genera (Table 1) were *Present address: Service de Mycologie, Institut Pasteur, Paris, France.
analyzed for their phospholipid composition (Table 2). The phospholipids were identified on the basis of (a) their relative mobility on TLC and reactions with ninhydrin, Dragendorff, anisealdhyde, Schiff and molybdate reagents; (b) the mobility of their phosphate esters produced by mild alkaline hydrolysis versus the phosphate esters from authentic compounds; and (c) the identity of the sugars, polyols and amino acids and alcohols yielded by acid hydrolysis. The fatty acids of the diphosphatidyl glycerols (DPG) or acyl phosphatidyl glycerols (where DPG was lacking) were also determined (Table 3). The fatty acids were identified byGLCon the basis of the Rt's of their methyl esters (FAMES) on polar and non-polar columns versus authentic compounds and, in the case of unsaturated fatty acids, by the Rt's of the methoxy derivatives i49]. Quantitation was carried out by measuring the areas under the various peaks of the resolved FAMES by multiplying the heights of the peaks by the widths at half height. Since no authentic material is available commercially, heptadecenoic acid, the major constituent of the fatty acids of the cardiolipins
(Received 18 April 1977; received for publication 18 July 1977) 249
250
MARY P. LECHEVALIER, CLAUDE DE BIEVRE AND HUBERT LECHEVALIER
TABLE 1. STRAINS USED IN THIS STUDY Taxon Actinomadura dassonvillei (Brocq-Rousseau) Lechevalier and Lechevalier
madurae (Vincent) Lechevalier and Lechevalier
pelletieri (Laveran) Lechevalier and Lechevalier sp.
ActinoManes missouriens/s Couch rectilineatus Lechevalier and Lechevalier spp.
Chamia nigra Thirumalachar olivacea Thirumalachar and Sukapure Corynebacterium aquaticum Liefson boris Bergey et al. diphtheriae (Kruse) Lehmann and Neumann Dactylosporangium aurantiacum Thiemann, Pagani and Beretta thailandense Thiemann. Pagani and Beretta Intraepotangium calvum Kalakoutskii, Kirillova and Krassil'nikov Microbispora amethystogenes Nonomura and Ohara rosea Nonomura and Ohara Microellobosporia flavea Cross, Lechevalier and Lechevalier violacea Tsyganov, Zhukova and Timofeeva Micromonospora coerulea Jensen narashinoensis (Shidara) (subsp. of) Arai and Kuroda purpurea Luedemann and Brodsky purpureochtomogenes (Waksman and Curtis) Luedemann Micropolyspora brevicatena Lechevalier, Solotorovsky and McDermont faeni Cross, Maciver and Lacey sp. Microtetraspora viridie var. intermedia Nonomura and Ohara Mycobacterium abcessus Moore and Frerichs
boris Karlson and Lessel fortuitum Cruz smegmatis (Trevisan) Lehmann and Neumann tuberculosis (Zopf) Lehmann and Neumann sp.
Nocardia aerocolonigenes (Shinobu and Kawato) Pridham amarae Lechevalier and Lechevalier asteroides (Eppinger) 81anchard autotrophica (Takamiya and Tubaki) Hirsch brasiliensis (Lindberg) (Castellani and Chalmers)
Strain No.* 1509 1575 I 1371 (LL-Botl 1) I 1328 (LL-L 13) I 1330 (LL-Cross, Fil) N 434 1507 1780 1953 LL-S4 LL-Sal 1 I 408 1610 1778 LL-N15
Sourcet
T. Cross
1824 (LL-E3-15) 13918 (LL-7-10) LL-K30 LL-Z20 LL- P 128 LL-15-7 LL-Su 12 LL-V17576
R. S. Sukapure E. Tejera
B 2252 NIRD 128 A 11913
D. Weaver R. Smith
A 23491 LL-9-41A LL-7 KIP
L. V. Katakoutskii
A 15740 (LL-37-6) 1864 13748 A 15839 (I 3858) LL-795 LL-V12328 A 10028 LL-817C2 NRRL B943
A. Kuchaeva E. T~era G. Luedemann
11086 LL-A 91 LL-V4066 LL-Y 39
P. H. Gregory P. H. Gregory
LL-Mt-2
H. Nonomura
A 19977 13061 A 9834 1788 11000 1375 1455 H37Ra NCTC 4524t 1550 A 27808 (I 3960; LL-Se 6) LL-Se 3 LL-Se 120 A 3318 A 19247 (I 727) 1433N A 19727 13520 1774B 11093
251
CHEMOTAXONOMY OF AEROBIC ACTINOMYCETES: PHOSPHOLIPID COMPOSITION TABLE 1--continued Taxon caviae (Erikson) Gordon and Mihm
Strain No. • I 1203
Sourcet
I 1934
See Mycobacterium sp, I 1107 I 1149 I 1240 I 3639 I W21
farcinica Travisan orientalis (Pittinger and Brigham) Pridham and Lyons rhodochrous (Overbeck) Lechevalier, Horan and Lachevalier
N 1621
LL- Mil 15 Nocardioides a/bus Prauser
IMET 7801 IMET 7807
Oemkovia turbata (Erikson) Prausero Lechevalier and Lechevalier
I 762 SSlC 891 A 27402 (I 3959; LL-G 62) LL-St/3P
xanthineolytica Lechevalier Pomicromonospora citrea (Krasil'nikov et al.) spp. Pseudonocardia thermophila Henssen Streptom¥ces a/bus (Rossi-Doria) Waksman and Hanrici fradiae (Waksman and Curtis) Waksman and Henrici griseus (Krainsky) Waksman and Henrici rimosus Sobin et al. somaliensis (Brumpt) Waksman and Henrici Streptosporangium roseum Couch sp. (rubrum type) (amethystoganes-type) sp. Streptoverticillium alboreticuli (Nakazawa) Locci et al. rubrireticuli (Waksman and Henrici) Baldacci • A=ATCC=American Type Culture Collection, Rockville, Md., U.S.A. B=CDC=Center for Disease Control, Atlanta, Ga., U.S.A. I=lMRU=Waksman Institute of Microbiology, Rutgers University. IMET=lnstitute f~r Mikrobiologie und Experimentelle Therapie, Jena, DDR. LL= Lechevaliers' Collection. N =NCTC=National Collection of Type Cultures, U.K. NIRD=National Institute for Research in Dairying, U.K. NRRL=Northern Regional Research Laboratory, Peoria, I11.,U.S.A. SSIC=Statens Serum Institute Collection, Denmark. t The source is given for LL strains not isolated in the Lechevaliars' laboratory. $ Formerly Nocardia farcinica.
of Streptosporangium and Micromonospora strains, was identified on the basis of the log of the Rt of its methyl ester on G LC and the Rt of its monomethoxy derivative. Heptadecenoic acid has never been reported in major amounts from actinomycetes, but occurs in other bacteria [50]. Strains belonging to 17 of the 20 genera contained phospholipids with nitrogenous constituents. The largest group contained only one major phospholipid of this type: phosphatidyl ethanolamine. This is referred to in Table 2 as phospholipid pattern type PII. Members of the genera Actinoplanes, Chainia, Dactylo-
sporangium, Microellobosporia, Micromono-
R. F. Lewis
LL-18 LL-G165 LL-G173
L. V. Kalakoutskii
LL-A18
A.Henssen
758 3004 3535 3475 3558 383 719 761 1298 A 12428 LL-J7 LL-9-26 LL-N6 LL-15-17 A 24213 LL-100-19
D. P. Stahly
spora, Micropo/yspora (brevicatena only), Mycobacterium, Nocardia (except autotrophica)o Streptomyces and Streptoverticillium showed this pattern. Another group contained phosphatidyl choline (type P III phospholipid pattern). Phosphatidyl methylethanolamine (PME) was also present in most strains falling into this category. Phosphatidyl cholinecontaining taxa included Actinomadura dassonvillei, Micropolyspora faeni and spo,Nocardia autotrophica and Pseudonocardia. The third and fourth groups were characterized by their content of certain, as yet unidentified, phospholipids containing glucosamine (GluNU). These include members of the genera Intrasporang-
252
MARY
TABLE
2--PHOSPHOLIPIDS*
OF
ACTINOMYCETES
P.
LECHEVALIER,
CLAUDE
DE
BIEVRE
AND
HUBERT
EXAMINED
Strain Taxon
No.
Actinomadura madurae
sp.
Actinoplanes missouriens/s rectilineatus
Chainia nigra olivacea Dactylosporangium aurantiacum thailandense Microellobosporia flavea violacea Micromonospora coerulea narashinoensis purpurea purpureochromogenes Micropolyspora brevicatena Mycobacterium abcessus bovis fortuitum 8megmatis var.
sp.
Nocardia aerocolonigenes amarae
PC
PE
.
GluNU
+
. tr.
+
+
.
.
.
.
.
+
+
+
.
.
.
.
.
+
+
+
.
.
.
.
.
+
408
+
+
.
.
.
.
.
+
610
+
+
.
778
+
+
.
.
.
.
.
N15
+
+
.
.
.
.
.
B2252
-
tr.
+
-
-
-
+
-
N 128
+
+
+
-
-
+
+
-
A11913
+
+
+
-
-
±
Mt
+
+
-
7801
±
+
+
-
-
7807
tr.
+
+
-
-
824
+
+
tr.
-
+
-
.
.
DPG
+
2
.
APG
+
1
.
PME
-
.
-
-
.
+
-
+
-
PA -
-
-
-
?
PI
-
-
-
±
-
-
+
-
-
+
.
.
.
.
-
+
.
.
.
.
-
-
+
-
-
hominis
-
+
+
-
3919
+
+
±
-
-
K30
+
+
-
-
+
-
-
+
Z20
±
+
-
-
+ §
-
-
+
-
-
P128
+
+
-
+
-
-
+
-
15-7
+
+
-
+
-
-
+
-
SU12
+
+
+§
-
-
+
-
V17676
+
+
+ §
-
-
+
-
A23491
±
+
9-41A
+
+
3858
±
+
795
+
+
V12328
tr.
+
A10026
±
±
817C2
+
B943
+
1086
+
A19977
±
+
-
-
+
-
+
-
-
+
-
-
+
-
-
+
-
-
+§
-
-
+
-
+
-
+
+
-
-
+
-
?
+
-
+
-
+
-
-
+
-
+
-
+
-
-
+
-
+
tr.
+
-
-
+
-
+
+
-
3061
+
+
-
9834
+
+
-
+
-
+
-
+
-
+
-
+
788
+
±
-
-
+
1000
+
+
-
-
+
+
-
-
+
-
-
+
-
375
+
+
-
+
-
-
+
455
+
+
-
+
-
-
+
-
H37Ra
+
+
-
+
-
-
+
-
4524
+
+
-
+
-
-
+
-
550
-
+
-
-
+ §
-
+
-
+
-
-
+
-
+
-
+
+
-
-
+
+
-
Se120
+
+
-
-
+
+
-
3318
+
+
-
-
+
+
-
19247
+
+
-
-
+
433N
+
+
-
-
+
brasiliensis
774B
+
-
+
-
+
1093
+
+
-
-
+
-
+
-
caviae
1203
+
+
-
-
+
-
+
-
1934
+
+
-
-
+
-
+
-
1107
tr.
+
-
-
+ §
-
1149
±
+
-
-
+§
-
+
-
1240
•
+
-
-
+
-
+
-
fradiae
-
-
-
+
Streptomyces albus
-
-
+
-
-
type
-
+
tr.
orientahs
?
