Antonie van Leeuwenhoek 74: 3–20, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.
3
Rhodococcal systematics: problems and developments Michael Goodfellow1∗, Grace Alderson2 & Jongsik Chun3 1 Department
of Agricultural and Environmental Science, University of Newcastle, Newcastle upon Tyne NE1 7RU, U.K.; 2 Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, U.K. and 3 Center for Marine Biotechnology, Columbus Center, 701 East Pratt Street, Baltimore, MD 21204, U.S.A. (∗ Author for correspondence: E-mail:
[email protected])
Key words: Rhodococcus, taxonomic history, polyphasic taxonomy, nomenclature
Abstract Various approaches that have been used in the development of a system of classification for the genus Rhodococcus are discussed. The application of chemotaxonomic, molecular systematic and numerical phenetic methods have greatly contributed to improvements in the systematics of rhodococci and related mycolic-acid containing actinomycetes. The genus currently encompasses twelve validly described species but improved diagnostic methods are needed to distinguish between them. In addition, evidence from 16S ribosomal RNA sequencing suggests that the genus is still heterogeneous.
Introduction The current renaissance in bacterial systematics can be traced back to the introduction of new taxonomic concepts and the application of novel methodologies in the late 1950s and 1960s. The developments that flowed from these activities became embodied in the emergence of chemotaxonomy, molecular systematics and numerical phenetic taxonomy (Goodfellow & O’Donnell 1993). The application of these methods revolutionised actinomycete systematics with broadly based polythetic classifications replacing the primitive morphologically constructed taxonomies that had held sway for over 100 years. The reliance placed on small numbers of morphological and staining characters caused such confusion in the classification of Corynebacterium, Mycobacterium and Nocardia that some species of these genera were seen to be virtually interchangeable. The importance of examining the morphological features of such organisms undisturbed on agar media was stressed by Ørskov (1923) who classified actinomycetes into three large groups in extensive morphological studies. Many of the organisms in his second group, which would now contain strains belonging to the genera Actinomyces, Nocardia and Rhodococcus, were
found to have morphological features in common with corynebacteria and mycobacteria. In 1931 Jensen proposed the genus Proactinomyces for Ørskov’s group II actinomycetes, but most of his contemporaries avoided the use of this name following the establishment of the priorities of the names Actinomyces and Nocardia for the anaerobic and aerobic actinomycetes, respectively (Waksman & Henrici 1943). A classification of actinomycetes based on the mode of cellular branching was proposed by Bisset & Moore in 1949. They described a new genus Jensenia (formally proposed in 1950) to encompass ‘soil diphtheroids’ which they considered to differ from corynebacteria, mycobacteria and nocardiae. Jensen (1952) raised the possibility of Arthrobacter and Jensenia being synonymous, but it is not clear whether Bisset & Moore examined any arthrobacters. In retrospect, it seems likely that most of Bisset & Moore’s diphtheroids would now be assigned to the genus Rhodococcus. Phillips (1953) suggested that Jensenia was synonymous with Nocardia; later the type species of Jensenia, J. canicruria, was considered to be similar to Nocardia rubra by Adams & McClung (1960) and to Nocardia erythropolis by Adams et al., (1970). The organism was labelled Nocardia canicruria by Adams & McClung (1962).
4 It was clear by the early 1950s that better taxonomic criteria were needed for the classification of corynebacteria, mycobacteria, nocardiae and related actinomycetes. The determination of major wall sugar and amino acid composition led to the assignment of actinomycetes to several wall chemotypes (Lechevalier & Lechevalier 1970) and peptidoglycan types (Schleifer & Kandler 1972). Simple wall composition analyses provided the first unambiguous evidence of a close relationship between corynebacteria, mycobacteria and nocardiae (Cummins & Harris 1958); all such strains contain major amounts of meso-diaminopimelic acid (meso-A2pm ), arabinose and galactose, that is, they have a wall chemotype IV sensu Lechevalier & Lechevalier (1970) and an A1γ peptidoglycan (Schleifer & Kandler 1972). The close relationship between these taxa was further underlined with the discovery that they all contained mycolic acids, that is, high-molecular weight 2-alkyl 3hydroxy branched chain fatty acids. The latter are covalently linked into a peptidoglycan–arabinogalactan– mycolic acid matrix (Sutcliffe 1998). Actinomycetes which contain mycolic acids are collectively referred to as the mycolata (Chun et al., 1996). The taxonomic history of the genus Rhodococcus was initially entwined with that of Gram-positive cocci and subsequently enmeshed with that of mycolic acid-containing actinomycetes. Indeed, the generic name Rhodococcus was introduced into the early bacteriological nomenclature three times (Buchanan 1925). It was first proposed by Zopf (1891) for two species of red bacteria which