(Rnase P) RNA Genes from LL-2,6- Diaminopimelic Acid-Containing Actinomycetes

International Journal of Systematic and Evolutionary Microbiology (2000), 50, 2021–2029 Printed in Great Britain

Comparative sequence analyses of the ribonuclease P (RNase P) RNA genes from LL-2,6- diaminopimelic acid-containing actinomycetes

Jung-Hoon Yoon and Yong-Ha Park

Author for correspondence: Yong-Ha Park. Tel: j82 42 860 4620. Fax: j82 42 860 4598. e-mail: yhpark!mail.kribb.re.kr

Environmental Bioresources Ribonuclease P (RNase P) RNA genes of LL-2,6-diaminopimelic acid (LL-A2pm)- Laboratory, Korea Research containing actinomycetes, except Streptomyces species, were sequenced after Institute of Bioscience and Biotechnology (KRIBB), PCR-ampliﬁcation and cloning. By using the sequence data, the relationships PO Box 115, Yusong, between species within genera and the relationships between taxa above Taejon, Korea genus level were investigated and the usefulness of the RNase P RNA gene as another phylogenetic marker was evaluated. RNase P RNA gene sequences of all strains used in this study contained relatively conserved regions along with highly variable regions. The mean RNase P RNA gene similarity value was approximately 82O18% and the mean RNase P RNA gene similarity value when gaps were included was approximately 76O24%. The nucleotide similarities between the RNase P RNA genes of different strains were mostly fewer than the 16S rDNA similarities. The RNase P RNA gene was more useful than 16S rDNA for clearly differentiating the relationships between species belonging to a genus and the relationships between some genera. However, nucleotide sequences of RNase P RNA genes were not necessarily appropriate for comparisons at all taxonomic levels (such as those between species, between genera and between families).

Keywords: RNase P RNA gene, -2,6-diaminopimelic acid, actinomycetes, phylogeny

INTRODUCTION these, 16S rRNA or 16S rDNA have been extensively studied and have become important as target genes The best means of investigating the genetic relationship in the phylogeny and classification of prokary- between organisms is to compare their total genomic otic organisms (Woese & Fox, 1977; Woese, 1987; sequences. However, this approach is not likely to be Stackebrandt et al., 1997). Nevertheless, there is a possible without innovation in the existing molecular general consensus that 16S rRNA gene sequences may biological and analytical techniques. In the meantime, be inappropriate for elucidating the relationships DNA–DNA hybridization and some useful gene frag- between closely related organisms and may be in- ments are used for elucidating genetic relationships or sufficient to guarantee species identity (Fox et al., in phylogenetic studies. Such gene fragments include 1992). DNA–DNA relatedness is generally recognized genes for rRNAs (Woese, 1987), the β-subunit of ATP- as being very important for defining species in synthase (Amann et al., 1988), tRNA (Hofle, 1990), current bacterial systematics (Wayne et al., 1987; glutamine synthetase (Brown et al., 1994), elongation Stackebrandt & Goebel, 1994). However, despite the factor Tu (Kamla et al., 1996), a 65 kDa heat-shock fact that it provides a decisive criterion in species protein (hsp65) (Swanson et al., 1997) and a histone- definition, DNA–DNA reassociation also has some like protein (hbb) (Valsangiacomo et al., 1997). Among experimental problems, being affected by various factors such as DNA concentration, DNA purity, size

...... of DNA fragments, incubation temperature, diﬀer- ences in the presence of RNA and so forth (Johnson, Abbreviations: LL-A2pm, LL-2,6-diaminopimelic acid; ITS, internally transcribed spacer. 1991); in addition, diﬀerent methods are used to The GenBank accession numbers for the sequences of the RNase P RNA determine the levels of DNA–DNA relatedness genes determined in this study are listed in Table 1. (Vandamme et al., 1996). Therefore, quantitatively