+
Se3
rhodochrous
PA
Unknown$
-
27808
asteroides
Other
-
Ptl
spp.
tuberculosis
PG
± 1"
Sal
Corynebacterium aquaticum bovis diphtheriae Microtetraspora viridis v a r . intermedia Nocardiodes albus
PI
507
953
pe//etieri
Phospholipid PIMs
780
$4
LECHEVALIER
+
-
-
-
+
-
+
-
+
-
-
3639
+
+
-
-
+
-
+
-
W21
+
+
-
-
+
-
+
-
1621
+
+
-
-
+
-
+
-
Mi115
+
+
-
-
+
-
+
-
+
-
+
-
-
+
-
-
+
3004
+
+
-
-
+
3535
758
+
+
+
±
-
-
-
+
-
-
d
CHEMOTAXONOMY
TABLE
OF AEROBIC
Strain No.
griseus rimosus somahensis
Streptoverticillium alboreticuli rubrireticufi Actinomadura dassonvillei
253
COMPOSITION
PtMs
PI
PG
PC
PE
PME
APG
DPG
3475
+
+
-
-
3558 383
+ +
+ +
-
-
719 761 1298
+ + +
+ + +
-
-
+ + +
24213
+
+
-
-
+ §
100-19
+
+
-
-
+
-
N434
tr.
4-
+
4-
-
4-
+
tr.
509
+
+
+
+
-
tr.
+
575 1328
+ 4-
tr. 4-
+ +
+ 4-
-
tr. +
1330
tr.
4-
+
+
-
1371
-
tr.
+
+
-
A91 V4086 Y39
4-
+ + +
+ + +
+ + +
19727 3520
tr. +
+ +
± 4-
A18
4-
+
7 KIP
+
II
15740 864 3748
sp.
Nocardia autotrophica Pseudonocardia thermophila Intrasporangium calvum Microbispora amethystogenes rosea
Other
Unknown~t
+ §
-
+
-
-
+
+ § + §
-
+ +
-
-
-
+ + +
-
-
+
+
-
-
-
+
-
-
-
-
-
PA ?
-
+ 4-
+ +
-
PA? -
-
4-
+
4-
-
-
-
+
+
+
-
-
-
_
+§ + +
-
+ + +
_
PA _
+
+ +
tr.
+ +
+ +
-
-
-
-
+
+ {
+
+
_
_
_
+
-
-
+
-
+
+
-
-
+ 11 +11 +ll
+ + +
-
-
+ § +§ +§
-
+ + +
+ 4+
PA? -
-
A12428 J7
+ U +ll
+ 4-
-
-
+ § +
+ +
+ +
+ +
-
-
9-26
+11
+
-
-
+§
tr.
+
+
-
-
N6 15-17
+11
+
-
-
+§
+
+
+
-
-
+11
+
-
-
-
+
+
+
-
-
762 891
+ Jl
Phospholipid type
_
PIV
Streptosporangium roseum sp.
Oerskovia turbata
-
PV
xanthineolytica Promicromonospora citrea spp.
4-
+
-
-
-
-
A27402
+11 +11
+
+
-
+
+
-
-
-
St/3P
+11
+
+
-
-
18 G165
+ II
+
+
-
-
-
+ll
+
+
-
-
-
G173
+ll
+
+
-
4-
-
ium, Microbispora,
and Streptosporangium P I V ) a n d Oerskovia a n d Promicromonospora ( p h o s p h o l i p i d type PV). The PV types were differentiated from PIV's by their content of phosphatidyl glycerol. Members of only four genera contained no type
major amounts of nitrogen-containing phospholipids. T h e s e w e r e Actinomadura (madurae a n d pe/letieri), Corynebacterium, Microtetraspora a n d Nocardioides ( P h o s p h o l i p i d T y p e I ) .
+
-
PIMs =Phosphatidyl inositol mannosides PI =Phosphatidyl inositol PG = Phosphatidyl glycerol PC =Phosphatidyl choline PE =Phosphatidyl ethanolamine PME =Phosphatidyl methyl ethanolamine APG =Acyl phosphatidyl glycerol DPG Diphosphatidyl glycerol Glu-NU =Glucosamine-containing unknowns PA =Phosphatidic acid + =major component; 4- = m i n o r component; tr=trace; - =component absent. All minor fractions having too little material for identification. Phosphatidyl ethanotamine acylated with hydroxy fatty acids also present. May or may not be present. Cannot currently be separated from the unknown
(phospholipid
GluNU
PIll
Micropolyspora faeni
I" :1: § 11
PHOSPHOLIPID
2--continued
Taxon
•
ACTINOMYCETES:
4-
+
-
4-
-
+
-
+
+
-
+
+
+
-
-
+
+
+
-
-
+
+
+
-
-
+
+
+
-
-
glucosamine-containing
phospholipids.
As judged by phospholipid and fatty acids patterns, the following genera among those examined,
appear
to
be
inhomogeneous:
Actinomadura, Corynebacterium, Micropolyspora a n d Nocardia. T h i s c o m e s a s n o s u r p r i s e since there is other chemical evidence of their heterogeneity (summarized in Table 4). Although all the corynebacteria examined fall into the phospholipid pattern type PIo an examination of Table 2 shows there to be
254
MARY P. LECHEVALIER, CLAUDE DE BIEVRE AND HUBERT LECHEVALIER
TABLE 3. FATTY ACIDS FOUND IN ACTINOMYCETE PHOSPHOLIPIDS I. ANTEISO/ISO BRANCHED CHAIN FATTY ACIDS PRESENT ( >10% OF TOTAL FATTY ACIDS) A. Principal component branched chain fatty acid of anteisoiso series
Actinomadura (dassonvillei) Actinoplanes (missouriensi$; 2 spp.) Chainla Corynebacterium (aquaticum) Dactylosporangium Micropolyspora (faeni; sp.) Micromonospora (coerulea; echinospora) Microbispora Microellobosporia Nocardia (autotrophica; orientalis) Nocardioides (7807) Oerskovia Promicromonospora Pseudonocardia Streptomyces (albus fradiae; griseus; fimosus) Streptoverticillium
Phospholipid fatty acid Type 1
B. Principal component unsaturated fatty acid other than heptadecanoic Actinomadura (madurae; pelletieri; sp.) Actinoplanes (rectilineatus; 2 spp.)
Microtetraspora Microellobosporia (flavea) Nocardioides (7801) Streptomyces (somaliensis) Nocardia (aerocolonigenes)
Phospholipid fatty acid Type 2
C. Principal component heptadecanoic acid
Micromonospora (fusca; purpureochromogenes) Streptosporangium
Phospholipid fatty acid Type 3
II. ANTEISO-ISO FATTY ACIDS USUALLY NOT PRESENT; IF SO, < 10% OF TOTAL FATTY ACIDS
Actinomadura (madurae; pelletieri ) Corynebacterium (diphtheriae; bovis) Micropolyspora (brevtcatena) Mycobacterium Nocardia (amarae; asteroides; brasiliensis; caviae; rhodochrous)
Phospholipid fatty acid Type 4
TABLE 4, CHEMICAL HETEROGENEITY OF VARIOUS ACTINOMYCETE TAXA Taxon
Actinomadura dassonvillei madurae pelletieri Corynebacterium aquaticum boris diphtheriae Micropolyspora brevicatena faeni Nocardia amarae, asteroides brasiliensis, caviae rhodochrous aerocolonigenes orientalis autotrophica
Phospholipid pattern*
Whole cell sugar/cell wall typet
Mycolic acids
Phospholipid fatty acid pattern~t
P Ill PI PI
C/Ill B/Ill B/Ill
-
1 2 2
P I§ PI PI
-/VI A/IV A/tV
+ +
1 4 4
P II P III
A/IV A/IV
+ -
4 1
P II P II P II P III
A/IV C/Ill A/IV A/IV
+ -
4 2 1 1
• See Table 2. 1" See Lechevalier and Lechevalier [2]. ~t=See Table 3. §=See text.
important differences between the C. bovis and C. diphtheriae strains and the C. aquaticum strain; namely, lack of phosphatidyl inositol mannosides (PIMs) and APG in the latter. In certain genera where phospholipid composition is homogeneous, there is evidence that DPG (or APG) fatty acid patterns may be
species-specific. These include the taxa listed in Table 5. In the course of our studies some unusual types of phospholipids were discerned. As previously mentioned, phospholipids containing glucosamine were found in members of the genera Intrasporangium, Microbispora, Oersko-
CHEMOTAXONOMY OF AEROBIC ACTINOMYCETES: PHOSPHOLIPID COMPOSITION
via, Promicromonospora and Streptosporangium. These compounds also contain mannose and inositol and in some cases, glucose, and occur as 2-5 presumably similar compounds, the total number depending on the strain analyzed. These compounds (GluNU's) give a strong green color with the anisealdehyde reagent and a positive blue reaction with the phosphorus reagent of Dittmer and Lester. Only a slowly reacting faint peach color is produced with ninhydrin even on prolonged heating. In acid or base-containing solvent systems on TLC, the GluNU's are appreciably retarded in their migration relative to diphosphatidyl glycerol (DPG) or phosphatidyl ethanolamine compared to that in a neutral system. For example, in the neutral System 1 (see Experimental), the GluNU's from Streptosporangium sp. J7 appear as five spots with RDPG'Sof 0.45, 0.48, 0.62, 0.73 and 0.80. In the acid-containing System 2, only four spots are resolved having RDPG'Sof 0.1 4, 0.21, 0.31 and 0.39. Except for the faint reaction with ninhydrin,theGluNU's cannot bedistinguished, by TLC from phosphatidyl inositol mannosides (PIMs) and acylated PIMs [23, 24, 51, 52]. Even the phosphate esters of the GluNU,s migrate in the area of those of the PIM's on PC; thus, it is not known whether conventional PIM's are also present in the Glu NU-containing strains. Glucosamine was identified by relative mobility versus authentic standards on PC in two systems and reactions with ninhydrin (brown-purple), acid aniline phthalate (brown), ammoniacal silver nitrate and the Elson-Morgan reagent (red). Its identity was confirmed by degradation to arabinose by treatment with ninhydrin. It is released only on hydrolysis of the GluNU's with 6N HCI at 100 ° for 18 h; none is released with 3N H2SO, in 3 h at 100 ° or by either mild (0.025 N methanolic LiOH at 37 ° for 20 min) or strong (2N aqueous KOH at 100 ° for 30 min) alkaline hydrolysis. Glucosamine has only been reported as a phospholipid constituent in glucosaminyl phosphatidyl glycerol (GPG). GPG has previously been reported from Bacillus megaterium de Ban/ [53] and Pseudomonas ova/is Chester [54]. Our compounds are clearly different from GPG not only by their content of mannose and inositol, but the fact that their phosphate esters derived from mild alkaline hydrolysis are ninhydrin-negative whereas that from GPG is ninhydrin-positive [55]. Other unusual phospholipids that were encountered in this study have been identified
255
TABLE 5. SPECIES SPECIFIC FATTY ACID PATTERNS IN GENERA WHERE THE PHOSPHOLIPID COMPOSITION IS HOMOGENEOUS Phospholipid fatty Taxon acid pattern"
Actlno,olanea missouriensis and 2 spp, rectilineatus and 2 spp.
1 2
Microellobosporia
flavea putpurea
2 1
Micromonospora coerulea and echinosDora fusca and purpureochromogenes Nocardio/des albus (7807) a/bus (7801)
1 3 1 2
Streptomyces a/bus, fradiae
griseus, rimosus somaliensis
1 1 2
*See Table 3.
