INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY Vol

CHEMICAL COMPOSITION AS A CRITERION IN THE CLASSIFICATION OF AEROBIC ACTINOMYCETES

Mary P. Lechevalier and Hubert Lechevalier

Institute of Microbiology, Rutgers University, The State University of New Jersey, New Brunswick, New Jersey

ABSTRACT. The information concerning the main compo- nents found in cell wall preparations and whole-cell hydrolysates of some 600 strains of aerobic actinomycetes is reviewed. The results show that whole-cell sugar patterns can usually be used to predict cell wall composition and that the combination of both criteria permit separation of aerobic actinomycetes into 10 taxonomically useful groups.

Eight years ago, mainly due to the interesting results published by Dr. Cummins, we started to consider cell wall composition as a criterion in the taxonomy of aerobic actinomycetes. We were at that time spending a year at the Pasteur Institute in Paris and it was in the congenial atmosphere generated by our French colleagues, their food and their wines, that our cell wall project was launched. The National Institutes of Health (Grant A1 05489) and the National Science Foundation (Grants G-9694, GB-511, GB-3397, GB-5810) have permitted us to study the morphology and chemical composition of some 600 strains of aerobic actinomycetes, It is thus some eight years and 600 strains later that we have the pleasure, through the initiative of the same Dr. Cummins, of reviewing our results. Cummins himself (1962) summarized, in the language of Pasteur, what Gas known at the time of the beginning of our studies. The present quote has been translated into that of Lister: "One can distinguish two types of aerobic actinomycetes that one may call, in a simplified fashion, the Streptomyces-type and the Nocardia-type. The cell walls of strains belonging to the Nocardia-type contain arabinose, galactose, alanine, glutamic acid and DL-diaminopimelic acid, whereas those belonging to the Streptomyces-type often do not harbor any sugar and contain alanine, glutamic acid, glycine, and LL-diaminopimelic acid or a mixture of the forms LL and DL of this acid." It seemed to us that, to be of value as a criterion in classification, cell wall composition would have to be estimated by simple and rapid methods. In other terms, we felt, and still feel, that the examination of whole cell hydrolysates would have to yield valuable information since one could not and still cannot expect cell wall preparations to be made by those working in diagnostic laboratories. In order to be able to learn how to use the information obtained in the analyses of whole cell hydrolysates, one had however to make and analyze cell watt preparations from a number of different aerobic actinomycetes. 436 INTERNATIONAL JOURNAL

In these studies we believed that the sampling should be large and the identification of the organisms involved should be reasonably secure. One of the first things that attracted our attention, as we proceeded with analyses of cell wall preparations made from representatives of the various genera of actinomycetes, was that strains of Micromono- spora did not contain appreciable quantities of LL-2, 6-diaminopimelic acid (LL-DAP). From the published information that we were aware of, we were expecting either an equal amount of meso and LL-forms of DAP (Hoare and Work, 1957) or mainly LL-DAP with some of the meso form (Cummins and Harris, 1958). We were not aware, at that time, of the work of Jaugstetter (1961) who had found meso-DAP in the cell wall hydrolysates of 5 strains of Micromonospora. Exchange of letters with Dr. Cummins revealed that a clerical error was responsible for the Cummins and Harris report of high con- centrations of LL-DAP in cell wall preparations of strains of Micro- monospora. It then became obvious that streptomycetes were almost unique among gram-positive bacteria with a well developed branching mycelium due to their high content of LL-DAP. Since most of the DAP found in cells is present in their cell wall and since this diamino acid moves slowly during paper chromatography and gives a characteristic olive - green ninhydrin reaction fading to yellow, it was possible to propose a rapid method of differentiation between the streptomycetes and most other actinomycetes by paper chromatography of whole cell hydrolv- sates (Becker g g. 1964). This method is especially valuable, of course, in the identification of streptomycetes which have lost their ability io produce a characteristic, sporulating aerial mycelium. Inversely, the method is useful in differentiating from streptomycetes those strains of Nocardia, such as many strains of g.asteriodes, which have a well developed and sporulating aerial mycelium. As analyses of cell wall preparations of aerobic actinomycetes accumulated, it was noted that they fell into four main groups (Becker --et at., 1965; Yamaguchi, 1965). Looking at actinomycetes and their relatives, aerobic and anaerobic, one can, at present, recognize 9 cell watt types (Table I). These do not correspond readily to any of the 9 types reported by Robinson (1968) for coryneform bacteria. For one thing, Robinson does not consider the presence of sugars; another difficulty is that he never reports the presence of more than one basic amino acid per cell wall type. Using the whole cell sugar analysis method of Lechevalier (1968), one can recognize four types of whole cell sugar patterns in aerobic actinomycetes which contain the meso-form of DAP (M. P. Lechevalier and Lechevalier, 1969 (Table 11). As a rule, it is possible to predict the cell wall type of a ms- DAP-containing aerobic actinomycete on the basis of its whole cell sugar group. Strains with a Type A whole cell sugar pattern have a cell watt of Type IV, those with sugar patterns of Types B and G have a cell wallaf Type 111 and, finally those with a Type D whole cell sugar pattern have a type I1 cell wall. This information is summarized in Table 111. It is not our intention to review critically all the generic names listed in Table 111. This has already been done by a number of authors Table I