01208 # 2000 IUMS 2021 J.-H. Yoon and Y.-H. Park identical results are not always obtained. This also its value as a marker for identification, classification makes it difficult to construct databases using and phylogeny. Such sequences will be used to extend DNA–DNA relatedness results. RNase P RNA gene-based systematic studies to other genera. It is also necessary to investigate whether the Although 16S rDNA sequencing and DNA–DNA RNase P RNA gene can be used to elucidate the relatedness tests are indispensable tools in current relationships between distantly related taxa (such as bacterial systematics, additional genetic markers also those between genera or families). may be necessary to support the results of 16S rDNA sequencing and DNA–DNA relatedness, in line with In this study, -2,6-diaminopimelic acid (-A#pm)- trends in polyphasic taxonomy. Recently, the se- containing actinomycete taxa, except Streptomyces quences of the 16S–23S rDNA (16S–23S) internally species, were chosen for extending the phylogenetic transcribed spacer (ITS) have been shown to be useful study based on RNase P RNA gene sequences. - for elucidating the relationships between closely re- A#pm-containing actinomycetes are a taxonomically lated strains or species (Leblond-Bourget et al., 1996; and biotechnologically interesting group. Representa- Yoon et al., 1997a, 1998b). However, the 16S–23S tives of -A#pm-containing coryneform and no- ITS also has some disadvantages as a phylogenetic cardioform actinomycetes, as well as the genus Strepto- marker. The large variation in sequence and length of myces, have the ability to accumulate phosphate 16S–23S ITSs make it difficult to infer relationships (Nakamura et al., 1995), to produce the antibiotic between distantly related taxa (e.g. between taxa above erythromycin (Miller et al., 1991) and to degrade some genus level). Also, heterogeneities in sequence and harmful aromatic compounds (Yoon et al., 1997c, length of 16S–23S ITSs within one organism have been 1999). Currently, -A#pm-containing actinomycetes found (Hain et al., 1997; Yoon et al., 1998b). Such fall into five phylogenetic lineages, as shown by 16S heterogeneities, caused by the existence of multiple rRNA gene sequence analyses (Stackebrandt et al., 16S–23S ITS alleles, may reduce the value of the 1997). The five lineages represent the following families 16S–23S ITS as a phylogenetic marker. Direct (Stackebrandt et al., 1997): the family Nocardioidaceae sequencing of heterogeneous PCR products of the comprises the genera Nocardioides (Prauser, 1976) and 16S–23S ITS yields unreadable mixed sequence data Aeromicrobium (Miller et al., 1991); the family Intra- above a certain position (Yoon et al., 1998b). If the sporangiaceae comprises the genera Terrabacter extent of heterogeneity in sequence and length is to be (Collins et al., 1989), Terracoccus (Prauser et al., 1997) determined, the PCR products should be cloned before and Intrasporangium (Kalakoutskii et al., 1967); the sequencing is begun. However, because the numbers of family Propionibacteriaceae comprises the genera rRNA gene clusters vary according to the organisms Propioniferax (Yokota et al., 1994), Luteococcus (Baylis & Bibb, 1988; Pernodet et al., 1989; Yoon et (Tamura et al., 1994), Microlunatus (Nakamura et al., al., 1996), it may not be easy to investigate the extent of 1995) and Friedmanniella (Schumann et al., 1997); the the heterogeneity. Such heterogeneity within strains family Streptomycetaceae comprises a single genus, makes phylogenetic analysis of the 16S–23S ITS namely Streptomyces; and the family Sporichthyaceae sequences rather complex. comprises a single genus, namely Sporichthya (Lechevalier et al., 1968). In addition to genera such as Recently, the ribonuclease P (RNase P) RNA gene was Streptomyces, Intrasporangium and Sporichthya, evaluated for its potential as a new phylogenetic which were described earlier, recently the numbers of marker (Cho et al., 1998); its application to the study -A pm-containing coryneform and nocardioform of the genus Saccharomonospora showed that it could # taxa have increased considerably. Nevertheless, until possibly be used effectively in systematic studies. The now, only approximately 22 validly described species mean nucleotide similarity of RNase P RNA gene of the 11 genera (except for the genus Streptomyces) sequences between Saccharomonospora species was have been validly described as -A pm-containing intermediate between those values obtained from the # actinomycete taxa. There is evidence that -A pm- sequences of 16S rDNA and the 16S–23S ITS (Cho et # containing coryneform bacteria may be relatively al., 1998). The members of the genus Saccharo- common in nature and that the majority of monospora formed a single cluster distinct from some such organisms have not been described (Collins Gram-positive bacteria in a dendrogram based on et al., 1994; Keddie et al., 1966). -A pm-containing comparative analysis of RNase P RNA gene se- # coryneform and nocardioform actinomycetes are im- quences. Unlike the 16S–23S ITS, RNase P RNA is portant taxa that have received attention from the known to be encoded by a single-copy gene (rnpB)in point of view of taxonomy, biodiversity and bio- Gram-positive bacteria (Haas et al., 1996) and its size technology. Therefore, additional useful taxonomic is relatively conserved (350–400 bp in bacteria; Pace & markers (whether genetic or chemotaxonomic) may be Brown, 1995). Despite the potential of the RNase P necessary to construct a polyphasic taxonomic ap- RNA gene as an additional phylogenetic marker, few proach to differentiating between existing -A pm- sequence data have been determined, especially for # containing taxa and to describing putative novel - actinomycetes, and few systematic studies based on A pm-containing taxa. RNase P RNA gene sequences have been performed. # Therefore, RNase P RNA gene sequences from diverse The aim of this study was to investigate the relation- taxa including actinomycetes are necessary to evaluate ships between taxa above genus level, as well as to