TABLE 6". GROUPING OF ACTINOMYCETES BASED ON PHOSPHOLIPID COMPOSITION I. CONTAIN NO NITROGENOUS PHOSPHOLIPIDS (Type PI) A. Contain only PIMs, PI end DPG. Actinomadura (madurae, pelletieri)
Microtetraspora B. Contain PIMs, PI, DPG, PG and APG's.
Corynebacter/um (bovis, dlphtheriae) C. Contain PIM, PI, PG and APG's.
Nocardioides D. Contain PG, and DPG, no PIMs, traces PI.
Corynebacterium aquat/cum II. CONTAIN NITROGENOUS PHOSPHOLIPIDS A. Contain PE, PIMs, PI and DPG (Type PII) i. Fatty acidst of Type 1
Chainia Dacty/osporangium Nocardia (oriental/s) S treptovertic///ium
Streptomyces (a/bus, frad/ae, griseu~ rimosus) ii. Fatty acids of Type 1 or 2
Actinoplanes Microellobosporia iii. Fatty acids of Type 2
Nocardia (aeroco/onigenes) Streptomyces (somaliensts) iv. Fatty acids of Type 1 or 3
Micromonospora v. Fatty acids of Type 4.
Micropolyspora (brevicatena) Mycobacteriurn Nocardia (amarae. asteroides, brasiliensis, caviae, orientalis, rhodochrous) B. Contain PC (PME, DPG, PIMs and PI variable) (Type PIll) i. Also contain PG and APGs
Actinomadura (dassonvillei)
ii. Also contain PG. No APG.
Micropo/yspora (faeni, sp.) iii. No APGs or PG. (or onlytraces of latter) Nocardia (autotrophica) Pseudonocardia C. Contain GluNU. PI and DPG. i. Contain PE or PME or both. No PG. (Type PIV)
Streptosporangium Microbispora
Intrasporangium
ii. Contain little to no PE or PME. Contain PG. (Type PV)
Oerskovia Promicromonospora "See Table 2 for abbreviations of phospholipids. tSee Table 3.
256
MARY P. LECHEVALIER, CLAUDE DE BIEVRE AND HUBERT LECHEVALIER
as acylated phosphatidyl glycerols (APGs). These were found in the following taxa: Actinomadura dassonvil/ei; Corynebacterium (bovis; diphtheriae); Nocardioides," and Promicromonospora. They are found only in organisms having phosphatidyl glycerol (PG) as a major component; however, not all PGcontaining strains formed APG. APGs had greater Rf's than PG on TLC plates and in a few cases replaced diphosphatidyl glycerol (DPG) altogether. In most cases both mono-acylated and diacylated PG were present. The latter chromatographed just in back of DPG on TLC plates in System 1; the mono-acylated form co-chromatographed with lyso DPG. In both casesglyceryl phosphoryl glycerol was obtained on mild deacylation and glycerol was the only major product from acid hydrolysis. Acylated phosphatidyl glycerols have previously been reported from Sa/mone//a typhimurium (Loeftier) Castellani Et Chalmers [56, 57], A/carlgenes aquamarinus (Zobell 8 U pham) Hendrie, Holding and Shewan [58], Arthrobacter marinus Cobet, Wirsen and Jones [58], various species of Vibrio Pacini [58] and Bifidobacterium Orla-Jensen [59]. Among the fatty acids which were found on deacylation of the DPG's (or APG's if no DPG was present) of the various actinomycetes studied, several deserve, special mention: (a) tubercu/ostearic acid (10-methyl-octadecanoic acid) was found in large amounts in strains of Actinomadura (madurae and pe//etieri), Corynebacterium bovis, Nocardia (amarae,asteroides,brasi/iens/s,caviae, rhodochrous but not aeroco/onigenes, autotrophica or orientalis) and Mycobacterium (all species examined except Mo abcessus). It was also found variably in Actinomadura dassonvi//ei and Nocardio/des. It was not present in the DPG's of C. diphtheriae or C. aquaticum. (b) Lower homo/ogs of tubercu/ostearic acid, previously reported in Microbispora [60], Micromonospora [37], and Streptornyces [1 9, 43], we have tentatively identified in the DPG's of some strains of Dacty/osporangium, Microbispora, Micromonospora, Micropo/yspora, Nocardioides and Streptosporangium. The identification was based on Rt's on two columns, and lack of reactivity to solvo-mercuration-demercuration. (c) Hydroxy fatty acids, previously reported to be acylated to phosphatidylethanolamine from certain Streptomyces [38, 43] were
identified in some of the strains of the following genera: Actinop/anes, Chainia, Microbispora, Microe//obosporia, Nocardia, Pseudonocardia,Streptomyces, Streptoverticillium and Streptosporangiurn (See Table 2). The methyl esters of these compounds were identified among the organic solvent-soluble deacylation products of the appropriate phosphatidyl ethanolamine fractions by means of TLC versus authentic ~-hydroxy methyl hexacosanoate. The phospholipids of such strains showed two spots containing phosphatidyl ethanolamine on TLC plates: one acylated with non-hydroxylated fatty acids and a more polar compound acylated, at least in part, with hydroxy fatty acids. (d) Unknown unsaturatedfatty acids probably having a branched-chain, were found, usually in small amounts, in certain strains of Actinomadura dassonvil/ei, Actinoplanes, Chainia, Dactylosporangium, Microe//obosporia, Micromonospora, Micropo/yspora faeni, Nocardia (autotrophica and orientalis) Nocardioides, Pseudonocardia, Streptomyces (especially somarlensis) and Streptoverticil/ium. It appears that the phospholipids of Actinomadura pe//etier/differ from those of the other actinomycetes studied in the linkage of some of their fatty constituents. Deacylation by the procedure (methanolic0.025 N LiOH) normally used in this study, releases fatty acids but produces no appreciable H20-soluble phosphate esters of the usual type. For example, deacylation of the "phosphatidyl inositol" fractions of A. pel/etieri 408 or 778 gives rise to three substances. The principal product (Product A) is a phosphorus-containing substance, soluble in both CHCI3 and MeOH but insoluble in H 20 and petrol. In the PC system used for the phosphate esters it is seen as a poorly visualized bluish spot (using the paper phosphorus reagent) running just in back of the front. On TLC in System No. 1, it migrates at an RpI of about 0.4 and reacts well with the phosphorus reagent used for TLC plates. It gives the typical brown-gray color of phosphatidyl inositol with Schiff reagent. The IR spectrum of Product A shows complete disappearance of the ester band at 1730-1740 cm-' but retention of the strong CH JCH3 bands at 2860 and 2930 cm-' of the parent compound. (Deacylation of the phosphatidyl inositol fraction of Micromonospora coeru/ea 12328 yields a typical phosphate ester soluble in MeOH and H20 whose
CHEMOTAXONOMY OF AEROBIC ACTINOMYCETES: PHOSPHOLIPID COMPOSITION
257
taxonomic studies and bring to light some new interesting interrelationships. Once again [68] we find that members of the genera Nocardia and Mycobacterium are indisputably linked. Micropolyspora brevicatena is also closely related to these two genera. It has recently been proposed [68] to limit members of the genus Nocardia to "Actinomycetes containing nocardomycolic acids, having Type IV cell walls and Type A whole cell sugar patterns and not capable of being sharply characterized by morphological criteria." Thus we conclude that on the basis of not only cell wall composition (in N. aerocolonigenes) and lack of nocardomycolic acids (Table 4), but of phospholipid composition, certain "nocardiae," including N. aerocolonigenes, N. autotrophica and N. orienta/is should be excluded from the genus. N. autotrophica strains have some morphological similarities to Pseudonocardia and their phospholipids are similar. As both taxa also lack mycolic acids and share the same cell wall type, a new genus for them might be warranted. Micropo/yspora faeni and our bisporate M. sp. Y 39 are unrelated to the type species M. brevicatena both on the basis of phospholipid composition and lack of nocardomycolic acids. As they are all nonetheless related by morphology and share the same cell wall type, it is proposed to leave them all together in Micropolyspora, at least for the time being. Corynebacteria having a Type IV cell wall appear to be set apart from the Mycobacterium-Nocardia complex by their content of phosphatidyl glycerol and acyl phosphatidyl glycerols. More organisms of this genus should be analyzed. C. aquaticum (Type VI cell wall), not unexpectedly, differs markedly from these corynebacteria. We could not isolate either phosphatidyl inositol mannosides or phosphatidyl ethanolamine from C. aquaticum although Khuller and Brennan [40], using the same strain, did report the presence of these phospholipids. The previously recognized close relationships of Promicromonospora and Oerskovia [69], Streptosporangium and Microbispora [2] and Actinomadura and Microtetraspora [1 ] are underlined by their similarities in phospholipid composition. The creation of the genus Nocardioides Prauser to accommodate nocardoform actinomycetes having a cell wall of Type I [13] and the recent removal of Actinomadura dassonvillei to the new genus Nocardiopsis Meyer [1 2] are also supported by Discussion The results of our studies on phospholipids out findings. As can be seen in Table 2, the underline old relationships based on other phospholipids of A. dassonvi/lei differ markedly
IR spectrum shows the loss of the ester band and marked reduction of the CH 3/CH 2 bands.) Two other minor products are formed. One, Product B, is phosphorus-negative and soluble in petrol. It migrates with batyl alcohol and just ahead of monopalmitin in System No. 1 on TLC and like the monoglycerides, it reacts rapidly with Schiff's reagent to give a bright purple color. The other minor product is a CHCI3soluble, phosphorus-positive compound with an Rel of about 1.2. None of these products are obtained when authentic PI or PI from other actinomycetes are deacylated. As it seemed that Product A might be the lyso form of either alkenyl-acyl (plasmalogen) or alkyl-acyl glycerophosphoryl inositol, it was subjected to the stronger deacylation procedure of Dittmer and Wells [61 ] reported to cleave the more resistant acyl groups of plasmalogens. This resulted in a more complete degradation of the parent compound with the same products produced. The strong deacylation was followed by an acid hydrolysis (hydrochloric acidmercuric chloride) known to cleave the alk-Ienyl link of plasmalogens [61 ]. A small amount of phosphate ester was formed but most of Product A was recovered unaltered. No aldehydes (the products of the cleavage of the alk-l-enyl groups of a plasmalogen by acid) could be demonstrated in the organic fraction from the acid hydrolysis procedure by TLC versus authentic palmitaldehyde. Neither the parent compound, Product A or any of the products from the acid hydrolysis reacted with 2,4-dinitrophenylhydrazine. This appears to eliminate the possibility that the A. pel/etieri phospholipids are plasmalogens. The parent compound did not give a positive reaction with the amido group reagent of Bishel and Austin [62]; thus, sphingolipids are probably not present. We conclude that Product A may be the lyso form of an alkyl-acyl phosphatidyl inositol and Product B a mono-alkyl glyceride. Alkyl-acyl phosphatides have been reported in Proteus mirabilis Hauser [63] and vertebrate tissues [64, 65]. Glycerophospholipids with ether linkages have been tentatively identified in mycobacteria [66] and mannophospholipids in ether linkage with long chain alcohols have also been reported from this taxon [67]. A summary of our findings is presented in Table 6.
258
MARY P. LECHEVALIER,CLAUDE DE BIEVREAND HUBERT LECHEVALIER
f r o m t h o s e o f A. madurae j u s t as t h o s e of Nocardioides d i f f e r f r o m Nocardia and Strepto-
myces.
Experimental Cultures The cultures examined are listed in Table 1.