had been described by Overbeck (1891). These organisms had been known as Micrococcus erythromyxa and Micrococcus rhodochrous. Winslow & Rogers (1906), apparently unaware of Zopf’s report, proposed Rhodococcus as a generic designation for red cocci. They recognised two species, R. fulvus and R. roseus. This proposal may well be regarded simply as an emendation of that of Zopf. Rhodococcus was independently introduced as a bacterial genus by Molisch (1907) who recognised a single species, R. capulatus. This organism is described as belonging with the Athiorhodaceae, a group of sulphur bacteria. This generic name is a homonym of Rhodococcus Zopf. The genus Rhodococcus, with R. rhodochrous as the type species, was recognised in the early editions of Bergey’s Manual of Determinative Bacteriology (Bergey et al., 1923, 1925, 1930, 1934). However, species described as R. agilis, R. cinnebareus, R. corallinus, R. rhodochrous, R. rosaceus and R. roseus
were transferred to the genus Micrococcus in the fifth edition of Bergey’s Manual of Determinative Bacteriology (Bergey et al., 1939), and remained there in the following edition (Breed et al., 1948). Rhodococcallike strains were also assigned to a plethera of other morphologically defined genera during this time. It was only with the sweeping changes that occurred in the classification of Gram-positive cocci and the mycolata, as a result of the application of modern taxonomic techniques, that the genus Rhodococcus was seen as a coherent taxon that encompassed actinomycetes of agricultural, clinical and industrial importance (Finnerty 1992). Recent studies on rhodococcal metabolism have revealed a unique spectrum of novel enzyme activities that have been applied to the industrial production of acrylic acid and acrylamide (Hughes et al., 1998), to stereo- and regio-specific transformations of nitriles (Bunch 1998) and sterols (Warhurst & Fewson 1994), and to enantioselective biotransformations of α-amine amides to (S) α-amino acids (Beard & Page 1998). The metabolic versatility of rhodococci is also illustrated by their ability to degrade xenobiotic pollutants (Dabbs 1998), desulphurise water soluble coal derived material (Denome et al., 1993), synthesise biosurfactants (Lang & Philp 1998), generate hydrogen (Grzeszik et al. 1997) and accumulate radioactive caesium (Tomioka et al., 1994). In addition, certain members of the genus are pathogenic for animals (Prescott 1991; Morton et al., 1998), humans (McNeil & Brown 1994) and plants (Vantomme et al. 1982; Stange et al., 1996). Bell et al. (1998) recently reviewed the biology of the genus. The present article charts the developments which led to the re-introduction of the genus Rhodococcus, reviews changes that have occurred in rhodococcal systematics over the past 20 years and outlines prospects for future developments. Additional improvements in the classification and identification of rhodococci can be expected to further facilitate our understanding of these organisms and thereby promote their use in biotechnology and bioremediation while helping to assuage their detrimental activities as animal and plant pathogens.
The new beginning In the period immediately following the publication of the sixth edition of Bergey’s Manual of Determinative Bacteriology (Breed et al., 1948), Ruth Gordon
5 realised that it was not possible to distinguish between corynebacteria, mycobacteria and nocardiae solely on the basis of morphological and staining properties. In place of the monothetic classifications that had existed up to 1948 she stressed the need to take a polythetic approach and base classifications on similarity of representative strains to overall patterns. The new strategy was applied in a comprehensive series of studies designed to clarify the taxonomy of mycobacteria, nocardiae and streptomycetes. During the course of this work a group of strains believed to represent a definite species was highlighted (Gordon & Mihm 1957, 1959). Members of this taxon had been received from different investigators as representatives of species belonging to the genera Bacillus, Bacterium, Erythrobacillus, Micrococcus, Mycobacterium, Nocardia, Proactinomyces, Rhodococcus, and later Jensenia (Gordon & Mihm 1961). The presumptive new species was provisionally assigned to the genus Mycobacterium because the colonial morphology of the strains resembled that of mycobacteria more closely than it did the colonial morphology of nocardiae, as typified by Nocardia asteroides, and because some of the strains when first isolated from soil were known to be as acid-fast as, for example, strains of Mycobacterium phlei. The oldest specific name carried by the strains was rhodochrous (Overbeck 1891); this name was assigned to the species in accordance with the rule of priority. Gordon (1966) reduced additional nomenclatural species to synonyms of Mycobacterium rhodochrous, noted some variation in the taxon but nevertheless described it as a good species in search of a genus. Corynebacterium, Mycobacterium and Nocardia were offered as possible niches. It soon became evident that Mycobacterium rhodochrous formed a recognisable, albeit heterogeneous, taxon which could be distinguished from the genera Corynebacterium, Mycobacterium and Nocardia. Impressive evidence to this effect came from diverse sources, including antibiotic sensitivity (Goodfellow & Orchard 1974), chemotaxonomic (Goodfellow et al., 1974; Alshamaony et al., 1976a,b; Ratledge & Patel 1976; Collins et al., 1977), DNA:DNA relatedness (Bradley 1973; Mordarski et al., 1976), phage susceptibility (Manion et al., 1964; Pietkiewicz et al., 1974) and serological studies (Magnusson 1962; Ridell & Norlin 1973). However, despite all of this activity it was not possible to resolve the internal taxonomic structure of the taxon as many of the test strains