Major constituents of cell walls of actinomycetes and relatives differentiation made between meso-DAP and DD-DAP. No --_-_ Cell wall as partic DAB lysine ornithine acid glycine arabinose galactose tYQe E-DAP DAPLL reaction

I t t + ** Ii t II1 t + IV t t * V t VI t t VII + t t VIII t

lY+ t l-

* Glycine is variably resent in these groups; many amino acids present; ** hydroxy DAP may afso be present. DAB = 2,4-diaminobutyric acid DAP = 2,6 diaminopimelic acid

A 11 preparations contained major amounts of alanine, glutamic acid, glucosamine, and muramic acid. 438 INTERNATIONAL JOURNAL

Table 11

Whole-cell sugar patterns af aerobic actinomycetes

with meso -diaminopimelic acid

-- Diagnostic Type sugars

A galactose, arabinose, no xylose

maduros e B no arabinose or xylose

C none

D xylose, arabinose

(Lechevalier and Lechevalier, 1967; Kiister, 1968, Williams gal. , 1968; H. A. Lechevalier and Lechevalier, 1970; Prauser, 1970). Let us discuss only some specific problems of classification, The only doctrine we would advance in classification is that of practicality. One should be able to locate an unknown entity in the spectrum of known organisms with the minimum amount of fuss and bother. This explains the popularity of morphology in taxonomy. How- ever, all that glitters is not gold and in the same way some very different organisms may look alike. For example, there is no basic morphological difference between some members of the genus Streptomyces and sporulating strains of Actinomadura. In addition, typical morphology is not always exhibited by all strains of a given genus. It is particularly true of strains isolated from clinical materials that the capacity to form characteristic morphological structures has been lost. Non- morphological criteria have to be used to help us place such strains in a proper niche. To help answer these questions, whole cell sugar patterns and cell wall composition are of great value and can be corre- lated with other criteria such as phage sensitivity (Prauser and Falta, 1968). Another problem is how to draw a line in cases where the morpho- logies meet. As we mentioned, there is no morphological difference between a Streptomyces species forming short chains of spores and Actinomadura madurae. Likewise, should an actinomycete forming longitudinal pairs of conidia both on the substrate and on the aerial mycelia be placed in the genus Microbispora or in the genus Micropoly- spora? Taking morphology and chemical composition into consideration, one can thus arrange some of the genera listed into Table I11 as indicated in Table IV. The word "euactinomycetes" refers to the morphologically most advanced actinomycetes. SYSTEMATIC BACTERIOLOGY 439

Table I11 Distribution of cell wall types and whole cell sugar patterns

in actinomycetes and relatives*

Cell wall Sugar I 1 Organisms type pattern

Stre tomyces (Stre toverticillium, Chainia, Actino- I N. c. pyc:dium, ActinoGorangium, Elytrosporangiumj; 1 1 Microellobosporia; Sporichthya; Intrasporangium.

