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Actinomyces As Actinomyces Suis Comb

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Actinomyces As Actinomyces Suis Comb INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1992, p. 161-165 Vol. 42, No. 1 0020-7713/92/010161-05$02.00/0 Phylogenetic Evidence for the Transfer of Eubacterium suis to the Genus Actinomyces as Actinomyces suis comb. nov. W. LUDWIG,l* G. KIRCHHOF,l M. WEIZENEGGER,l AND N. WEISS2 Lehrstuhl fur Mikrobiologie, Technische Universitat, 0-8000 Munich, and Deutsche Sammlung von Mikroorganismen und Zellkulturen, 0-3300 Braunschweig, Germany The 16s rRNA primary structures of Eubacterium suis DSM 20639T (T = type strain) and Bijidobacterium bijidum DSM 20456T were determined by sequencing in vitro amplified rDNA. Sequence comparisons indicated that B. bijidum is moderately related to representatives of the genera Actinomyces and Mobiluncus. The closest relative of E. suis is Actinomyces pyogenes. E. suis and A. pyogenes are more closely related phylogenetically to one another than to the other Actinomyces species that have been investigated by using comparative 16s rRNA analysis. Therefore, we propose that E. suis should be transferred to the genus Actinomyces as Actinomyces suis comb. nov. Eubacterium suis Wegienek and Reddy 1982, a commonly MATERIALS AND METHODS occurring swine pathogen (23), was originally isolated by Soltys and Spratling in 1957 (20). The name “Corynebacte- Bacterial strains and culture conditions. B. bifidum DSM rium suis” was proposed for this bacterium because of the 20456T (T = type strain) was cultured anaerobically at 37°C diphtheroid morphology of the anaerobic organism. In 1982 in a medium which contained (per liter of distilled water) 10 in a taxonomic study, Wegienek and Reddy described the g of tryptone, 5 g of meat extract, 5 g of yeast extract, 10 g morphological, cultural, and biochemical characteristics of of glucose, 3 g of K2HP0,, 5 g of NaC1, 10 ml of Tween 80, this organism and proposed the name Eubacterium suis. 10 g of sodium ascorbate, and 0.5 g of cysteine hydrochloride Corynebacteria are aerobic to facultatively anaerobic, con- (pH 6.8). Eubacterium suis DSM 20639T was grown anaer- tain arabinose, galactose, and meso-diaminopimelic acid as obically at 37°C in peptone yeast extract maltose medium; the major cell wall components, and produce major amounts the peptone yeast extract glucose medium described in the of propionate in carbohydrate metabolism; in contrast, Eu- 1990 Catalog of strains of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (6a) was modified by bacterium suis is anaerobic and contains rhamnose and replacing glucose with 5 g of maltose per liter. lysine as the major cell wall components, and propionate is Cell wall analysis. The analysis of cell wall components not among the end products of carbohydrate metabolism. was performed as described by Schleifer and Kandler (19). Despite differences in cell wall composition and DNA base Sequence analysis. Genomic DNA was purified by using ratios (55 mol% G+C for Eubacterium suis versus 31 to 45 the Marmur method (12). 