were incorrectly identified and few were common to all of the studies. The most impressive advance towards resolving the taxonomic status of Mycobacterium rhodochrous came from the application of numerical taxonomy. This approach to classification was designed to assign sizeable numbers of individual strains to homogeneous clusters (taxospecies) using large sets of phenotypic data where all features are given equal weight (Sneath 1962). As stated above, Ruth Gordon had brought this conceptual approach to her vision of a bacterial species (Gordon & Smith 1953, 1955; Gordon & Mihm 1957, 1959) but had not presented it in a way that could exploit the use of electronic computers which were becoming available at the time. In contrast, Sneath developed the mathematical concepts and methods needed to harness the power of computers to sift and sort large sets of phenotypic data. Numerical taxonomic surveys showed that Mycobacterium rhodochrous formed a taxon equivalent in rank with aggregate clusters equated with the genera Corynebacterium, Mycobacterium and Nocardia (Cerbón 1967; Bradley 1971; Goodfellow 1971; Tacquet et al., 1971; Goodfellow et al., 1972, 1974; Kubica et al., 1972; Jones 1975; Tsukamura et al., 1979), although no attempt was made to erect a new genus. An already complex taxonomic situation was further confused following the proposal of the genus Gordona for slightly acid-fast bacteria isolated from soil and from the sputa of patients with pulmonary disease (Tsukamura 1971). As originally proposed this genus contained three species, Gordona bronchialis, the type species, Gordona rubra and Gordona terrae; a few reference Mycobacterium rhodochrous strains included in the study differed by only one or two characters from the organisms isolated from sputum and soil. A further species, Gordona aurantiacus, was described by Tsukamura & Mizuno (1971). Subsequently, additional rhodochrous strains were classified as Gordona rhodochroa, Gordona rosea and Gordona rubropertincta (syn. G. rubra) by Tsukamura (1973, 1974). Strains provisionally classified as Nocardia rubra (Tsukamura 1969) were assigned to a new species, Gordona lentifragmenta (Tsukamura et al., 1975). The taxonomic dilemma raging around what was variously known as the rhodochrous taxon, the rhodochrous complex and Mycobacterium rhodochrous was not resolved in the seventh edition of Bergey’s Manual of Determinative Bacteriology (McClung 1974) where rhodochrous strains were assigned to sev-
6 eral Nocardia species with the proviso that further work was needed to clarify their taxonomic position. In the corresponding section on Mycobacterium, Runyon et al. (1974) excluded rhodochrous strains due to their complete or almost complete lack of acid-fastness and on distinctive biochemical, immunological and phage susceptibility properties. Gordona Tsukamura 1971 and Jensenia Bisset & Moore 1950 were cited as genera incertae sedis by McClung (1974). The taxonomic position of strains assigned to the rhodochrous complex was considered by Bousfield & Goodfellow (1976) who suggested that they might be classified as Mycobacterium rhodochrous, reclassified in an alternative established genus or considered as a genus in their own right. Possible generic names, cautiously intimated by Cross & Goodfellow (1973), were Rhodococcus Zopf 1891, Proactinomyces (Jensen 1931) Bradley & Bond 1974, Jensenia Bisset & Moore 1950 and Gordona Tsukamura 1971. Of these, Rhodococcus, had priority as the only authentic strain of the type species, Rhodococcus rhodochrous, was a member of the rhodochrous taxon (Gordon & Mihm 1957).
The return of the genus Rhodococcus Tsukamura (1974) re-established the genus Rhodococcus to accommodate six species, namely R. aurantiacus, R. bronchialis, R. rhodochrous, R. roseus, R. rubropertinctus and R. terrae, for organisms previously classified in the genus Gordona. Similar conclusions were drawn from the more comprehensive numerical taxonomic study by Goodfellow & Alderson (1977) who gave a comprehensive description of the genus and recognised nine species, R. rhodochrous, the type species, and R. bronchialis, R. coprophilus, R. corallinus, R. equi, R. erythropolis, R. ruber, R. rubropertinctus and R. terrae. They also noted that their R. rhodochrous taxon encompassed two strains which had originally carried the rhodochrous epithet: ATCC 13808 (N54) (R. rhodochrous Zopf 1891) and N31 (Micrococcus rhodochrous Overbeck 1891); the former had been designated the type strain of Mycobacterium rhodochrous (Gordon & Mihm, 1957). The separation of rhodococci from representatives of other mycolic acid-containing genera was also supported by rRNA:DNA similarity data (Mordarski et al. 1980a).