Growth of Cells All strains, except as noted below, were grown in yeast extract-dextrose broth (1% Difco yeast extract; 1% dextrose in tap H 20; pH 7.2 before autoclaving) on a reciprocal shaker (3 in. stroke; 60 strokes per min) at 28 ° or 37 ° . The cells were harvested in early to mid stationary phase. The broth cultures were autoclaved; the cells, separated by centrifugation, were held at - 2 5 ° until used. M/cropolyspora brevicatena I 1086 was grown in N-Z Amine-glycerol broth (NZG) [70] in static culture, Mycobacterium tuberculosis var. hominis H37Ra in Sauton's medium or TB broth [6] in static culture, and Actinoplanes rectilineatus LL-7-10 in NZG (shaken).
Extraction of Lipids Cells were defrosted at room temperature and broken by sonication for 2-10 min in a Raytheon 10 kc sonic oscillator. The broken cells were separated from the aqueous supernatant by centrifugation at 4600-18, 400 x g and extracted 3 x by shaking with CHCI~-MeOH (2:1) for 2-3 days on a reciprocal shaker at 28 ° . The aqueous supnatant from the centrifugation was extracted once with CHCI3. The extracts were combined and taken to dryness at 50 ° in a rotary evaporator. In some cases, for comparison, phospholipids were also extracted from whole cells using phenol according to the method of De Siervo [71], or directly from membranes isolated from cell sonicates by differential centrifugation.
Purification of the Phospholipids The crude extract was dissolved in CHCI3 and separated into neutral lipid, glycolipid and phospholipid fractions by column chromatography on activated silica gel (J. T. Baker Chemical, Phillipsburg, N, J., 80-200 mesh or equivalent) by the method of Rouser eta/. [72]. The crude methanolic phospholipid fraction from the coloumn was taken to dryness in a rotary evaporator as before, the residue dissolved in CHCI3 and added to 5 x volume of cold (4 ° ) dry Me2CO saturated with MgCI2. The precipitate was centrifuged off and washed once with cold Me2CO-MgCI2 and taken up in 5-10 ml of CHCI3-MeOH-H=O (60:30:4.5) with mild heat if necessary to effect solution, and purified of contaminating amino acids and sugars by passage through a Sephadex G 25 column according to Wells and Dittmer [73]. The eluate from this column was taken by dryness under N2 with mild heat, taken up in 5 ml of CHCI3 and washed twice with 5 ml of 0.5% aqueous NaCI. The CHCI3 layer was taken to dryness as before under N2, then 100 mg spotted on 10 PF254 (Brinkmann Instruments, Westbury, N. Y., U.S.A.) silica gel TLC plates (10 g of silica per one 10 x 20 cm plate) and developed in CHCI3-MeOH-H20 (70:35:7) (System 1). The center of the plate was masked and the outer edges of
the various bands visualized by spraying with the reagent for phosphorus-containing compounds of Dittmer and Lester [74]. The unsprayed portions of the bands were scraped off and eluted with 75 ml of CHCI3-MeOH (2:1) followed by the same solvents in the proportions (1:2). The eluates from each band were combined and brought to dryness under N2 to yield the purified phospholipids.
Analysis of the Phospho//pids The phospholipids were characterized by spraying with various reagents including the Munier and Macheboeuf modification of Dragendorff's reagent (for choline, methylethanolamine and ethanolamine-containing phospholipids), ninhydrin (amino groups) and anisealdehyde (for sugar-containing compounds) [74]. Schiff reagent was also used to verify the identity of phosphatidyl inositol (gray-brown color) and phosphatidyl glycerol (bright rapid purple) [75]. In the latter case, the phospholipids were separated by two-way TLC using System 1 (above) in one direction followed by a CHCI3-HOAc-MeOH-H20 system (80:18:12:5) (System 2) at right angles to the first development. Further identification of the phospholipids was carried out by mild deacylation of the purified fractions according to Dawson [76]. modified as follows: 2-4 mg of pure phospholipid is dissolved in 0.4 ml of petrol (b.p. 40-55°). In some cases it is necessary to add 1-2 drops of MeOH, To this solution is added 3.75 ml MeOH, 0.37 ml distilled H20 and 0.12 ml 1N LiOH.The whole is mixed and incubated in a waterbath at 37 ° for 20 min. The deacylated mixture was neutralized to pH 5.0-6.0 by addition of BioRex 70 (H+) (Calbiochem, LaJolla, Calif., U.S.A.) and extracted with 10 ml of petrol. The petrol, which contained the fatty acids (as methyl esters ) released by the deacylation, was analyzed by GLC (FID) on 3% OV-1 or 15% DEGS (Stabilized diethylene glycol succinate; Analabs, North Haven, Conn., U.S.A.) columns. Unsaturation of the fatty acids was verified by the gas chromatography of the methoxy derivatives made using the solvo-mercuration demercuration technique [49]. The BioRex resin was separated from the methanolic phase and eluted with 80% aqueous EtOH. The ethanolic eluates were combined with the methanolic phase and this mixture taken to dryness under N 2. These residues, which contained the phosphate esters from the deacylation of the phospholipids, were dissolved in MeOH-H 20 (1:1) and spotted on Whatman No. 1 paper along with the phosphate esters prepared in the same way from authentic phospholipid standards (Supelco, Beltefonte, Pa.; Applied Science Labs, State College, Pa.; Analabs, North Haven, Conn., U.S.A.). Following development in IpOH-NH~ (2:1) for 18 hr, the dried papergrams were sprayed first with ninhydrin, heated at 105 ° to visualize glyceryl phosphoryl ethanolamine or glyceryl phosphory methylethanolamine (purple spots) and then with the Dawson modification of the Haynes-lshervvood molybdate reagent [76] and irradiated with UV to develop the blue spots corresponding to the phosphate esters. The pure phospholipids were also analyzed by acid hydrolysis. For amino acids, 1-2 mg of phospholipid
CHEMOTAXONOMYOF AEROBICACTINOMYCETES:PHOSPHOLIPIDCOMPOSITION were hydrolyzed with 6N HCI for 18 h at 100 ° in a sealed tube. The hydrolyzate was extracted with chloroform to remove the fatty materials and the HCI removed from the sample as described by Becker et al. [4]. The samples were spotted on Whatman No. 1 paper and developed in n-BuOH-pyridine-H20-HOAc (60:40:30:3) for 48 h, then dried, sprayed with ninhydrin and heated at 105 ° to detect amino acids, amino sugars and amino alcohols followed by the Dragendorff reagent for choline. Sugars and polyols were obtained by hydrolysis of 2 mg of phospholipid in 3N H2SO4 for 3 h at 100 °. The H2SO4 was removed as the BaSO4 salt by addition of saturated Ba(OH)2 until the pH was 5.0, and centrifuging. The supernatant was taken to dryness in vacuo at room temperature, red/ssolved in a small amount of H20, spotted on Whatman No. 1 paper and developed in freshly prepared n-BuOHpyridine-H20-toluene (5:3:3:4, upper) system for 48 h or in MeOH-H20 (9:1) for 6 h or whenever the front reached the bottom of the paper. Visualization of sugars and polyols was made by spraying with acid aniline phthalate and heating to 105 ° for reducing sugars followed by dipping in the tolidine reagent of White and Frerman [77] for polyols. Other reactions used included ammoniacal silver nitrate, the Elson-Morgan reagent and the ninhydrin degradation for glucosamine [78]. Acknowledgements--We wish to thank Eva Fekete and Magda Gagliardi for technical assistance, and D. Minnikin who suggested the utility of two-way TLC in confirming the presence of certain phospholipids. This study was supported in part by National Science Foundation Grant DEB 73-01 692. Claude de Bi~vrewas the holder of a Fellowship from the French Ministry of Foreign Affairs.
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260
MARY P. LECHEVALIER,CLAUDEDE BIEVREAND HUBERTLECHEVALIER
45. Dhariwal, K. R., Chander, A. and Venkitasubramanian, T. A. (1 975) Experientia 31,776. 46. Gochnauer, M. B., Leppard, G. G., Komaratat, P., Kates, M., Novitsky, T. and Kushner, D. J. (1975) Canad, J. Microbiol. 21, 1500. 47. Hackett, J. A. and Brennan, P. J. (1975) Biochem. J. 148, 253. 48. Komura, I., Yamada, K., Otsuka, S.-I. and Komagata, K. (1975) J. Gen. Appl. Microbiol. 21,251. 49. Lechevalier, M. P. and Lechevalier, H. (1974). Int. J. Syst. Bacteriol. 24, 278. 50. O'Leary, W. M. (1973) In Handbook of Microbiology, Vol. II. (Laskin, A. I. and Lechevalier, H. A., eds.), p. 275, CRC Press, Cleveland. 51. Vilkas, E. and Lederer, E. (1956) Bull. Soc. Chim. Biol. 38, 111. 52. Ballou, C. E.,Vilkas, E. and Lederer, E. (1963) J. Biol. Chem. 238, 69. 53. Op den Kamp, J. A. F. and van Deenen, L. L. M. (1966) Chem. Phys. Lipids 1, 86. 54. Phizackerley, P. J. R., MacDougall, J. C. and Francis, M. J. O. (1966) Biochem, J. 99, 21C. 55. MacDougall, J. C. and Phizackerley, P. J. R. (1969) Biochem. J. 114, 361. 56. Ames, G. F. (1968) J. Bacterio/. 95, 833. 57. Olsen, R. W. and Ballou, C. E. (1971 ) J. Biol. Chem. 248, 3305. 58. Oliver, J. D. and Colwell, R. R. (1973) J. Bacteriol. 114, 897. 59. Exterkate, F. A., Otten, B. J., Wassenberg, H. W. and Veerkamp, J. H. (1971 ) J. Bacteriol, 108, 824. 60. Oshima, M., Ueta, N. and Yamakawa. T. (1969) Jap. J. Exp. Med. 39, 77. 61. Dittmer, J. C. and Wells, M. A. (1969) In Methodsin Enzymo/ogy (Colowick, S. P. and Kaplan, N. O., eds.), Vol. 14, pp. 482-530, Academic Press, New York. 62. Kates, M. (1972) Techniques of Lipido/ogy. Isolation, Analysis and Identification of Lipids, p. 439, American Elsevier, New York.
63. Rebel, G. (1972) Arch. Mikrobiol. 81,333. 64. Renkonen, O. and Hirvisalo, E. L. (1963) Biochem. J. 89, 29P. 65. Horrocks, L. A. and Ansell, G. B. (1967) Biochim. Biophys. Acta 137, 90, 66. Chandramouli, V., $arma, G. R. and Venikitasubramanian, T. A. (1971 ) Indian J. Biochem. 5" Biophys. 8,55. 67. Takayama, K., Schnoes, H. K. and Semmler, E. J. (1973) Biochim. Biophys. Acta 318, 212. 68. Lechevalier, M. P. (1976) In The Biology of the Nocardiae (Goodfellow, M., Brownell, G. H. and Serrano, J. A., eds.), pp. 1-38, Academic Press, London. 69. Lechevalier, M. P. (1972) Int. J. Syst. Bactenol. 22, 260. 70. Lechevatier, H. A., Solotorovsky, M.and McDurmon McDurmont, C. I. (1961 ) J. gen. Microbiol. 26, 11. 71. De Siervo, A. J. (1969) J. Bacteriol. 100, 1342. 72. Rouser, G., Kritchevsky, G. and Yamamoto, A. (1 967) In Lipid Chromatographic Analysis (Marinetti, G. V., ed.), Vol. 1, pp. 99-162. Marcel Dekker, New York. 73. Wells, M. A. and Dittmer, J. C. (1963) Biochemistry 2, 1259. 74. Randerath, K. (1968) Thin Layer Chromatography, pp. 154, 235, Academic Press, New York. 75. Shaw, N. (1968) Biochem. Biophys. Acta 164, 435. 76. Dawson, R. M. C. (1967) In Lipid Chromatographic Analysis (Marinetti, G. V., ed.), Vol. 1, pp. 1 63-189, Marcel Dekker, New York. 77. White, D. C. and Frerman, F. E. (1967) J. Bacteriol. 94, 1 854. 78. Block, R. J., Durrum, E. L. and Zweig, G. (1958) A Manual of Paper Chromatography and Paper Electrophoresis, pp. 178, 209, Academic Press, New York.