16s rDNA was amplified in vitro mol% G+C for other members of the genus Eubacterium) by using the polymerase chain reaction technique (14) in (11, 13) and on the basis of a rather limited number of combination with oligonucleotide primers identical or com- common characteristics, such as anaerobiosis, morphology, plementary to highly conserved regions of the rRNAs. The similar cultural conditions, and the absence of propionate, amplified rDNA fragments were cloned in the vector pBlue- lactate, and succinate among the end products of carbohy- script (Stratagene, LaJolla, Calif.) and Escherichia coli drate metabolism, Wegienek and Reddy proposed that the JM83. The cloned rDNA and the amplified rDNA were organism should be included in the genus Eubacterium. sequenced by using the chain termination method (4, 16) and The genus Eubacterium is mainly defined by negative site-specific oligonucleotide primers. The oligonucleotides characteristics and represents a heterogeneous collection of gram-positive, nonsporeforming, obligately anaerobic rods (11,13). In particular, the wide range of genomic DNA base - MurNAc - GlcNAC - ratios indicates that the genus includes organisms that are 1 not related phylogenetically. L-Ala At present, comparative sequence analysis of rRNA is one of the most powerful methods for determining higher phylo- genetic relationships among bacteria. In order to elucidate + j L-Ala c L-Lys j the phylogenetic affiliations of Eubacterium suis, we se- D-Ala j I D-Glu 1 t D-Ala quenced the greater part of its 16s genes coding for rRNA t (rDNA). The 16s rDNA of Bifidobacterium bllfdum was L-Lys analyzed as a reference rDNA in addition to the previously D-Glu pubfished sequences for gram-positive bacteria with high DNA G+C contents. T,-Ala t - GlcNAc - MurNAc - FIG. 1. Fragment of the peptidoglycan primary structure of Eubacteriurn suis. Abbreviations: Ala, alanine; GlcNAc, N-acetyl- glucosamine; Glu, glutamic acid; Lys, lysine; MurNAc, N-acetyl- * Corresponding author. muramic acid. 161 162 LUDWIG Er AL. INT.J. SYST.BACTERIOL. 1 92 mmmnagagtttgatmtggctcag~GGCGTGCTTMCACATGCMGTCGAACGGAAAGGCTTCTTTCTTTTTTGATT TTTGTGGAGAGTTTnATCCTGGCTCAGGAC~CGCTGGCGGCGTGCTTMCACATGCAAGTCGAACGAT~GCCGGGn-C~TTT--GTT mmmnagagtttgatmtggctcagGATCAACGCTGGCGGCGTGCTTMCACATGCMGTCGAACGGTGATCCATCAAGCTTG----CTT 93 184 GGAGTGCTCGAGTGGCGAACGGGTGAGTMTACGTGGGTMCCTGCCCTCAACTGTGGGATMGCTTGGCAAACTGGGTCTMTACTGCATA TTGGTGGATTAGTGGCGAACGGGTGAGTMnACGTGAGTMCCTGCCCTTGTCTTTGGGATMGCCTGG~CTGGGTCTMTACCGGATA GGTGGTGATGAGTGGCGAACGGGTGAGTMTGCGTGACCCTG 185 276 TTCTTCTGTCCTCGCATGGGGGTGGTTGGAMG-TTTTTTCGGTTGGGGATGGGCTCGCGGCCTATCAGTTTGTTGGTGGGGTGATGGCCTA TTCTGCTTTTGCCGCATGGTGGGGGTTGG~AAGATTTTTT-GGATGGGMTGG~TCACGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTA TTCCAC-ATGATCGCATGTGATT-GTGGGAMGATTCTATCGGCGTGGGATGGGGTCGCGTCCTAT~GCTTGTTGGTGAGGTMCGGCTCA 277 368 CCMGGCGTTGACGGGTAGCCGGCCTGAGAGGGTGGTCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGG~GGCAGCAGTGGGG CCAAGGCGTCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACATTGGGACTGAGATACGGCC~CTCCTACGGGAGGCAGCAGTGGGG CCMGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGG 369 460 GATATTGCACAATGGGCGGAAGCTTGATGCAGCGACGCCGCGTGGGGGATGAAGGCTTTCGGGTTGTACTTCTTTTGGTGCAGAACMGG MTATTGCACAATGWCGCAAGTCTGATGCAGCGACGCCGCGTGGGGGATGAAGGCTTTCGGGTTGTACTCCTTTCAGTACAGAAGAAGC MTATTGCACMTGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTACCTCTTTTGTTTGGGAGCAAGC LA1 552 C~CGCACTTGGGGTGGGTTGAGGGTATGTGCTGAATMGCGCCGGCTMCTACGTGCCAGCAGCCGCGGTMTACGTAGGGCGCGAGCGTTG