The taxonomic integrity of most of the Rhodococcus species cited above was underpinned by DNA:DNA relatedness data (Mordarski et al., 1976, 1977) which showed that representatives of numerically defined taxospecies shared 70% or more DNA:DNA relatedness with low 1Tm(e) values thereby falling within the guidelines set for the circumscription of bacterial species recommended in the celebrated paper of Wayne et al., (1987). However, subsequent DNA:DNA pairing data showed that the type strains of R. corallinus and R. rubropertinctus represented a single genomic species (Mordarski et al., 1980b). Since R. rubropertinctus (Hefferan 1904) Goodfellow & Alderson 1977 had priority over R. corallinus Goodfellow & Alderson 1977, the latter became a subjective synonym of the former. The re-establishment of the genus Rhodococcus represented a significant milestone in unravelling the complex taxonomic variation encompassed by the mycolata. The genus accommodated strains which showed a wide range of morphological diversity and was defined primarily on the basis of wall envelope composition. The taxon was restricted to actinomycetes that had: (a) a peptidoglycan consisting of Nacetylglucosamine, N-glycolylmuramic acid, D- and L -alanine and D-glutamic acid with meso-A2pm as the diamino acid; (b) arabinose and galactose as diagnostic sugars; (c) a phospholipid pattern consisting of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides (i.e. phospholipid pattern type 11 sensu Lechevalier et al., 1977); (d) a fatty acid profile containing major amounts of straight-chain unsaturated and tuberculostearic acids (i.e. a type IV fatty acid pattern sensu Lechevalier et al., 1977); (e) mycolic acids with 32–66 carbons (Minnikin & Goodfellow 1980, 1981); and (f) dihydrogenated menaquinones with either eight or nine isoprene units (Collins et al., 1977, 1985). Rhodococci were not seen to have any distinctive morphological features other than the ability of many strains to form hyphae that fragmented into rods and cocci, but they did show considerable heterogeneity (Locci 1976, 1981; Williams et al., 1976; Locci & Sharples, 1984). Thus, strains of R. bronchialis, R. rubropertinctus, and R. terrae are amycelial, and show a rod-coccus cycle, but further differentiation is represented by R. erythropolis, R. rhodnii and R. rhodochrous, strains of which exhibit elementary branching prior to fragmentation. A third group was formed by R. coprophilus, R. fascians, and R. ru-
7 ber strains of which produce well-branched substrate mycelia. Finally, R. equi strains show traces of elementary branching at early stages of growth. The time taken to complete the developmental cycle ranges from 24 h in relatively undifferentiated forms such as R. equi to several days for those like R. coprophilus that show the most pronounced morphological differentiation. Members of the genus do not usually form aerial hyphae, but exceptions are R. coprophilus and R. ruber, which produce feeble aerial hyphae, and R. bronchialis strains, which exhibit aerial synnenata (Locci & Sharples 1984). More convincing evidence of the heterogeneity of the re-established genus Rhodococcus came from chemotaxonomic and serological data (Alshamaony et al. 1976a,b; Collins et al., 1977, 1985). All of the species originally assigned to the genus Gordona Tsukamura 1971, that is, R. bronchialis, R. rubropertinctus and R. terrae contained mycolic acids with between 48 and 66 carbon atoms and major amounts of dihydrogenated menaquinones with nine isoprene units whereas R. coprophilus, R. equi, R. erythropolis, R. rhodochrous and R. ruber where characterised by the presence of shorter mycolic acids and dihydrogenated menaquinones with eight isoprene units as the major isoprenologue. The two aggregate groups were also recognised on the basis of antibiotic sensitivity patterns (Goodfellow & Orchard 1974), delayed skin reactions on sensitized guinea pigs, and polyacrylamide gel electrophoresis of cell extracts (Hyman & Chaparas 1977). Additional species added to the genus Rhodococcus over the decade following its re-introduction were R. aichiensis Tsukamura 1982, R. chlorophenolicus Apajalahti et al., 1986, R. chubuensis Tsukamura 1982, R. globerulus Goodfellow et al., 1982, R. luteus (Söhngen 1913) Nesterenko et al., 1982, R. marinonascens Helmke & Weyland 1984, R. maris (Harrison 1929) Nesterenko et al., 1982, R. obuensis Tsukamura 1982 and R. sputi Tsukamura 1978. It was also proposed, though not accepted, that R. lentifragmentus (Kruse 1896), Tsukamura 1978 had priority over R. ruber (Kruse 1896) Goodfellow & Alderson 1977EP (effective publication). In addition, the position of R. aurantiacus (neé Gordona aurantiaca Tsukamura & Mizuno 1971) was seen to be questionable. Early numerical taxonomic data indicated the equivocal position of Gordona aurantiaca in the genus Gordona (Tsukamura 1974, 1975) and the type strain fell outside the Rhodococcus aggregate cluster recog-
nised by Goodfellow & Alderson (1977). Aurantiaca strains were later found to contain characteristic mycolic acids, completely unsaturated menaquinones with nine isoprene units (MK9) and formed a numerically defined taxon equivalent in rank to aggregate clusters corresponding to the genera Corynebacterium, Mycobacterium, Nocardia and Rhodococcus (Goodfellow et al., 1978). Corynebacterium paurometabolum Steinhaus 1941 and Mycobacterium album Söhngen 1913 were seen to have many properties in common with the aurantiaca taxon (Goodfellow & Minnikin 1980; Collins & Jones 1982). The genus Rhodococcus was cited in the Approved Lists of Bacterial Names (Skerman et al. 1980) and is recognised in the current edition of Bergey’s Manual of Systematic Bacteriology (Goodfellow 1989). Five out of the 20 species listed under Nocardia in the Approved Lists of Bacteria Names were classified in the genus Rhodococcus by Goodfellow (1989). Nocardia calcarea Metcalf & Brown 1957 was seen as a synonym of R. erythropolis (Gray & Thornton 1928) Goodfellow & Alderson 1977EP ; N. globerula (Gray 1928) Waksman & Henrici 1948 was reclassified as R. globerulus Goodfellow et al., 1982, as was N. corynebacteroides Serrano et al., 1972; N. restricta Turfitt 1944 was cited as a synonym of R. equi (Magnusson 1923) Goodfellow & Alderson 1977; and N. coeliaca (Gray & Thornton 1928) Waksman & Henrici 1948 had properties consistent with its inclusion in the genus Rhodococcus (Gordon et al., 1974).