2022 International Journal of Systematic and Evolutionary Microbiology 50 Comparison of RNase P RNA genes of actinomycetes study the interspecific relationships of -A#pm-con- Amplification and cloning of the RNase P RNA gene. The taining actinomycetes, using RNase P RNA gene RNase P RNA gene was amplified using two primers sequences. -A#pm-containing taxa were also used to described previously (Cho et al., 1998). Amplification was determine if the RNase P RNA gene is appropriate for performed under the same conditions as those described clarifying the phylogenetic relationships between previously (Yoon et al., 1997b). The PCR products were distantly related organisms (e.g. between genera or purified using the GeneClean II kit (Bio 101) and cloned into T-vector prepared with pBluescript (Stratagene) (Marchuk families). et al., 1991). General techniques for cloning were followed, as described previously (Kim et al., 1995). METHODS Sequencing of the RNase P RNA gene. Nucleotide sequences were determined using the T7 Sequenase version 2.0 DNA Bacterial strains and culture conditions. Table 1 summarizes sequencing kit (Amersham) with primers T3 (5h-ATTAA- the strains used in this study. Most strains were grown in CCCTCACTAAAG-3h) and T7 (5h-TAATACGACTCAC- shake flasks containing trypticase soy broth (BBL) supple- TATAGGGCGA-3h). In some cases, reactions with dITP mented with glucose (0n75%, w\v) at suitable temperatures. and the SequiTherm EXCEL II DNA sequencing kit Propioniferax innocua was cultivated in shake flasks con- (Epicentre Technologies) were performed to relieve com- taining trypticase soy broth supplemented with yeast extract pression artifacts. (0n3%, w\v) at 37 mC. Friedmanniella antarctica was cultivated in shake flasks containing R-medium at 22 mC. Data analysis. The sequences of the RNase P RNA genes Microlunatus phosphovorus was cultivated in shake flasks determined in this study were aligned by using   containing microlunatus medium (Nakamura et al., 1995) at software (Thompson et al., 1994). Phylogenetic trees were 25 mC. The broth cultures were checked for purity before inferred by using three tree-making algorithms, namely the being harvested by centrifugation. neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge Isolation of DNA. Chromosomal DNA was isolated and & Farris, 1969) methods, all of which were performed using purified according to the method described previously (Yoon the  package (Felsenstein, 1993). Evolutionary dis- et al., 1996). tance matrices for the neighbour-joining method were

Table 1. Strains used in this study and accession numbers of RNase P RNA gene sequences

Species Source* Accession no.