Chemotaxonomy of Aerobic Actinomycetes: Phospholipid Composition MARY P. LECHEVALIER, CLAUDE DE BIEVRE* and HUBERT LECHEVALIER Waksman Institute of Microbiology, Rutgers, The State University, P.O. Box 759, Piscataway, NJ 08854, USA Key Word Index--Actinoplanaceae; Micromonosporaceae; Mycobacteriaceae; Nocardiaceae; cetaceae; Thermomonosporaceae; Actinomycetales; phospholipids; fatty acids; chemotaxonomy.
Streptomy-
A b s t r a c t - - A survey of the phospholipid composition of 97 strains representing 20 genera of the Actinomycetales showed that five groups could be distinguished on the basis of the presence or absence of certain nitrogenous phospholipids. Phospholipid type PI (no nitrogenous phospholipids) is characteristic of the genera Actinomadura (madurae, pelletieri), Corynebacterium, Microtetraspora and Nocardioides. Actinomycetes of Type PII contain only one nitrogenous phospholipid, phosphatidyl ethanolamine. These include members of the genera Actinoplanes,
Chainia, Dactylosporangium, Microellobosporia, Micromonospora, Micropolyspora (brevicatena), Mycobacterium, Nocardia (all species examined but autotrophica), Streptomyces and Streptoverticillium. Phospholipid pattern type PIII (characteristic phospholipid, phosphatidyl choline) was found in Actinomadura (dassonvillei), Micropolyspora (faeni), Nocardia (autotrophica), and Pseudonocardia. Actinomycetes having a type P IV pattern contain an unknown, previously undescribed phospholipid containing glucosamine (GluNU) which was found to be characteristic of members of the genera Intrasporangium, Microbispora and Streptosporangium. Actinomycetes of type PV contain phosphatidyl glycerol in addition to GluNU and include members of the genera Promicromonospora and Oerskovia. Other phospholipids found variably in all groups included phosphatidyl inositol, phosphatidyl inositol mannosides, phosphatidyl methylethanolamine, acyl phosphatidyl glycerol (APG) and diphosphatidyl glycerol (DPG). The fatty acids present in DPG (or APG when DPG was absent) may be species-specific. The chemical heterogeneity of the genera Actinomadura, Corynebacterium, Micropolyspora and Nocardia is discussed.
Introduction The chemistry of cell constituents of actinomycetes has played an increasingly important role in the taxonomy of these economically important, gram-positive branching bacteria [1]. Cell wall composition [2, 3], whole cell amino acids [4], whole cell sugars r5] and lipids such as mycolic acids r6-8] have enabled taxonomists to clarify relationships among different groups of actinomycetes and has led to the proposal of at least five new genera [9-13]. The phospholipid composition of actinomycetes has been the subject of a number of reports [14-48], but no systematic attempt has been made to apply this criterion to the taxonomy of broad groups of actinomycetes. In this paper we wish to report on the phospholipid composition of 97 strains representing species of 20 genera of actinomycetes. Results
More than ninety strains of actinomycetes representing twenty genera (Table 1) were *Present address: Service de Mycologie, Institut Pasteur, Paris, France.
analyzed for their phospholipid composition (Table 2). The phospholipids were identified on the basis of (a) their relative mobility on TLC and reactions with ninhydrin, Dragendorff, anisealdhyde, Schiff and molybdate reagents; (b) the mobility of their phosphate esters produced by mild alkaline hydrolysis versus the phosphate esters from authentic compounds; and (c) the identity of the sugars, polyols and amino acids and alcohols yielded by acid hydrolysis. The fatty acids of the diphosphatidyl glycerols (DPG) or acyl phosphatidyl glycerols (where DPG was lacking) were also determined (Table 3). The fatty acids were identified byGLCon the basis of the Rt's of their methyl esters (FAMES) on polar and non-polar columns versus authentic compounds and, in the case of unsaturated fatty acids, by the Rt's of the methoxy derivatives i49]. Quantitation was carried out by measuring the areas under the various peaks of the resolved FAMES by multiplying the heights of the peaks by the widths at half height. Since no authentic material is available commercially, heptadecenoic acid, the major constituent of the fatty acids of the cardiolipins
(Received 18 April 1977; received for publication 18 July 1977) 249
250
MARY P. LECHEVALIER, CLAUDE DE BIEVRE AND HUBERT LECHEVALIER
TABLE 1. STRAINS USED IN THIS STUDY Taxon Actinomadura dassonvillei (Brocq-Rousseau) Lechevalier and Lechevalier
madurae (Vincent) Lechevalier and Lechevalier
pelletieri (Laveran) Lechevalier and Lechevalier sp.
ActinoManes missouriens/s Couch rectilineatus Lechevalier and Lechevalier spp.
Chamia nigra Thirumalachar olivacea Thirumalachar and Sukapure Corynebacterium aquaticum Liefson boris Bergey et al. diphtheriae (Kruse) Lehmann and Neumann Dactylosporangium aurantiacum Thiemann, Pagani and Beretta thailandense Thiemann. Pagani and Beretta Intraepotangium calvum Kalakoutskii, Kirillova and Krassil'nikov Microbispora amethystogenes Nonomura and Ohara rosea Nonomura and Ohara Microellobosporia flavea Cross, Lechevalier and Lechevalier violacea Tsyganov, Zhukova and Timofeeva Micromonospora coerulea Jensen narashinoensis (Shidara) (subsp. of) Arai and Kuroda purpurea Luedemann and Brodsky purpureochtomogenes (Waksman and Curtis) Luedemann Micropolyspora brevicatena Lechevalier, Solotorovsky and McDermont faeni Cross, Maciver and Lacey sp. Microtetraspora viridie var. intermedia Nonomura and Ohara Mycobacterium abcessus Moore and Frerichs
boris Karlson and Lessel fortuitum Cruz smegmatis (Trevisan) Lehmann and Neumann tuberculosis (Zopf) Lehmann and Neumann sp.
Nocardia aerocolonigenes (Shinobu and Kawato) Pridham amarae Lechevalier and Lechevalier asteroides (Eppinger) 81anchard autotrophica (Takamiya and Tubaki) Hirsch brasiliensis (Lindberg) (Castellani and Chalmers)
Strain No.* 1509 1575 I 1371 (LL-Botl 1) I 1328 (LL-L 13) I 1330 (LL-Cross, Fil) N 434 1507 1780 1953 LL-S4 LL-Sal 1 I 408 1610 1778 LL-N15
Sourcet
T. Cross
1824 (LL-E3-15) 13918 (LL-7-10) LL-K30 LL-Z20 LL- P 128 LL-15-7 LL-Su 12 LL-V17576
R. S. Sukapure E. Tejera
B 2252 NIRD 128 A 11913
D. Weaver R. Smith
A 23491 LL-9-41A LL-7 KIP
L. V. Katakoutskii
A 15740 (LL-37-6) 1864 13748 A 15839 (I 3858) LL-795 LL-V12328 A 10028 LL-817C2 NRRL B943
A. Kuchaeva E. T~era G. Luedemann
11086 LL-A 91 LL-V4066 LL-Y 39
P. H. Gregory P. H. Gregory
LL-Mt-2
H. Nonomura
A 19977 13061 A 9834 1788 11000 1375 1455 H37Ra NCTC 4524t 1550 A 27808 (I 3960; LL-Se 6) LL-Se 3 LL-Se 120 A 3318 A 19247 (I 727) 1433N A 19727 13520 1774B 11093
251
CHEMOTAXONOMY OF AEROBIC ACTINOMYCETES: PHOSPHOLIPID COMPOSITION TABLE 1--continued Taxon caviae (Erikson) Gordon and Mihm
Strain No. • I 1203
Sourcet
I 1934
See Mycobacterium sp, I 1107 I 1149 I 1240 I 3639 I W21
farcinica Travisan orientalis (Pittinger and Brigham) Pridham and Lyons rhodochrous (Overbeck) Lechevalier, Horan and Lachevalier
N 1621
LL- Mil 15 Nocardioides a/bus Prauser
IMET 7801 IMET 7807
Oemkovia turbata (Erikson) Prausero Lechevalier and Lechevalier
I 762 SSlC 891 A 27402 (I 3959; LL-G 62) LL-St/3P
xanthineolytica Lechevalier Pomicromonospora citrea (Krasil'nikov et al.) spp. Pseudonocardia thermophila Henssen Streptom¥ces a/bus (Rossi-Doria) Waksman and Hanrici fradiae (Waksman and Curtis) Waksman and Henrici griseus (Krainsky) Waksman and Henrici rimosus Sobin et al. somaliensis (Brumpt) Waksman and Henrici Streptosporangium roseum Couch sp. (rubrum type) (amethystoganes-type) sp. Streptoverticillium alboreticuli (Nakazawa) Locci et al. rubrireticuli (Waksman and Henrici) Baldacci • A=ATCC=American Type Culture Collection, Rockville, Md., U.S.A. B=CDC=Center for Disease Control, Atlanta, Ga., U.S.A. I=lMRU=Waksman Institute of Microbiology, Rutgers University. IMET=lnstitute f~r Mikrobiologie und Experimentelle Therapie, Jena, DDR. LL= Lechevaliers' Collection. N =NCTC=National Collection of Type Cultures, U.K. NIRD=National Institute for Research in Dairying, U.K. NRRL=Northern Regional Research Laboratory, Peoria, I11.,U.S.A. SSIC=Statens Serum Institute Collection, Denmark. t The source is given for LL strains not isolated in the Lechevaliars' laboratory. $ Formerly Nocardia farcinica.
of Streptosporangium and Micromonospora strains, was identified on the basis of the log of the Rt of its methyl ester on G LC and the Rt of its monomethoxy derivative. Heptadecenoic acid has never been reported in major amounts from actinomycetes, but occurs in other bacteria [50]. Strains belonging to 17 of the 20 genera contained phospholipids with nitrogenous constituents. The largest group contained only one major phospholipid of this type: phosphatidyl ethanolamine. This is referred to in Table 2 as phospholipid pattern type PII. Members of the genera Actinoplanes, Chainia, Dactylo-
sporangium, Microellobosporia, Micromono-
R. F. Lewis
LL-18 LL-G165 LL-G173
L. V. Kalakoutskii
LL-A18
A.Henssen
758 3004 3535 3475 3558 383 719 761 1298 A 12428 LL-J7 LL-9-26 LL-N6 LL-15-17 A 24213 LL-100-19
D. P. Stahly
spora, Micropo/yspora (brevicatena only), Mycobacterium, Nocardia (except autotrophica)o Streptomyces and Streptoverticillium showed this pattern. Another group contained phosphatidyl choline (type P III phospholipid pattern). Phosphatidyl methylethanolamine (PME) was also present in most strains falling into this category. Phosphatidyl cholinecontaining taxa included Actinomadura dassonvillei, Micropolyspora faeni and spo,Nocardia autotrophica and Pseudonocardia. The third and fourth groups were characterized by their content of certain, as yet unidentified, phospholipids containing glucosamine (GluNU). These include members of the genera Intrasporang-
252
MARY
TABLE
2--PHOSPHOLIPIDS*
OF
ACTINOMYCETES
P.