16S rRNA sequencing rules okay — or does it? The studies outlined above indicated that while DNA:DNA relatedness and numerical phenetic methods provided valuable data for the circumscription of mycolata species they were of much less value in establishing relationships at and above the genus level or in clarifying the taxonomy of heterogeneous taxa such as Rhodococcus. These limitations became apparent in the early 1980s when methods for establishing quantitative measurements of evolutionary distances between diverse taxa were becoming available. In prokaryotic systematics, comparisons of 16S rRNA sequences were being used to clarify relationships at and above the genus level, not least among actinomycetes (Embley & Stackebrandt 1994). The reasons for choosing 16S rRNA, and its useful features for classification and identification of prokaryotes, have been fully discussed by Carl Woese
8 and his colleagues who pioneered this field of study (Woese 1987; Woese et al. 1990). Two fundamental assumptions underline this approach, namely that lateral gene transfer is rare between 16S rRNA genes, and that the amount of evolution or dissimilarity between 16S rRNA sequences of a given pair of organisms is representative of the variation shown by the corresponding genomes. In general, these assumptions are well placed though evolutionary relationships need to be interpreted with care as phylogenetic reconstruction is based on simple assumptions which can be violated by the data to a greater or lesser extent (Swoffold & Olsen 1990; Hillis et al., 1993). None of the available methods of phylogenetic inference can be relied upon to give the ‘correct tree’ topology due to factors such as lineage dependent inequalities in the rates of change (O’Donnell et al., 1993). 16S rRNA sequence data confirmed the close relationship between the genera Corynebacterium, Mycobacterium, Nocardia and Rhodococcus and showed that these taxa formed a suprageneric group within the evolutionary radiation encompassed by actinomycetes (Ruimy et al., 1994, 1995; Rainey et al., 1995a; Chun & Goodfellow 1995; Chun et al. 1996). Additional studies helped to clarify the internal taxonomic structure of the established mycolata taxa and led to the recognition of several new genera (Rogall et al., 1990; Stahl & Urbance 1990; Pascual et al. 1995; Rainey et al., 1995b; Ruimy et al., 1995; Chun et al., 1997). In particular, 16S rRNA sequence data gave credence to the view that Rhodococcus species could be assigned to two aggregate groups each of which merited generic status (Goodfellow 1989). It was also shown that the aurantiaca taxon formed a genus in its own right as intimated by Goodfellow et al. (1978). The genus Gordona Tsukamura 1971 was reestablished by Stackebrandt et al. (1988) for actinomycetes classified as R. bronchialis, R. rubropertinctus, R. sputi and R. terrae. A further species, R. obuensis, was subsequently found to be a subjective synonym of R. sputi (Zakrzewska-Czerwinska et al., 1988). Similarly, the genus Tsukamurella Collins et al., 1988a was proposed when organisms classified as Corynebacterium paurometabolum Steinhaus 1941 and Rhodococcus aurantiacus Tsukamura & Yano 1985 were merged to form a single species, T. paurometabolum; the specific epithet was later corrected to paurometabola by Yassin et al. (1995). These proposals left the genus Rhodococcus as a more homogeneous taxon encompassing 12 validly described species, namely, R. chlorophenolicus, R. coprophilus,
R. equi, R. erythropolis, R. fascians, R. globerulus, R. luteus, R. marinonascens, R. maris, R. rhodnii, R. rhodochrous and R. ruber. The taxonomic status of the species was supported by numerical phenetic and DNA:DNA relatedness data (Mordarski et al., 1980b; Zakrzewska-Czerwinska et al., 1988; Goodfellow et al., 1990). The speed and enthusiasm by which early practitioners of 16S rRNA sequencing reclassified established taxa and described novel mycolata species was tempered with the realisation that no single 16S rRNA interstrain nucleotide sequence difference value could be set to unequivocally define species boundaries (Stackebrandt & Goebel 1994; Wayne et al., 1996). Indeed, strains of some closely related bacterial species were found to have identical, or almost identical, nucleotide sequences, even though they belonged to different genomic species (Fox et al., 1992; MartinezMurcia et al., 1992). Amongst actinomycetes, this situation is exemplified by Tsukamurella species (Chun et al., unpubl.). These observations indicate that the resolution of DNA:DNA pairing is higher than that of 16S rRNA sequence analysis when closely related species are being compared. Nevertheless, even in this context 16S rRNA sequence data can be used to select appropriate reference strains for the more exacting DNA:DNA relatedness studies, thereby reducing the number of reference strains that need to be examined. Fox et al. (1992) proposed the term rRNA superspecies for organisms which have virtually identical 16S rRNA sequences but can be distinguished using DNA:DNA relatedness data.