Nocardioides albus KCTC 9186T AF110036 Nocardioides luteus KCTC 9575T AF110037 Nocardioides jensenii KCTC 9134T AF110038 Nocardioides plantarum NCIMB 12834T AF110039 Nocardioides simplex KCTC 9106T AF110040 Nocardioides pyridinolyticus KCTC 0074BPT AF110041 Nocardioides nitrophenolicus KCTC 0457BPT AF110042 ‘Nocardioides ﬂavus’ IFO 14396T AF110043 ‘Nocardioides fulvus’ JCM 3335T AF110044 Kribbella ﬂavida IFO 14399T (KCTC 9580T) AF110045 Kribbella sandramycini ATCC 39419T (KCTC 9609T) AF110046 Aeromicrobium erythreum NRRL B-3381T AF110047 Aeromicrobium fastidiosum KCTC 9576T AF110048 Terrabacter tumescens KCTC 9133T AF110049 Terracoccus luteus DSM 44267T AF110050 Intrasporangium calvum IFO 12989T (KCTC 9796T) AF110051 Friedmanniella antarctica DSM 11053T AF110052 Microlunatus phosphovorus JCM 9379T AF110053 Microlunatus phosphovorus JCM 9380 AF110054 Propioniferax innocua DSM 8251T AF110055 Luteococcus japonicus IFO 12422T (KCTC 9794T) AF110056 Luteococcus japonicus IFO 15385 (KCTC 9795) AF110057 Sporichthya polymorpha IFO 12702T (KCTC 9797T) AF110058 * The following abbreviations are used: KCTC, Korean Collection for Type Cultures, Taejon, Korea; JCM, Japan Collection of Microorganisms, Institute of Physical and Chemical Research, Saitama, Japan; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; NCIMB, National Collection of Industrial and Marine Bacteria, NCIMB, Aberdeen, UK; ATCC, American Type Culture Collection, Manassas, VA, USA; IFO, Institute for Fermentation, Osaka, Japan; NRRL, Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, IL, USA.

International Journal of Systematic and Evolutionary Microbiology 50 2023 J.-H. Yoon and Y.-H. Park generated with the algorithm of Jukes & Cantor (1969) using confirm that the genuine sequence from each strain the  program. The stability of relationships was had been obtained. assessed by a bootstrap analysis of 100 data sets by using the  package. The lengths of the RNase P RNA gene sequences determined ranged from 266 bp (in Nocardioides luteus Nocardioides flavus T RESULTS AND DISCUSSION ,‘ ’ IFO 14396 and ‘Nocardioides fulvus’ JCM 3335T) to 326 bp (in The genomic DNAs of strains used in this study were Nocardioides jensenii and Luteococcus japonicus). The checked for contamination. The genomic DNAs of the nucleotide sequences corresponding to annealing Nocardioides species, the Aeromicrobium species and regions of PCR primers were excluded from sub- Terrabacter tumescens used in this work were those sequent analyses, since their sequences may be different shown to be pure, by previous 16S rDNA analysis from the original sequences of the genes. The sequences (Yoon et al., 1998a). The DNAs extracted from other determined correspond to the region between positions strains were identified as being uncontaminated, by 78 and 346 (approx. 71%), by comparison with the direct determination of partial sequences of 16S RNase P RNA gene (377 bp long) of Escherichia coli rDNAs. The gene encoding the RNase P RNA (rnpB) (Reed et al., 1982; Lawrence et al., 1987), and the was amplified by the PCR, using primers described region between positions 68 and 376 (approx. 75%), previously (Cho et al., 1998), and was cloned before by comparison with the RNase P RNA gene (413 bp) being sequenced. At least two to three clones con- of Streptomyces bikiniensis (Morse & Schmidt, 1992). taining RNase P RNA gene from each strain were The 5h-region of the RNase P RNA gene before the sequenced to obtain exact sequence data and to annealing region of the forward primer is not con-