A ctinoplanes ; A mor phosporangium; Ampulla riella; Dactylos porangium; Micr omonos pora.

Actinomadura (madurae-type); Microbispora; Strepto- sporangium; Spirillospora; Pla_no-monospora; Dermato- B philus; 111 A ctinomadura (dass onvi.llei -type) ; T hermoactinomyces ; Actinobifida; Geodermatophilus.

I I IcI I I M cobacterium; Nocardia; some corynebacteria; IV I A I MTcropolyspora; Pseudonocardia; Thermomonospora. Actinom ces (israeli-type); some corynebacteria; some N' 1 celluloAnads. Actinomyces (bovis-type); Rothia; Oerskovia~ Arthrobacter.

Erysipelothrix; some corynebacteria.

Cellulomonas

Myc o plana vlllIx I N.C. = No characteristic sugar pattern. * Based on data published in Becker et al., 1965; Cummins, 1965; Lechevalier, --et al., 1966; Lechevalier, 1968; Luedemann, 1968; M. P. Lechevalier and Lechevalier, 1970; H. A. Lechevalier and Lechevalier, 1970; Sukapure et al. 1970.

Characterization of cell wall types I to IV is based on numerous analyses (see Table IV). Characterization of other cell wall types is tentative. 440 INTERNATIONAL JOURNAL

Table IV. Proposed classification of the euactinomycetes

First figure = number of strains from which cell wall preparations have been analyzed; second figure = number of strains the whole cell sugar pattern of which have been determined.

A Mycelium dividing in all planes. Cell wall type III. Sugar patterns B or C, Dermatophilaceae Dermatophilus 9/5 Geodermatophilus 7/7 B. Mycelium dividing only perpendicularly to the long axis of the hyphae. a) Sporangia A ct ino planac eae 1) cell wall type 11. Sugar pattern D. Actinoplanes 27/2 1 Ampullariella 4/3 Dactylosporangium I1/7

2) cell wall type LII. Sugar pattern.B. Planomonospora 1/ 1 Spirillospora 2 /2 Streptosporangium 13/20

b) No sporangia. Cell wall type 11. Sugar pattern D. Micromonosporaceae Micromonospora 48/26

c) Sporangia or no sporangia. Cell wall type 1. (N. C.) St r e pt omyc etac ea e Streptomyces 128/111 Str eptoverticillium 7/3 Microellobosporia 5 /5 Sporichthya 3 /3

Sugar patterns B or C. d) No sporangia. Cell wall type ILI. T hermoa ctinomycetac ea e Thermoactinomyc e s 14/9 A ctinobifida 1 7 / 17 Microbispora 3 1/67 Actinomadura 113 /I .70 e) No sporangia. Cell wall type IV. Sugar pattern A. Nocardiaceae T hermomonos pora 15/ 12 Micropolyspora 36 /2 6 Nocardia 70/134 Pseudonocardia 15 /13

(Modified from H. A. Lechevalier and Lechevalier, 1970).