LECHEVALIER,
CLAUDE
DE
BIEVRE
AND
HUBERT
EXAMINED
Strain Taxon
No.
Actinomadura madurae
sp.
Actinoplanes missouriens/s rectilineatus
Chainia nigra olivacea Dactylosporangium aurantiacum thailandense Microellobosporia flavea violacea Micromonospora coerulea narashinoensis purpurea purpureochromogenes Micropolyspora brevicatena Mycobacterium abcessus bovis fortuitum 8megmatis var.
sp.
Nocardia aerocolonigenes amarae
PC
PE
.
GluNU
+
. tr.
+
+
.
.
.
.
.
+
+
+
.
.
.
.
.
+
+
+
.
.
.
.
.
+
408
+
+
.
.
.
.
.
+
610
+
+
.
778
+
+
.
.
.
.
.
N15
+
+
.
.
.
.
.
B2252
-
tr.
+
-
-
-
+
-
N 128
+
+
+
-
-
+
+
-
A11913
+
+
+
-
-
±
Mt
+
+
-
7801
±
+
+
-
-
7807
tr.
+
+
-
-
824
+
+
tr.
-
+
-
.
.
DPG
+
2
.
APG
+
1
.
PME
-
.
-
-
.
+
-
+
-
PA -
-
-
-
?
PI
-
-
-
±
-
-
+
-
-
+
.
.
.
.
-
+
.
.
.
.
-
-
+
-
-
hominis
-
+
+
-
3919
+
+
±
-
-
K30
+
+
-
-
+
-
-
+
Z20
±
+
-
-
+ §
-
-
+
-
-
P128
+
+
-
+
-
-
+
-
15-7
+
+
-
+
-
-
+
-
SU12
+
+
+§
-
-
+
-
V17676
+
+
+ §
-
-
+
-
A23491
±
+
9-41A
+
+
3858
±
+
795
+
+
V12328
tr.
+
A10026
±
±
817C2
+
B943
+
1086
+
A19977
±
+
-
-
+
-
+
-
-
+
-
-
+
-
-
+
-
-
+§
-
-
+
-
+
-
+
+
-
-
+
-
?
+
-
+
-
+
-
-
+
-
+
-
+
-
-
+
-
+
tr.
+
-
-
+
-
+
+
-
3061
+
+
-
9834
+
+
-
+
-
+
-
+
-
+
-
+
788
+
±
-
-
+
1000
+
+
-
-
+
+
-
-
+
-
-
+
-
375
+
+
-
+
-
-
+
455
+
+
-
+
-
-
+
-
H37Ra
+
+
-
+
-
-
+
-
4524
+
+
-
+
-
-
+
-
550
-
+
-
-
+ §
-
+
-
+
-
-
+
-
+
-
+
+
-
-
+
+
-
Se120
+
+
-
-
+
+
-
3318
+
+
-
-
+
+
-
19247
+
+
-
-
+
433N
+
+
-
-
+
brasiliensis
774B
+
-
+
-
+
1093
+
+
-
-
+
-
+
-
caviae
1203
+
+
-
-
+
-
+
-
1934
+
+
-
-
+
-
+
-
1107
tr.
+
-
-
+ §
-
1149
±
+
-
-
+§
-
+
-
1240
•
+
-
-
+
-
+
-
fradiae
-
-
-
+
Streptomyces albus
-
-
+
-
-
type
-
+
tr.
orientahs
?
+
Se3
rhodochrous
PA
Unknown$
-
27808
asteroides
Other
-
Ptl
spp.
tuberculosis
PG
± 1"
Sal
Corynebacterium aquaticum bovis diphtheriae Microtetraspora viridis v a r . intermedia Nocardiodes albus
PI
507
953
pe//etieri
Phospholipid PIMs
780
$4
LECHEVALIER
+
-
-
-
+
-
+
-
+
-
-
3639
+
+
-
-
+
-
+
-
W21
+
+
-
-
+
-
+
-
1621
+
+
-
-
+
-
+
-
Mi115
+
+
-
-
+
-
+
-
+
-
+
-
-
+
-
-
+
3004
+
+
-
-
+
3535
758
+
+
+
±
-
-
-
+
-
-
d
CHEMOTAXONOMY
TABLE
OF AEROBIC
Strain No.
griseus rimosus somahensis
Streptoverticillium alboreticuli rubrireticufi Actinomadura dassonvillei
253
COMPOSITION
PtMs
PI
PG
PC
PE
PME
APG
DPG
3475
+
+
-
-
3558 383
+ +
+ +
-
-
719 761 1298
+ + +
+ + +
-
-
+ + +
24213
+
+
-
-
+ §
100-19
+
+
-
-
+
-
N434
tr.
4-
+
4-
-
4-
+
tr.
509
+
+
+
+
-
tr.
+
575 1328
+ 4-
tr. 4-
+ +
+ 4-
-
tr. +
1330
tr.
4-
+
+
-
1371
-
tr.
+
+
-
A91 V4086 Y39
4-
+ + +
+ + +
+ + +
19727 3520
tr. +
+ +
± 4-
A18
4-
+
7 KIP
+
II
15740 864 3748
sp.
Nocardia autotrophica Pseudonocardia thermophila Intrasporangium calvum Microbispora amethystogenes rosea
Other
Unknown~t
+ §
-
+
-
-
+
+ § + §
-
+ +
-
-
-
+ + +
-
-
+
+
-
-
-
+
-
-
-
-
-
PA ?
-
+ 4-
+ +
-
PA? -
-
4-
+
4-
-
-
-
+
+
+
-
-
-
_
+§ + +
-
+ + +
_
PA _
+
+ +
tr.
+ +
+ +
-
-
-
-
+
+ {
+
+
_
_
_
+
-
-
+
-
+
+
-
-
+ 11 +11 +ll
+ + +
-
-
+ § +§ +§
-
+ + +
+ 4+
PA? -
-
A12428 J7
+ U +ll
+ 4-
-
-
+ § +
+ +
+ +
+ +
-
-
9-26
+11
+
-
-
+§
tr.
+
+
-
-
N6 15-17
+11
+
-
-
+§
+
+
+
-
-
+11
+
-
-
-
+
+
+
-
-
762 891
+ Jl
Phospholipid type
_
PIV
Streptosporangium roseum sp.
Oerskovia turbata
-
PV
xanthineolytica Promicromonospora citrea spp.
4-
+
-
-
-
-
A27402
+11 +11
+
+
-
+
+
-
-
-
St/3P
+11
+
+
-
-
18 G165
+ II
+
+
-
-
-
+ll
+
+
-
-
-
G173
+ll
+
+
-
4-
-
ium, Microbispora,
and Streptosporangium P I V ) a n d Oerskovia a n d Promicromonospora ( p h o s p h o l i p i d type PV). The PV types were differentiated from PIV's by their content of phosphatidyl glycerol. Members of only four genera contained no type
major amounts of nitrogen-containing phospholipids. T h e s e w e r e Actinomadura (madurae a n d pe/letieri), Corynebacterium, Microtetraspora a n d Nocardioides ( P h o s p h o l i p i d T y p e I ) .
+
-
PIMs =Phosphatidyl inositol mannosides PI =Phosphatidyl inositol PG = Phosphatidyl glycerol PC =Phosphatidyl choline PE =Phosphatidyl ethanolamine PME =Phosphatidyl methyl ethanolamine APG =Acyl phosphatidyl glycerol DPG Diphosphatidyl glycerol Glu-NU =Glucosamine-containing unknowns PA =Phosphatidic acid + =major component; 4- = m i n o r component; tr=trace; - =component absent. All minor fractions having too little material for identification. Phosphatidyl ethanotamine acylated with hydroxy fatty acids also present. May or may not be present. Cannot currently be separated from the unknown
(phospholipid
GluNU
PIll
Micropolyspora faeni
I" :1: § 11
PHOSPHOLIPID
2--continued
Taxon
•
ACTINOMYCETES:
4-
+
-
4-
-
+
-
+
+
-
+
+
+
-
-
+
+
+
-
-
+
+
+
-
-
+
+
+
-
-
glucosamine-containing
phospholipids.
As judged by phospholipid and fatty acids patterns, the following genera among those examined,
appear
to
be
inhomogeneous:
Actinomadura, Corynebacterium, Micropolyspora a n d Nocardia. T h i s c o m e s a s n o s u r p r i s e since there is other chemical evidence of their heterogeneity (summarized in Table 4). Although all the corynebacteria examined fall into the phospholipid pattern type PIo an examination of Table 2 shows there to be
254
MARY P. LECHEVALIER, CLAUDE DE BIEVRE AND HUBERT LECHEVALIER
TABLE 3. FATTY ACIDS FOUND IN ACTINOMYCETE PHOSPHOLIPIDS I. ANTEISO/ISO BRANCHED CHAIN FATTY ACIDS PRESENT ( >10% OF TOTAL FATTY ACIDS) A. Principal component branched chain fatty acid of anteisoiso series
Actinomadura (dassonvillei) Actinoplanes (missouriensi$; 2 spp.) Chainla Corynebacterium (aquaticum) Dactylosporangium Micropolyspora (faeni; sp.) Micromonospora (coerulea; echinospora) Microbispora Microellobosporia Nocardia (autotrophica; orientalis) Nocardioides (7807) Oerskovia Promicromonospora Pseudonocardia Streptomyces (albus fradiae; griseus; fimosus) Streptoverticillium
Phospholipid fatty acid Type 1
B. Principal component unsaturated fatty acid other than heptadecanoic Actinomadura (madurae; pelletieri; sp.) Actinoplanes (rectilineatus; 2 spp.)