Table 2. Percentage nucleotide similarity, with or without gaps included, for the RNase P RNA genes of the strains used in this study and Streptomyces bikiniensis ...... The values on the lower left are levels of nucleotide similarity and the values on the upper right are those with gaps included. Strains: 1, Nocardioides albus;2,Nocardioides luteus;3,Nocardioides jensenii;4,Nocardioides plantarum;5,Nocardioides simplex; 6, Nocardioides pyridinolyticus;7,Nocardioides nitrophenolicus;8,‘Nocardioides fulvus’ JCM 3335T;9,Kribbella ﬂavida; 10, Kribbella sandramycini; 11, Aeromicrobium erythreum; 12, Aeromicrobium fastidiosum; 13, Terrabacter tumescens; 14, Terracoccus luteus; 15, Intrasporangium calvum; 16, Friedmanniella antarctica; 17, Microlunatus phosphovorus JCM 9379T; 18, Microlunatus phosphovorus JCM 9380; 19, Propioniferax innocua; 20, Luteococcus japonicus; 21, Sporichthya polymorpha; 22, Streptomyces bikiniensis.

Strain 1 2* 34567891011121314151617181920† 21 22

1 k 89n066n970n880n171n376n889n368n671n667n071n872n371n470n967n867n267n072n567n256n570n6 2*94n0 k 64n169n178n069n078n499n669n369n465n967n370n067n771n566n066n666n375n764n458n869n0 3 77n678n6 k 75n168n474n373n364n770n469n167n269n069n670n567n972n674n074n362n899n756n574n2 4 80n179n781n2 k 77n572n778n569n167n166n872n466n570n171n471n273n875n476n070n575n460n073n6 5 82n482n481n787n2 k 76n985n878n372n771n067n271n973n974n473n071n871n170n974n468n758n770n9 6 78n478n583n582n983n4 k 73n868n773n373n466n768n171n672n169n872n474n775n467n274n358n373n4 7 84n682n086n086n794n783n2 k 78n867n167n469n067n074n472n377n973n774n373n272n373n661n172n6 8 94n499n679n379n782n878n282n3 k 68n969n066n267n670n067n771n566n366n966n776n164n758n568n7 9 75n780n880n476n782n182n681n880n4 k 91n068n172n365n264n064n371n469n870n269n970n156n769n2 10 79n080n879n475n880n683n381n680n592n2 k 66n174n666n863n965n071n870n370n768n169n455n668n5 11 76n477n474n875n576n876n277n077n776n073n7 k 78n669n667n967n968n368n769n665n867n259n672n2 12 79n078n277n675n782n077n680n878n674n977n388n0 k 67n664n963n169n968n268n667n169n357n967n2 13 80n682n575n376n883n276n883n382n577n078n878n079n5 k 89n787n770n470n469n765n069n959n274n4 14 80n179n876n378n884n278n481n579n877n177n476n677n991n4 k 84n866n968n267n965n470n858n573n6 15 82n278n877n178n883n680n481n678n881n181n976n979n293n891n3 k 67n569n768n868n368n258n873n9 16 76n377n476n881n682n177n482n777n880n881n276n277n375n873n076n9 k 80n980n666n472n960n573n0 17 74n576n379n681n079n579n682n676n778n879n974n076n274n273n878n283n8 k 98n768n774n358n473n6 18 74n576n379n781n479n580n680n676n779n580n674n776n973n273n276n983n299n0 k 67n874n658n673n2 19 82n982n276n380n184n080n980n182n681n379n275n076n880n381n480n281n081n480n7 k 62n852n466n4 20† 77n978n999n781n682n183n586n379n380n179n774n877n975n776n677n477n179n980n076n3 k 56n573n9 21 63n968n464n866n266n165n168n068n169n768n466n870n964n963n765n167n465n764n564n364n8 k 59n3 22 78n280n878n380n680n279n281n280n481n679n780n878n076n376n579n577n277n076n481n978n065n9 k * Identical results were obtained for Nocardioides luteus and ‘Nocardioides ﬂavus’ IFO 14396T. † Identical results were obtained for Luteococcus japonicus strains IFO 12422T and IFO 15385.