The rise of polyphasic taxonomy It is evident from what has been said that the individual approaches to classifying bacteria have various strengths and weaknesses, especially when applied at different levels in the taxonomic hierarchy. 16S rRNA sequencing is commonly used to infer evolutionary relationships but these need to be evaluated and refined using other taxonomic criteria, notably from chemotaxonomic and DNA:DNA pairing studies. Phenotypic tests also provide valuable information for separating closely related species and for identifying unknown strains to validly described taxa. This all-embracing approach is known as polyphasic taxonomy and was introduced by Rita Colwell (1970a,b) to signify successive or simultaneous taxonomic studies on groups of organisms using a combination of genotypic and
9 Table 1. Characteristics of the genus Rhodococcus and other mycolata generaa Characteristics
Corynebacterium
Dietzia
Gordonia
Mycobacterium
Nocardia
Rhodococcus
Skermania
Tsukamurella
Cell morphology
Pleomorphic rods, often club-shaped; commonly in angular and palisade arrangement
Short rods and cocci
Rods and cocci or moderately branching hyphae
Rods, occasionally branched filaments that fragment into rods and coccoid elements
Mycelium which later fragments into rods and cocci
Rods to extensively branched mycelium; the latter fragments into irregular rods and cocci
In early stages of growth (24 h) substrate mycelium resembles a pine tree
Rods occurring singly, in pairs or in masses; coccobacillary forms produced
Aerial hyphae Times for visible colonies (days) Degree of acid-fastness (not necessarily also alcohol fastness)
Absent 1–2
Absent 1–3
Absent 1–3
Usually absentb 2–40
Present 1–5
Absent 1–3
Present 9–21
Absent 1–3
Sometime weakly acid-fast
Not acid fast
Often partially acid-fast
Usually strongly acid-fast
Often partially acid-fast
Often partially acid-fast
Not acid fast strongly
Weak to acid-fast
Strictly aerobic Peptidoglycan typec
Alγ
+ Alγ
+ Alγ
+ Alγ
+ Alγ
+ Alγ
+ Alγ
+ Alγ
Acyl group of muramic acidd
N-acetylated
N-acetylated
N-glycolated
N-glycolated
N-glycolated
N-glycolated
N-glycolated
N-glycolated
Fatty acid typese
S, U, (T)f
S, U, T
S, U, T
S, U, T g
S, U, T
S, U, T
S, U, T
S, U, T
Mycolic acid typeh Overall size (number of carbons)
Single spot 22–38
Single spot 34–38
Single spot 46–66
Multiple spots 60–90
Single spot 48–60
Single spot 30–54
Single spot 58-64
Two spots 64–78
Number of double bondsi
0–2
ND
1–4
1–3
0–3
0–2
2–6
1–6
- Fatty acid esters released on - pyrolysis (number of carbons)
8–18
ND
16–18
22–26
12–18
12–16
16–20
20-22
Phospholipid typej
1
2
2
2
2
2
2
2
Predominant menaquinone(s)k
MK-8(H2 ) or
MK-8(H2 )
MK-9(H2 )
MK-9(H2 )
MK-8(H4 , ω-
MK-8(H2 )
MK-8(H4 ,ω−
MK-9
51–59 -
73 (-)
63–69 -
62–70 +
64–72 (-)
67–73 -
67.5 (-)
67–74 -
ND ND ND
ND ND ND
+ + +
ND ND
-
+ + +
+ + +
-
MK-9(H2 ) G+C content of DNA (mol%) Arylsulphatase produced Sensitivity to: - 5-Fluorouracil (20µg/ml) - Lysozyme (50µg/ml) - Mitomycin C(5µg/ml)
cycl)l
Symbols: +, positive, -, negative, (-),some strains give positive results, ND, not determined. a Data taken from Chun et al. (1997), Goodfellow & Magee (1997) and Yassin et al. (1997). b Mycobacterium farcinogenes and Mycobacterium xenopi may occasionally produce aerial hyphae. c A, cross-linkage between positions 3 and 4 of adjacent peptide subunits, 1, peptide bridge absent; γ , meso-A 2pm at position 3 of the tetrapeptide subunits (Schleifer & Kandler, 1972). d Acyl group detected using a simple glycolate test (Uchida & Aida, 1979). e Abbreviations: S, straight chain; U, monounsaturated; T, tuberculostearic acid (10-methyloctadecanoc acid; parentheses indicate variable occurrence. f Corynebacterium bovis contains tuberculostearic acid (Lechevalier et al. 1977). g Mycobacterium gordonae only contains traces of tuberculostearic acid (Minnikin et al. 1985). h Number of mycolic acid spots produced from whole organism methanolysates (Minnikin et al. 1975, 1984a,b; Yassin et al. 1997). i In mycobacterial mycolic acids, double bonds may be converted to cyclopropane rings; methyl branches and other oxygen functions may be present (Dobson et al., 1985; Minnikin et al., 1984a,b). j Phospholipid types: 1, phosphatidylglycerol (variable) and phosphatidylinositol; 2, phosphatidylethanolamine (Lechevalier et al., 1977). k Example of abbreviations: MK-9( ), menaquinone having two of the nine isprene units hydrogenated. 2 l Nocardiae were originally reported to have predominant amounts of MK-8(H ). However, the major component was later shown to 4 correspond to a hexahydrogenated menaquinone with eight isoprene units in which the end two units of the multiprenyl side chain were cyclized (Howarth et al., 1986; Collins et al., 1987).