Microtetraspora Microellobosporia (flavea) Nocardioides (7801) Streptomyces (somaliensis) Nocardia (aerocolonigenes)
Phospholipid fatty acid Type 2
C. Principal component heptadecanoic acid
Micromonospora (fusca; purpureochromogenes) Streptosporangium
Phospholipid fatty acid Type 3
II. ANTEISO-ISO FATTY ACIDS USUALLY NOT PRESENT; IF SO, < 10% OF TOTAL FATTY ACIDS
Actinomadura (madurae; pelletieri ) Corynebacterium (diphtheriae; bovis) Micropolyspora (brevtcatena) Mycobacterium Nocardia (amarae; asteroides; brasiliensis; caviae; rhodochrous)
Phospholipid fatty acid Type 4
TABLE 4, CHEMICAL HETEROGENEITY OF VARIOUS ACTINOMYCETE TAXA Taxon
Actinomadura dassonvillei madurae pelletieri Corynebacterium aquaticum boris diphtheriae Micropolyspora brevicatena faeni Nocardia amarae, asteroides brasiliensis, caviae rhodochrous aerocolonigenes orientalis autotrophica
Phospholipid pattern*
Whole cell sugar/cell wall typet
Mycolic acids
Phospholipid fatty acid pattern~t
P Ill PI PI
C/Ill B/Ill B/Ill
-
1 2 2
P I§ PI PI
-/VI A/IV A/tV
+ +
1 4 4
P II P III
A/IV A/IV
+ -
4 1
P II P II P II P III
A/IV C/Ill A/IV A/IV
+ -
4 2 1 1
• See Table 2. 1" See Lechevalier and Lechevalier [2]. ~t=See Table 3. §=See text.
important differences between the C. bovis and C. diphtheriae strains and the C. aquaticum strain; namely, lack of phosphatidyl inositol mannosides (PIMs) and APG in the latter. In certain genera where phospholipid composition is homogeneous, there is evidence that DPG (or APG) fatty acid patterns may be
species-specific. These include the taxa listed in Table 5. In the course of our studies some unusual types of phospholipids were discerned. As previously mentioned, phospholipids containing glucosamine were found in members of the genera Intrasporangium, Microbispora, Oersko-
CHEMOTAXONOMY OF AEROBIC ACTINOMYCETES: PHOSPHOLIPID COMPOSITION
via, Promicromonospora and Streptosporangium. These compounds also contain mannose and inositol and in some cases, glucose, and occur as 2-5 presumably similar compounds, the total number depending on the strain analyzed. These compounds (GluNU's) give a strong green color with the anisealdehyde reagent and a positive blue reaction with the phosphorus reagent of Dittmer and Lester. Only a slowly reacting faint peach color is produced with ninhydrin even on prolonged heating. In acid or base-containing solvent systems on TLC, the GluNU's are appreciably retarded in their migration relative to diphosphatidyl glycerol (DPG) or phosphatidyl ethanolamine compared to that in a neutral system. For example, in the neutral System 1 (see Experimental), the GluNU's from Streptosporangium sp. J7 appear as five spots with RDPG'Sof 0.45, 0.48, 0.62, 0.73 and 0.80. In the acid-containing System 2, only four spots are resolved having RDPG'Sof 0.1 4, 0.21, 0.31 and 0.39. Except for the faint reaction with ninhydrin,theGluNU's cannot bedistinguished, by TLC from phosphatidyl inositol mannosides (PIMs) and acylated PIMs [23, 24, 51, 52]. Even the phosphate esters of the GluNU,s migrate in the area of those of the PIM's on PC; thus, it is not known whether conventional PIM's are also present in the Glu NU-containing strains. Glucosamine was identified by relative mobility versus authentic standards on PC in two systems and reactions with ninhydrin (brown-purple), acid aniline phthalate (brown), ammoniacal silver nitrate and the Elson-Morgan reagent (red). Its identity was confirmed by degradation to arabinose by treatment with ninhydrin. It is released only on hydrolysis of the GluNU's with 6N HCI at 100 ° for 18 h; none is released with 3N H2SO, in 3 h at 100 ° or by either mild (0.025 N methanolic LiOH at 37 ° for 20 min) or strong (2N aqueous KOH at 100 ° for 30 min) alkaline hydrolysis. Glucosamine has only been reported as a phospholipid constituent in glucosaminyl phosphatidyl glycerol (GPG). GPG has previously been reported from Bacillus megaterium de Ban/ [53] and Pseudomonas ova/is Chester [54]. Our compounds are clearly different from GPG not only by their content of mannose and inositol, but the fact that their phosphate esters derived from mild alkaline hydrolysis are ninhydrin-negative whereas that from GPG is ninhydrin-positive [55]. Other unusual phospholipids that were encountered in this study have been identified
255
TABLE 5. SPECIES SPECIFIC FATTY ACID PATTERNS IN GENERA WHERE THE PHOSPHOLIPID COMPOSITION IS HOMOGENEOUS Phospholipid fatty Taxon acid pattern"
Actlno,olanea missouriensis and 2 spp, rectilineatus and 2 spp.
1 2
Microellobosporia
flavea putpurea
2 1
Micromonospora coerulea and echinosDora fusca and purpureochromogenes Nocardio/des albus (7807) a/bus (7801)
1 3 1 2
Streptomyces a/bus, fradiae
griseus, rimosus somaliensis
1 1 2
*See Table 3.
TABLE 6". GROUPING OF ACTINOMYCETES BASED ON PHOSPHOLIPID COMPOSITION I. CONTAIN NO NITROGENOUS PHOSPHOLIPIDS (Type PI) A. Contain only PIMs, PI end DPG. Actinomadura (madurae, pelletieri)
Microtetraspora B. Contain PIMs, PI, DPG, PG and APG's.
Corynebacter/um (bovis, dlphtheriae) C. Contain PIM, PI, PG and APG's.
Nocardioides D. Contain PG, and DPG, no PIMs, traces PI.
Corynebacterium aquat/cum II. CONTAIN NITROGENOUS PHOSPHOLIPIDS A. Contain PE, PIMs, PI and DPG (Type PII) i. Fatty acidst of Type 1
Chainia Dacty/osporangium Nocardia (oriental/s) S treptovertic///ium
Streptomyces (a/bus, frad/ae, griseu~ rimosus) ii. Fatty acids of Type 1 or 2
Actinoplanes Microellobosporia iii. Fatty acids of Type 2
Nocardia (aeroco/onigenes) Streptomyces (somaliensts) iv. Fatty acids of Type 1 or 3
Micromonospora v. Fatty acids of Type 4.
Micropolyspora (brevicatena) Mycobacteriurn Nocardia (amarae. asteroides, brasiliensis, caviae, orientalis, rhodochrous) B. Contain PC (PME, DPG, PIMs and PI variable) (Type PIll) i. Also contain PG and APGs
Actinomadura (dassonvillei)
ii. Also contain PG. No APG.
Micropo/yspora (faeni, sp.) iii. No APGs or PG. (or onlytraces of latter) Nocardia (autotrophica) Pseudonocardia C. Contain GluNU. PI and DPG. i. Contain PE or PME or both. No PG. (Type PIV)
Streptosporangium Microbispora
Intrasporangium
ii. Contain little to no PE or PME. Contain PG. (Type PV)
Oerskovia Promicromonospora "See Table 2 for abbreviations of phospholipids. tSee Table 3.
256
MARY P. LECHEVALIER, CLAUDE DE BIEVRE AND HUBERT LECHEVALIER
as acylated phosphatidyl glycerols (APGs). These were found in the following taxa: Actinomadura dassonvil/ei; Corynebacterium (bovis; diphtheriae); Nocardioides," and Promicromonospora. They are found only in organisms having phosphatidyl glycerol (PG) as a major component; however, not all PGcontaining strains formed APG. APGs had greater Rf's than PG on TLC plates and in a few cases replaced diphosphatidyl glycerol (DPG) altogether. In most cases both mono-acylated and diacylated PG were present. The latter chromatographed just in back of DPG on TLC plates in System 1; the mono-acylated form co-chromatographed with lyso DPG. In both casesglyceryl phosphoryl glycerol was obtained on mild deacylation and glycerol was the only major product from acid hydrolysis. Acylated phosphatidyl glycerols have previously been reported from Sa/mone//a typhimurium (Loeftier) Castellani Et Chalmers [56, 57], A/carlgenes aquamarinus (Zobell 8 U pham) Hendrie, Holding and Shewan [58], Arthrobacter marinus Cobet, Wirsen and Jones [58], various species of Vibrio Pacini [58] and Bifidobacterium Orla-Jensen [59]. Among the fatty acids which were found on deacylation of the DPG's (or APG's if no DPG was present) of the various actinomycetes studied, several deserve, special mention: (a) tubercu/ostearic acid (10-methyl-octadecanoic acid) was found in large amounts in strains of Actinomadura (madurae and pe//etieri), Corynebacterium bovis, Nocardia (amarae,asteroides,brasi/iens/s,caviae, rhodochrous but not aeroco/onigenes, autotrophica or orientalis) and Mycobacterium (all species examined except Mo abcessus). It was also found variably in Actinomadura dassonvi//ei and Nocardio/des. It was not present in the DPG's of C. diphtheriae or C. aquaticum. (b) Lower homo/ogs of tubercu/ostearic acid, previously reported in Microbispora [60], Micromonospora [37], and Streptornyces [1 9, 43], we have tentatively identified in the DPG's of some strains of Dacty/osporangium, Microbispora, Micromonospora, Micropo/yspora, Nocardioides and Streptosporangium. The identification was based on Rt's on two columns, and lack of reactivity to solvo-mercuration-demercuration. (c) Hydroxy fatty acids, previously reported to be acylated to phosphatidylethanolamine from certain Streptomyces [38, 43] were
identified in some of the strains of the following genera: Actinop/anes, Chainia, Microbispora, Microe//obosporia, Nocardia, Pseudonocardia,Streptomyces, Streptoverticillium and Streptosporangiurn (See Table 2). The methyl esters of these compounds were identified among the organic solvent-soluble deacylation products of the appropriate phosphatidyl ethanolamine fractions by means of TLC versus authentic ~-hydroxy methyl hexacosanoate. The phospholipids of such strains showed two spots containing phosphatidyl ethanolamine on TLC plates: one acylated with non-hydroxylated fatty acids and a more polar compound acylated, at least in part, with hydroxy fatty acids. (d) Unknown unsaturatedfatty acids probably having a branched-chain, were found, usually in small amounts, in certain strains of Actinomadura dassonvil/ei, Actinoplanes, Chainia, Dactylosporangium, Microe//obosporia, Micromonospora, Micropo/yspora faeni, Nocardia (autotrophica and orientalis) Nocardioides, Pseudonocardia, Streptomyces (especially somarlensis) and Streptoverticil/ium. It appears that the phospholipids of Actinomadura pe//etier/differ from those of the other actinomycetes studied in the linkage of some of their fatty constituents. Deacylation by the procedure (methanolic0.025 N LiOH) normally used in this study, releases fatty acids but produces no appreciable H20-soluble phosphate esters of the usual type. For example, deacylation of the "phosphatidyl inositol" fractions of A. pel/etieri 408 or 778 gives rise to three substances. The principal product (Product A) is a phosphorus-containing substance, soluble in both CHCI3 and MeOH but insoluble in H 20 and petrol. In the PC system used for the phosphate esters it is seen as a poorly visualized bluish spot (using the paper phosphorus reagent) running just in back of the front. On TLC in System No. 1, it migrates at an RpI of about 0.4 and reacts well with the phosphorus reagent used for TLC plates. It gives the typical brown-gray color of phosphatidyl inositol with Schiff reagent. The IR spectrum of Product A shows complete disappearance of the ester band at 1730-1740 cm-' but retention of the strong CH JCH3 bands at 2860 and 2930 cm-' of the parent compound. (Deacylation of the phosphatidyl inositol fraction of Micromonospora coeru/ea 12328 yields a typical phosphate ester soluble in MeOH and H20 whose
CHEMOTAXONOMY OF AEROBIC ACTINOMYCETES: PHOSPHOLIPID COMPOSITION
257