2024 International Journal of Systematic and Evolutionary Microbiology 50 Comparison of RNase P RNA genes of actinomycetes served in sequence and length (Brown, 1998), unlike the 16S rRNA gene. Therefore, it was difficult to design a primer for the region close to the 5h-end of the RNase P RNA gene. In some recent studies, most primers used for PCR amplification of the RNase P RNA gene also have been designed around the annealing regions of the PCR primers used in this study (Haas et al., 1996; Brown et al., 1996). The alignment of the RNase P RNA gene sequences of all strains exhibited relatively conserved regions along with highly variable regions and nucleotide gap- containing regions (data not shown). The regions corresponding to nt 116–138, 159–189 and 214–235, by comparison with the RNase P RNA gene sequence of Streptomyces bikiniensis, were relatively conserved, whereas the regions at nt 139–154, 239–275 and 350–376 were highly variable. The RNase P RNA gene sequence of Sporichthya polymorpha KCTC 9797T exhibited many mismatched nucleotides when compared with those of other strains, as is also demon- strated by nucleotide similarities (Table 2). When the nucleotide sequence of Sporichthya polymorpha KCTC ...... T 9797 was excluded from the alignment, the numbers Fig. 1. Neighbour-joining tree showing the relationships of matched nucleotides were somewhat increased. among Nocardioides species based on RNase P RNA gene sequences. Bootstrap values are shown at the branch points. The mean RNase P RNA gene similarity value of - Bar, 5 nucleotide substitutions per 100 nucleotides. A#pm-containing strains used in this study was approximately 82p18% and the mean RNase P RNA gene-similarity value when gaps were included was approximately 76p24% (Table 2). The between-strain similarity including gaps of the type strains of all valid nucleotide similarities of RNase P RNA genes were Nocardioides species was approximately 76n6p12n5% mostly fewer than the corresponding 16S rDNA (Table 2). The relationships between Nocardioides similarities. The nucleotide sequences of RNase P species, based on the RNase P RNA gene sequences, RNA genes are unlikely to be appropriate for com- were examined with the trees inferred using the three parisons at all taxonomic levels (e.g. between species, different algorithms (Fig. 1). The topologies of the between genera and between families). In particular, resultant trees were compared with the topology of a the nucleotide similarities between most species be- previously constructed tree based on 16S rDNA longing to the genus Nocardioides did not show sequences (Yoon et al., 1998a). The close relationships conspicuous differences when compared with those between some species in the tree based on the RNase P between them and members of other genera; more- RNA gene sequences (Fig. 1) are mostly consistent over, some species produced examples showing rather with the result obtained from 16S rDNA sequences higher nucleotide similarities to members of other (Yoon et al., 1998a). The type strains of Nocardioides genera than to Nocardioides species (Table 2). It albus and N. luteus were phylogenetic neighbours with appears that the RNase P RNA gene does not a nucleotide similarity of 94%. The clustering of Nocardioides simplex KCTC 9106T and Nocardioides necessarily show much higher sequence differences T between higher-ranked taxa than between lower- nitrophenolicus KCTC 0457BP was supported with ranked taxa in the -A#pm-containing strains used in low bootstrap resampling values only when the this study. A phylogenetic tree based on the RNase P neighbour-joining and maximum-likelihood methods RNA gene sequences of all strains differed from that were used. N. jensenii, Nocardioides plantarum and obtained using their 16S rDNA sequences. Therefore, Nocardioides pyridinolyticus formed phylogenetic phylogenetic trees based on RNase P RNA gene lineages distinct from each species. However, in a sequences of -A#pm-containing strains used in this phylogenetic analysis including other taxa used in study were omitted because of the recognized limita- this study, Nocardioides species did not form a tions of RNase P RNA gene as a molecular clock. monophyletic cluster encompassed by the genus Nocardioides. N. pyridinolyticus and N. jensenii ex- The RNase P RNA gene sequences of validly described hibited phylogenetic lineages independent of the Nocardioides species were relatively divergent when cluster containing the type species of the genus compared with levels of 16S rDNA similarity. The Nocardioides, N. albus and most Nocardioides species. mean RNase P RNA gene similarity of the type strains In particular, it was interesting that N. jensenii showed of all validly described Nocardioides species was only a 1 bp sequence difference with respect to L. 85n8p8n2% (Table 2). The mean RNase P RNA gene japonicus. It was confirmed that the RNase P RNA