N.C. = No characteristic sugar pattern. SYSTEMATIC BACTERIOLOGY 44 1

Left out of Table IV are members of the genus Actinomyces and relatives to be discussed by Dr. Pine and an assortment of llnocardioidll organisms, as they are called by Prauser (1967). These have long been taxonomic trouble-makers (Jensen, 1952). Among the latter are some corynebacteria which have the same cell wall and whole cell sugar patterns as mycobacteria and nocardiae. These are the true corynebacteria, since they include the type species of the genus (Cummins, 1965a). In our estimation, based on the information available at present, in the genera Corynebacterium, Mycobacter - -ium and Nocardia, should be placed only strains with a Type IV cell wall. Differentiation between these three genera cannot be made by cell wall composition but Lipid composition may be useful (Asselineau, 1966). In the case of Nocardia, we have recently proposed the genera Actinomadura (Lecheva lier and Lechevalier, 1970) and QerskoOia (Sukapure et at., 1970) thus removing from this previously hetero- geneous group strains with Type 111 and Type VI cell walls. We believe that suitable generic assignments will eventually be made for those organisms, now called corynebacteria, which have Type V and VII cell walls. Only time will tell if the genus Cellulomonas is at present satisfactorily defined. Our limited studies indicate that this genus harbors at least two groups with different types of cell walls. We have attempted to test the stability of cell wall composition as a criterion in classification (&put g c., 1967). Six representatives of aerobic actinomycetes (Nocardia asteriodes and Micropolyspora brevicatena, cell wall type 1V;A. madurae, Microbispora rosea, cell wall type III; Actinoplanes sp., cell wall type 11; Streptomyces griseus, cell wall type I) were subjected to selecting agents which permitted the isolation of stable variants morphologically different from the parent strain. Whole cell analyses of 134 substrains from the six parents revealed no significant change in the isomeric form of diaminopimelic acid or in sugar constituents. Analyses of cell wall preparations from 52 of these did not reveal any change in the diagnostic constituents of their murein or polysaccharides. It should be emphasized that it was not only the arabo-gatactans from strains having a whole cell sugar pattern of Type A which did not vary; the polysaccharides of strains having whole cell sugar patterns of types B and D were also unchanged. Unlike the first, the latter polysaccharides are not an integral part of the murein, but are removed during its purification. Actually, during the clean-up of the cell wall, they disappear little by little, rather than at a single point in the pro- cedure. That they are relatively firmly bound to the cell is underlined by the fact that they are not removed by extraction with boiling water or by surface-active agents such as sodium dodecyl sulfate. Rather, mild hydrolytic conditions such as extraction with 10 per cent TCA at 4°C or saturated picric acid at 100" are required to separate the polysaccharide from the cell. Formamide and organic solvents dre not effective. We have not attempted to purify these polysaccharides; rather we have concentrated on identifying the unknown sugar llmadurosellcharac- teristic of strains having a whole cell sugar pattern of Type B. This sugar, which is non-fermentable, migrates just ahead of arabinose in many chromatographic systems. It gives a brown color 442 INTERNATIONAL JOURNAL

characteristic of C-even sugars with acid aniline phthalate but no reaction with ninhydrin, maphthorescorcinol or boric acid. Hence, it is probably not an amino sugar, and it is not a ketose or 2-deoxy sugar. We have isolated madurose from the polysaccharide of a soil isolate of Actinomadura madurae by hydrolysis of whole cells followed by purification by ion-exchange and preparative paper chromatography. The trimethyl silyl derivative has been isolated by preparative gas- chromatography. Mass spectrographic analyses are currently being run by Dr. N. N. Gerber of our laboratory. Under the conditions used, the retention time of the silylated madurose is the same as that of attrose (Pigman, 1957), at least it is reported in the literature (Pierce, 1968). In some paper chromatographic systems, however, madurose migrates slightly ahead of a presumably authentic sample of D-altrose kindly supplied by Dr. H. S. Isbell of the National Bureau of Standards. Thus, at present we are still unsure of the identity of madurose. We may thus conclude that the combination of morphology and chemical composition permits the assignment of most aerobic actinomycetes to a number of large groups which are reasonably easy to recognize. The coryneform, or nocardioid bacteria, are less amen- able to characterization by these methods. But we are not giving up, other chemical criteria may eventually help us in the classification of these organisms .

ADDENDUM

Since this paper was presented, the sugar madurose has been identified as 3-0-methvl-D-galattose. (Lechevalier, M. P. and N. N. Gerber. Carbohydrate Research 13(3):451-454. 1970)

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