A----TTTT------- TGTGACGGTATGTGCAGAAGGCGCCGGCTMCTACGTGCCAGCAGCCGCGGTMTACGTAGGGC~TAGCGTTG C----TTCG------- GGTGAGTGTACCTTTCGAATMGCGCCGGCTMCTACGTGCCAGCAGCCGCGGTMTACGTAGGGCGCAAGCGTTA 553 644 TCCGGAATTATTGGGCGTAGGGCTTGTAGGCGGCTTGTTGCGCCTGCTGT~CGCGGGGCTTMCTTCGCGCGTGCAGTGGGTACGG TCCGCAATTATTGGGCGTAGAGCTCGTAGGCGGTTTGTTGCGCCTGCTGTCAAAGACCGGGGCTTMCTTCGGffiTTGCAGTGGGTACGG TCCGGATTTATTGGGCGTAGGGCTCGTAGGCGGCTCGTCGCGTCCGGTGT~GTCCATCGCTTAACGGTGGATCTGCGCCGGGTACGG 645 736 GCAGGCTTGAGTGTGGTAGGGGTGACTGGAATTCCAGGTGTAGCGGTGGAATGCGCA~TATCTGGAGGAATACCGATGGCGAAGGCAGGTC GCAGACTAGAGTGTGGTAGmGATTGGnATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAG~CACCGATGGCGAAGG~GmT GCGGGCTGGAGTGCGGTAGGGGAGACTGGAATfCCCGGTGTMCGGTGGAATGTGTA~TATCGG~~CACCGATGGCGAAGGCAGGTC 737 828 ACTGGGCCATTACTGACGCTGAGGAGCGAMGCGTGGGTAGC~CAG~TTAGATACCCTGGTAGTCCATGCTGTACGTTGGGAACTAG ACTGGGCCATTACTGACGCTGAGGAGCGTGGGTACGTTGGGCACTAG TCTGGGCCGTCACTGACGCTGAGGAGCCAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGT~CGGTG~CGCTGG 829 920 ATGTGGGGCACGTTCCACGTGTTCCGTGTCGGAGCTMCGCGTTMGCGTCCCGCCTGGGGAGTACGGCCGCAAGGCT~CTCA~ 921 1012 TTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTMTTCGATGCAACGCGAAGAACCTTACCMGGCTTGACATACATTGCGATATTC TTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTMTTCGATGCMCGCGAAGAACCTTACCMGGCTTGACATACACTGCGATGTGC TTGACGGGGGCCCGCACAAGCGGCGGAG~TGCGGATTMTTCGATG~CGC~~CCTTACCTGGGCTTGACATGTTCCCGAC~CGC 1013 1104 CAGAGATGGGGTAGCT-TTTTT-GGTGGTGTACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG CAGAGATGGTGCAGCC-TTCGG-GGTGGTGTACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTMGTCCCGCMCG CAGAGATGGCGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTMGTCCCGCAACG 1105 1196 AGCGCAACCCTTGTCTTGTGTTGCCAGCGGTTTTGGCCGGGGACTCACGGGAGACTGCCGGGGTTMCTCGGAG~GGTGGGGATGACGTC AGCGCAACCCTTGTCCTGTGTTGCCAGCATGTTGTGGTGGGGACTCACGGGAGACTGCCGGGGTTAACTCG~G~GGTGGGGAT~CGTC AGCGCAACCCTCGCCCCGTGTTGCCAGCACGTTATGGTGG~CTCACGGGGGACCGCCGGGGTTMCTCGGAGGAAGGTGGGGATGACGTC 1197 1288 AAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACMTGGCCGGTACAGTGGGTTGCGATACTGTGAGGTG~GCGAATCTCTGA AAATCATCATGCCCnTTATGTCTTGGGCTTCACGCATGCTACMTGGCCGGTACAGAGGGTTGC~GCCTGTGAGGGTGAGCTAATCCCTTA AGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAGCGGGATGCGACATGGCGACATGGAGCG~TCCCT~ 1289 1380 MGCCGGTCTCAGTTCGGATCGGGGTCTGCMCTCGACCTCGT~GTGGGAGTCGCTAGTMTCGCGGATCAG~TTGCCGCGGT~TAC MGCTnGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATG~GnCGGAGTCGCTAGTMTCGCAGATCAG~CGCTGCGGT~TAC AAACCGGTCTCAGTTCGGATCGGAGCCTGCMCCCGGCTCCGTGAAGGCGGAGTCGCTAGTMTCGCGGATCAGCAGCGCCGCGGTGAATGC 1381 1472 GTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGCGGTCG GTm-p GTTCCCGGGCCTTGTACACACCGCCCGTCMGTCAT~GTGGGCAGCACCC~GCCGGTGGCCTMCCCCTTGTGGGATGGAGCCGTCT 1473 1549 AAGGTGGGGTTGGCGATTGGGAC~GTCGTM~GCTAGCCGTACCG~GGTGCGGCTGGATCACCTCCTTTCT MGGTGAGGCTCGTGATTGGGACTMGTCGTMCAAGGTAGCCGTACCGGAAGGTGCGGCTggatcacctcctttct FIG. 2. Alignment of the 16s rRNA primary structures of Eubacteriurn suis (Es), B. bifidurn (Bb), and A. pyogenes (Ap) (22). Lower-case letters indicate target sites of amplification primers; n indicates a base that was not determined or was not unambiguously determined. VOL.42, 1992 ACTINOMYCES SUIS COMB. NOV. 163 TABLE 1. Similarity matrbf % of similarity with: Escherichia coli
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