International Journal of Systematic and Evolutionary Microbiology 50 2025 J.-H. Yoon and Y.-H. Park gene sequences of N. jensenii and L. japonicus were been shown with 16S rDNA sequences (Prauser et al., correctly determined, by using repeated tests. The type 1997; Park et al., 1999). The RNase P RNA gene can strains of two invalidly described Nocardioides species, be considered to be a more useful marker than 16S ‘N. flavus’ IFO 14396T and ‘N. fulvus’ JCM 3335T, rRNA for differentiation of the three taxa at the genus were used to infer the relationships with other level. On the other hand, it was shown in an original Nocardioides species. ‘N. flavus IFO 14396T had an description that Terracoccus luteus has two types of RNase P RNA gene sequence identical to that of N. colonies, i.e. the rough form (DSM 44275) and the luteus and ‘N. fulvus’ JCM 3335T showed only a 1 bp smooth form (DSM 44274) (Prauser et al., 1997). difference with respect to N. luteus.‘N. flavus’ IFO These forms were shown to be identical from a 14396T has already been described as a strain of N. taxonomic point of view. We also isolated colonies of luteus (Prauser, 1989) and this has been confirmed the rough form and the smooth form from the original recently by 16S rDNA and 16S–23S ITS sequence culture (DSM 44267T) and determined the RNase P analyses (Yoon et al., 1998a, b). ‘N. fulvus’ JCM 3335T RNA gene sequences from each colony type. The was not clearly defined taxonomically until the 16S RNase P RNA gene sequences from the two types of rDNA and 16S–23S ITS sequences were determined. colonies were found to be identical. Therefore, RNase P RNA gene sequences also support previous data showing that ‘N. flavus’ IFO 14396T and The genera Friedmanniella, Luteococcus, Microlunatus ‘N. fulvus’ JCM 3335T are suitable for description as and Propioniferax, assigned to the family Propioni- members of N. luteus. Another strain (IFO 14399) of bacteriaceae, also were compared. These genera also ‘N. fulvus’ has been classified in a new genus (Kribbella) consist of single species. Among the four genera, as Kribbella flavida, together with Nocardioides sp. Luteococcus japonicus formed a line of descent that is ATCC 39419, classified as Kribbella sandramycini clearly different from the other three genera. As shown (Park et al., 1999). The two species of the new genus above, interestingly, L. japonicus had a nucleotide Kribbella formed a cluster independent of other strains sequence nearly identical to that of N. jensenii. F. used in this study and exhibited nucleotide similarity antarctica, M. phosphovorus and P. innocua formed a of 92n2%. line of descent that can be considered as one phylogenetic cluster. Although the three genera form a Two Aeromicrobium species formed a phylogenetic phylogenetic cluster, they exhibit levels of nucleotide cluster that is clearly distinct from other taxa used in similarity (81n0–83n8%) that makes possible their this study. The nucleotide similarity between the two differentiation (Table 2). species was 88% (78n6% when nucleotide gaps were included). The RNase P RNA genes of the two species Sporichthya polymorpha, one genus and one species are likely to be appropriate, as another genetic marker that has been assigned to the family Sporichthyaceae, together with the 16S–23S ITS (Yoon et al., 1998b), formed a line of descent that can be clearly dis- for clarifying the relationship between the two species. tinguished from other -A#pm-acid-containing taxa. The two Aeromicrobium species were compared with Sporichthya polymorpha exhibited the lowest levels of the members of the genus Nocardioides, a phylogenetic nucleotide similarity to all strains when compared with relative (Tamura & Yokota, 1994; Yoon et al., 1998a). those between other strains (Table 2). Aeromicrobium species formed a phylogenetic lineage From the above results it is clear that the RNase P distinguishable from the members of the genus RNA gene was useful for indicating phylogenetic Nocardioides and exhibited less than 80% nucleotide relationships between species belonging to a genus and similarity to Nocardioides species, except in the case of relationships between several genera, being target the relationship between the type strains of Aero- genetic material that exhibits lower nucleotide simi- microbium fastidiosum and N. simplex (Table 2). larity than the 16S rRNA gene. Intraspecific variation Therefore, the RNase P RNA gene can be considered of this gene was investigated for two species in this as a useful molecule that not only clearly differentiates study. L. japonicus did not show intraspecific variation Aeromicrobium species from Nocardioides species but and two strains of M. phosphovorus showed relatively also shows the correct variation between the two minor variation of 4 bp differences. It was previously Aeromicrobium species. reported that the RNase P RNA genes of Saccharo- Three genera, Intrasporangium, Terrabacter and monospora species also exhibited little or no intra- Terracoccus, which belong to the family Intra- specific variation (Cho et al., 1998). The RNase P sporangiaceae and comprise only single species, formed RNA gene was found to have sequence variation a cluster distinct from other taxa, as shown in a intermediate between that of 16S rDNA and the phylogenetic analysis based on 16S rDNA sequences 16S–23S ITS in data concerning the genera (Prauser et al., 1997). Intrasporangium calvum, Terra- Nocardioides, Aeromicrobium and Kribbella (Yoon et bacter tumescens and Terracoccus luteus exhibited al., 1998a, b), as was also shown for the genus relatively high nucleotide similarity (91n3–93n8%) to Saccharomonospora (Cho et al., 1998). Generally, 16S each other, despite being of different genera, when rDNA sequences have been known to be appropriate compared with results between different genera or for comparisons between species that are not closely different species used in this study. Phylogenetically related and between taxa above genus level, and close relatedness among the three genera has already 16S–23S ITS sequences are thought to be appropriate