phenotypic data. The case for polyphasic taxonomy has been reiterated and extended by Vandamme et al. (1996) and Goodfellow and his colleagues (1997), and the approach widely used to further clarify the taxonomy of mycolata taxa. At the genus level, the polyphasic taxonomic approach led to proposals for the genera Dietzia (Rainey et al., 1995b) and Skermania (Chun et al. 1997) for actinomycetes previously classified as R. maris (Harrison 1929) Nesterenko et al., 1982 and N. pinensis
Blackall et al., 1989, respectively. These proposals raise the number of mycolata genera to eight (Figure 1). All of these taxa can be distinguished from one another using a combination of biochemical, chemical and morphological properties (Table 1). Representatives of the genera Gordonia, (formerly Gordona) Nocardia, Rhodococcus and Tsukamurella can also be distinguished by two-dimensional polyacrylamide gel electrophoresis of ribosomal protein AT-L30 (Ochi 1992).
10
Figure 1. Unrooted neighbour-joining tree based on 1394 unambiguously aligned nucleotides. The asterisks indicate branches that were also recovered with the Fitch-Margoliash, maximum-likelihood and maximum-parsimony algorithms. The numbers at the nodes indicate the level of bootstrap support based on a neighbour-joining analysis of 1000 re-sampled databases. The scale bar indicates 0.01 substitutions per nucleotide position.
11
Figure 2. Secondary structure of 16S rDNA between nucleotide positions 76 and 85 using numbering based on Nocardia otitidiscaviarum.
The congruence between the discontinuous distribution of the chemical markers and the emerging phylogeny of the mycolata led Chun et al. (1996, 1997) to classify mycolata genera into two suprageneric groups, namely the families Corynebacteriaceae and Mycobacteriaceae. The genera Corynebacterium and Dietzia were assigned to the emended family Corynebacteriaceae, and the genera Gordonia, Mycobacterium, Nocardia, Rhodococcus, Skermania and Tsukamurella to the revised family Mycobacteriaceae. Corynebacterium amycolatum Collins et al., 1988b and Turicella otitidis Funke et al., 1994, which lack mycolic acids, fall within the evolutionary radiation of the family Corynebacteriaceae (Figure 1). The hierarchic classification of actinomycetes proposed by Stackebrandt et al. (1997) is based on 16S rDNA/rRNA sequence clustering and the distribution of taxon specific signature nucleotides. In this system, the genera Corynebacterium and Turicella are classified in an emended family Corynebacteriaceae; the genus Dietzia in Dietziaceae fam. nov., the genus Gordonia in Gordoniaceae fam. nov., the genera Nocardia and Rhodococcus in the family Nocardiaceae, and the genus Tsukamurella in Tsukamurellaceae fam. nov. These taxa were assigned to the suborder Corynebacterineae subordo.nov. of the order Actinomycetes Buchanan 1918. Not all of the families proposed by Stackebrandt and his colleagues can be distinguished using chemical markers known to be of value in mycolata taxonomy. Several mycolata species have been reclassified and others reduced to synonyms of previously described taxa in polyphasic taxonomic studies focused on establishing relationships below the genus level. Rhodococcus aichiensis Tsukamura 1982 and R. chlorophenolicus Apajalahti et al., 1986 have been reclassified as Gordonia aichiensis (Klatte et al. 1994a)
and Mycobacterium chlorophenolicum Häggblom et al. (1994), respectively. In addition, R. chubuensis Tsukamura 1982, R. luteus Nesterenko et al., 1982 and R. roseus Tsukamura et al., 1991 have become synonyms of Gordonia sputi (Tsukamura 1985) Stackebrandt et al., 1988EP (Riegel et al. 1994), Rhodococcus fascians Goodfellow 1984 (Klatte et al., 1994b) and Rhodococcus rhodochrous (Zopf 1891) Tsukamura 1974 (Rainey et al., 1995c), respectively. Rainey and his colleagues also confirmed that Nocardia calcarea Metcalf & Brown 1957 should be seen as a synonym of Rhodococcus erythropolis (Gray & Thornton 1928) Goodfellow & Alderson 1977EP and Nocardia restricta Turfitt 1944 as a synonym of Rhodococcus equi (Magnusson 1923) Goodfellow & Alderson 1977. These proposals, when taken with the reclassification of Nocardia amarae Lechevalier & Lechevalier 1974 and Nocardia pinensis Blackall et al. 1989 as Gordonia amarae (Klatte et al., 1994a) and Skermania piniformis (Chun et al. 1997), leave the genus Nocardia as a homogeneous taxon for the first time in its long taxonomic history. During this period three new Rhodococcus species were described, that is, R. opacus (Klatte et al., 1994c), R. percolatus (Briglia et al., 1996) and R. zopfii (Stoecker et al., 1994). It is also clear from Figure 1 that T. wratislaviensis Goodfellow et al. 1991 belongs to the genus Rhodococcus.