taxonomic studies and bring to light some new interesting interrelationships. Once again [68] we find that members of the genera Nocardia and Mycobacterium are indisputably linked. Micropolyspora brevicatena is also closely related to these two genera. It has recently been proposed [68] to limit members of the genus Nocardia to "Actinomycetes containing nocardomycolic acids, having Type IV cell walls and Type A whole cell sugar patterns and not capable of being sharply characterized by morphological criteria." Thus we conclude that on the basis of not only cell wall composition (in N. aerocolonigenes) and lack of nocardomycolic acids (Table 4), but of phospholipid composition, certain "nocardiae," including N. aerocolonigenes, N. autotrophica and N. orienta/is should be excluded from the genus. N. autotrophica strains have some morphological similarities to Pseudonocardia and their phospholipids are similar. As both taxa also lack mycolic acids and share the same cell wall type, a new genus for them might be warranted. Micropo/yspora faeni and our bisporate M. sp. Y 39 are unrelated to the type species M. brevicatena both on the basis of phospholipid composition and lack of nocardomycolic acids. As they are all nonetheless related by morphology and share the same cell wall type, it is proposed to leave them all together in Micropolyspora, at least for the time being. Corynebacteria having a Type IV cell wall appear to be set apart from the Mycobacterium-Nocardia complex by their content of phosphatidyl glycerol and acyl phosphatidyl glycerols. More organisms of this genus should be analyzed. C. aquaticum (Type VI cell wall), not unexpectedly, differs markedly from these corynebacteria. We could not isolate either phosphatidyl inositol mannosides or phosphatidyl ethanolamine from C. aquaticum although Khuller and Brennan [40], using the same strain, did report the presence of these phospholipids. The previously recognized close relationships of Promicromonospora and Oerskovia [69], Streptosporangium and Microbispora [2] and Actinomadura and Microtetraspora [1 ] are underlined by their similarities in phospholipid composition. The creation of the genus Nocardioides Prauser to accommodate nocardoform actinomycetes having a cell wall of Type I [13] and the recent removal of Actinomadura dassonvillei to the new genus Nocardiopsis Meyer [1 2] are also supported by Discussion The results of our studies on phospholipids out findings. As can be seen in Table 2, the underline old relationships based on other phospholipids of A. dassonvi/lei differ markedly
IR spectrum shows the loss of the ester band and marked reduction of the CH 3/CH 2 bands.) Two other minor products are formed. One, Product B, is phosphorus-negative and soluble in petrol. It migrates with batyl alcohol and just ahead of monopalmitin in System No. 1 on TLC and like the monoglycerides, it reacts rapidly with Schiff's reagent to give a bright purple color. The other minor product is a CHCI3soluble, phosphorus-positive compound with an Rel of about 1.2. None of these products are obtained when authentic PI or PI from other actinomycetes are deacylated. As it seemed that Product A might be the lyso form of either alkenyl-acyl (plasmalogen) or alkyl-acyl glycerophosphoryl inositol, it was subjected to the stronger deacylation procedure of Dittmer and Wells [61 ] reported to cleave the more resistant acyl groups of plasmalogens. This resulted in a more complete degradation of the parent compound with the same products produced. The strong deacylation was followed by an acid hydrolysis (hydrochloric acidmercuric chloride) known to cleave the alk-Ienyl link of plasmalogens [61 ]. A small amount of phosphate ester was formed but most of Product A was recovered unaltered. No aldehydes (the products of the cleavage of the alk-l-enyl groups of a plasmalogen by acid) could be demonstrated in the organic fraction from the acid hydrolysis procedure by TLC versus authentic palmitaldehyde. Neither the parent compound, Product A or any of the products from the acid hydrolysis reacted with 2,4-dinitrophenylhydrazine. This appears to eliminate the possibility that the A. pel/etieri phospholipids are plasmalogens. The parent compound did not give a positive reaction with the amido group reagent of Bishel and Austin [62]; thus, sphingolipids are probably not present. We conclude that Product A may be the lyso form of an alkyl-acyl phosphatidyl inositol and Product B a mono-alkyl glyceride. Alkyl-acyl phosphatides have been reported in Proteus mirabilis Hauser [63] and vertebrate tissues [64, 65]. Glycerophospholipids with ether linkages have been tentatively identified in mycobacteria [66] and mannophospholipids in ether linkage with long chain alcohols have also been reported from this taxon [67]. A summary of our findings is presented in Table 6.
258
MARY P. LECHEVALIER,CLAUDE DE BIEVREAND HUBERT LECHEVALIER
f r o m t h o s e o f A. madurae j u s t as t h o s e of Nocardioides d i f f e r f r o m Nocardia and Strepto-
myces.
Experimental Cultures The cultures examined are listed in Table 1.
Growth of Cells All strains, except as noted below, were grown in yeast extract-dextrose broth (1% Difco yeast extract; 1% dextrose in tap H 20; pH 7.2 before autoclaving) on a reciprocal shaker (3 in. stroke; 60 strokes per min) at 28 ° or 37 ° . The cells were harvested in early to mid stationary phase. The broth cultures were autoclaved; the cells, separated by centrifugation, were held at - 2 5 ° until used. M/cropolyspora brevicatena I 1086 was grown in N-Z Amine-glycerol broth (NZG) [70] in static culture, Mycobacterium tuberculosis var. hominis H37Ra in Sauton's medium or TB broth [6] in static culture, and Actinoplanes rectilineatus LL-7-10 in NZG (shaken).
Extraction of Lipids Cells were defrosted at room temperature and broken by sonication for 2-10 min in a Raytheon 10 kc sonic oscillator. The broken cells were separated from the aqueous supernatant by centrifugation at 4600-18, 400 x g and extracted 3 x by shaking with CHCI~-MeOH (2:1) for 2-3 days on a reciprocal shaker at 28 ° . The aqueous supnatant from the centrifugation was extracted once with CHCI3. The extracts were combined and taken to dryness at 50 ° in a rotary evaporator. In some cases, for comparison, phospholipids were also extracted from whole cells using phenol according to the method of De Siervo [71], or directly from membranes isolated from cell sonicates by differential centrifugation.
Purification of the Phospholipids The crude extract was dissolved in CHCI3 and separated into neutral lipid, glycolipid and phospholipid fractions by column chromatography on activated silica gel (J. T. Baker Chemical, Phillipsburg, N, J., 80-200 mesh or equivalent) by the method of Rouser eta/. [72]. The crude methanolic phospholipid fraction from the coloumn was taken to dryness in a rotary evaporator as before, the residue dissolved in CHCI3 and added to 5 x volume of cold (4 ° ) dry Me2CO saturated with MgCI2. The precipitate was centrifuged off and washed once with cold Me2CO-MgCI2 and taken up in 5-10 ml of CHCI3-MeOH-H=O (60:30:4.5) with mild heat if necessary to effect solution, and purified of contaminating amino acids and sugars by passage through a Sephadex G 25 column according to Wells and Dittmer [73]. The eluate from this column was taken by dryness under N2 with mild heat, taken up in 5 ml of CHCI3 and washed twice with 5 ml of 0.5% aqueous NaCI. The CHCI3 layer was taken to dryness as before under N2, then 100 mg spotted on 10 PF254 (Brinkmann Instruments, Westbury, N. Y., U.S.A.) silica gel TLC plates (10 g of silica per one 10 x 20 cm plate) and developed in CHCI3-MeOH-H20 (70:35:7) (System 1). The center of the plate was masked and the outer edges of
the various bands visualized by spraying with the reagent for phosphorus-containing compounds of Dittmer and Lester [74]. The unsprayed portions of the bands were scraped off and eluted with 75 ml of CHCI3-MeOH (2:1) followed by the same solvents in the proportions (1:2). The eluates from each band were combined and brought to dryness under N2 to yield the purified phospholipids.
Analysis of the Phospho//pids The phospholipids were characterized by spraying with various reagents including the Munier and Macheboeuf modification of Dragendorff's reagent (for choline, methylethanolamine and ethanolamine-containing phospholipids), ninhydrin (amino groups) and anisealdehyde (for sugar-containing compounds) [74]. Schiff reagent was also used to verify the identity of phosphatidyl inositol (gray-brown color) and phosphatidyl glycerol (bright rapid purple) [75]. In the latter case, the phospholipids were separated by two-way TLC using System 1 (above) in one direction followed by a CHCI3-HOAc-MeOH-H20 system (80:18:12:5) (System 2) at right angles to the first development. Further identification of the phospholipids was carried out by mild deacylation of the purified fractions according to Dawson [76]. modified as follows: 2-4 mg of pure phospholipid is dissolved in 0.4 ml of petrol (b.p. 40-55°). In some cases it is necessary to add 1-2 drops of MeOH, To this solution is added 3.75 ml MeOH, 0.37 ml distilled H20 and 0.12 ml 1N LiOH.The whole is mixed and incubated in a waterbath at 37 ° for 20 min. The deacylated mixture was neutralized to pH 5.0-6.0 by addition of BioRex 70 (H+) (Calbiochem, LaJolla, Calif., U.S.A.) and extracted with 10 ml of petrol. The petrol, which contained the fatty acids (as methyl esters ) released by the deacylation, was analyzed by GLC (FID) on 3% OV-1 or 15% DEGS (Stabilized diethylene glycol succinate; Analabs, North Haven, Conn., U.S.A.) columns. Unsaturation of the fatty acids was verified by the gas chromatography of the methoxy derivatives made using the solvo-mercuration demercuration technique [49]. The BioRex resin was separated from the methanolic phase and eluted with 80% aqueous EtOH. The ethanolic eluates were combined with the methanolic phase and this mixture taken to dryness under N 2. These residues, which contained the phosphate esters from the deacylation of the phospholipids, were dissolved in MeOH-H 20 (1:1) and spotted on Whatman No. 1 paper along with the phosphate esters prepared in the same way from authentic phospholipid standards (Supelco, Beltefonte, Pa.; Applied Science Labs, State College, Pa.; Analabs, North Haven, Conn., U.S.A.). Following development in IpOH-NH~ (2:1) for 18 hr, the dried papergrams were sprayed first with ninhydrin, heated at 105 ° to visualize glyceryl phosphoryl ethanolamine or glyceryl phosphory methylethanolamine (purple spots) and then with the Dawson modification of the Haynes-lshervvood molybdate reagent [76] and irradiated with UV to develop the blue spots corresponding to the phosphate esters. The pure phospholipids were also analyzed by acid hydrolysis. For amino acids, 1-2 mg of phospholipid
CHEMOTAXONOMYOF AEROBICACTINOMYCETES:PHOSPHOLIPIDCOMPOSITION were hydrolyzed with 6N HCI for 18 h at 100 ° in a sealed tube. The hydrolyzate was extracted with chloroform to remove the fatty materials and the HCI removed from the sample as described by Becker et al. [4]. The samples were spotted on Whatman No. 1 paper and developed in n-BuOH-pyridine-H20-HOAc (60:40:30:3) for 48 h, then dried, sprayed with ninhydrin and heated at 105 ° to detect amino acids, amino sugars and amino alcohols followed by the Dragendorff reagent for choline. Sugars and polyols were obtained by hydrolysis of 2 mg of phospholipid in 3N H2SO4 for 3 h at 100 °. The H2SO4 was removed as the BaSO4 salt by addition of saturated Ba(OH)2 until the pH was 5.0, and centrifuging. The supernatant was taken to dryness in vacuo at room temperature, red/ssolved in a small amount of H20, spotted on Whatman No. 1 paper and developed in freshly prepared n-BuOHpyridine-H20-toluene (5:3:3:4, upper) system for 48 h or in MeOH-H20 (9:1) for 6 h or whenever the front reached the bottom of the paper. Visualization of sugars and polyols was made by spraying with acid aniline phthalate and heating to 105 ° for reducing sugars followed by dipping in the tolidine reagent of White and Frerman [77] for polyols. Other reactions used included ammoniacal silver nitrate, the Elson-Morgan reagent and the ninhydrin degradation for glucosamine [78]. Acknowledgements--We wish to thank Eva Fekete and Magda Gagliardi for technical assistance, and D. Minnikin who suggested the utility of two-way TLC in confirming the presence of certain phospholipids. This study was supported in part by National Science Foundation Grant DEB 73-01 692. Claude de Bi~vrewas the holder of a Fellowship from the French Ministry of Foreign Affairs.
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