2026 International Journal of Systematic and Evolutionary Microbiology 50 Comparison of RNase P RNA genes of actinomycetes for indicating the relationships between closely related a useful taxonomic marker that can be applied to a species and between strains belonging to a species. The variety of taxa. Extension of the RNase P RNA gene RNase P RNA gene appears to be most suitable for database will contribute both to systematic study and indicating the relationships between species belonging the study of the function and secondary structure of to a genus and the relationships between several the gene itself. genera, because it is a genetic marker that shows sequence variation intermediate between that of 16S ACKNOWLEDGEMENTS rDNA and the 16S–23S ITS. Most genera among the -A#pm-containing actinomycetes have only a single This work was supported by grants HS2321 and KG1295 species representative. Therefore, RNase P RNA gene from the Ministry of Science and Technology (MOST) of the Republic of Korea. We are grateful to Dr Yong Kook Shin sequences may be very useful for differentiation be- for helpful discussion and to Seok-Seong Kang for practical tween existing species and putatively novel species of assistance. We are also very grateful to the JCM (the Japan -A#pm-containing genera, as it is one of several Collection of Microorganisms) and the IFO (the Institute sources of valuable data in the polyphasic approach to for Fermentation, Osaka) for providing some of the strains taxonomy. Nevertheless, the RNase P RNA gene used in this study. may be inappropriate for determining phylogenetic relationships between some genera of -A#pm-con- REFERENCES taining taxa. It has already been shown that Nocardioides species did not form a monophyletic Amann, R., Ludwig, W. & Schleifer, K. H. (1988). 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