Emended description of Rhodococcus Zopf 1891 The changes outlined above leave the genus Rhodococcus as a more cohesive taxon that encompasses 12 validly described species, namely R. coprophilus, R. equi, R. erythropolis, R. fascians, R. globerulus, R. marinonascens, R. opacus, R. percolatus, R. rhodnii, R. rhodochrous, R. ruber and R. zopfii. The key properties of these organisms are given below. Rhodococci are aerobic, Gram-positive to Gramvariable, non-motile, catalase-positive actinomycetes that are usually partially acid-alcohol fast. In all strains the life cycle starts with the coccus or short rod stage, with different organisms showing a succession of more or less complex morphological stages by which the completion of the growth cycle is achieved: cocci may germinate only into short rods, or form filaments with side projections, or show elementary branching, or in the most differentiated forms produce extensively branched hyphae. The next generation of cocci or short rods is formed by fragmentation of the rods, filaments and hyphae. Some strains produce feeble, microscopi-
12 Table 2. Characters differentiating species of the genus Rhodococcus R. R. coprophilus equi
Morphogenetic H-R-C sequence Yellow colonies
R. zopfii
R-C
EB-R-C
H-R-C EB-R-C
H-R-C R-C
EB-R-C
EB-R-C EB-R-C
H-R-C H-R-C
-
-
+
-
-
-
-
-
-
-
-
+ -
+ v
+
+ -
v
ND ND
ND ND
-
+ +
-
ND ND
+ + + + -
v + v + + + + -
+ + + + + + -
+ + + + + + v +
+ v v -
+ + + + ND -
+ + + + + ND + ND + + ND
+ + + + + + + ND +
+ + +
+ + + + + -
v + + + + + -
ND ND + + ND ND ND ND
38–48
30–38 34–38
38–52
ND
ND
48–53 46–54
38–52
36–50
40–50 33–36
Degradation of: Adenine Tyrosine + Growth on sole carbon source (1%, w/v) Cellobiose Galactose Inositol Maltose Mannose Mannitol Ribose Sorbitol Sucrose Turanose Xylose Mycolic acids (number of carbons)
R. R. R. R. R. R. R. R. R. erythropolis fascians globerulus marino- opacus percolatus rhodnii rhodochrous ruber nascens
Symbols: +, ≥90% of strains positive; 90% of strains negative; v, variable; ND, not determined. Data taken from Goodfellow (1989), Goodfellow et al. (1991), Klatte et al. (1994c), Stoecker et al. (1994), and Briglia et al. (1996).
cally visible aerial hyphae, which may be branched or aerial synnemata that consist of unbranched filaments that coalesce and project upwards. Most strains grow well on standard laboratory media at 30◦C, though some require thiamine for growth. Colonies may be mucoid, rough or smooth and pigmented buff, cream, yellow, orange or red, although colourless variants do occur. The organism has an oxidative carbohydrate metabolism, is sensitive to lysozyme, does not produce mycobactins, is arylsulphatase negative and is unable to degrade casein, cellulose, chitin, elastin, xanthine or xylan. Rhodococci are able to use a wide range of organic compounds as sole sources of carbon and energy for growth. The peptidoglycan is of the A1γ type, contains meso-A2pm as the diamino acid and muramic acid
with N-glycolyl residues. The polysaccharide fraction of the wall is rich in arabinose and galactose. The wall envelope contains mycolic acids with 30– 54 carbon atoms and up to three double bonds and major proportions of straight-chain saturated, unsaturated, and 10-methyl (tuberculostearic)-branched fatty acids. Fatty acid esters released on pyrolysis gas chromatography of mycolic acid esters have 12–18 carbon atoms. Cells contain diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides as major phospholipids. Dihydrogenated menaquinones with eight isoprene units (MK
