Verrucomicrobium spinosum, a Eubacterium Representing an Ancient Line of Descent

System. Appl. Microbiol. 10, 57-62 (1987)

Verrucomicrobium spinosum, a Eubacterium Representing an Ancient Line of Descent WOLFGANG ALBRECHT, ANGELIKA FISCHER, JAN SMIDA, and ERKO STACKEBRANDT Institut fur Allgemeine Mikrobiologie, Christian-Albrechts-Universitat, 2300 Kiel, Federal Republic of Germany Received February 26, 1987

Summary 16S Ribosomal RNA of Verrucomicrobium spinosum was analysed by oligonucleotide cataloguing and reverse transcriptase sequencing. Similarity coefficients (SAB values) calculated for the RNase T1 catalogue of V. spinosum and about 460 catalogues of eubacterial strains were found to be as low as those separating individual eubacterial phyla (as defined by Woese et aI., 1985), indicating that V. spinosum represents a new division. The deep branching point of V. spinosum within the dendrogram of relationships is in accord with its position in the phlyogenetic tree derived from comparisons of two long 16S rRNA sequences of size 285 and 230 nucleotides from V. spinosum and the homologous regions of a variety of eubacteria. V. spinosum is most distantly related only to other eubacteria carrying prosthecates, e. g. members of Prosthecomicrobium, Ancalomicrobium, Stella, Caulobacter, Hyphomicrobium, Pedomicrobium, Hyphomonas and Prosthecochloris.

Key words: Verrucomicrobium spinosum - Prosthecate eubacteria - 165 rRNA Cataloguing - Reverse transcriptase sequencing - Phylogeny

Introduction The genus Verrucomicrobium with V. spinosum as the type species has been described (Schlesner, 1987) to harbor a prosthecate nonbudding organism which morphologically resembles members of Prosthecomicrobium and Ancalomicrobium but differing from those in a single phenotypic property, i. e. the presence of fimbriae. The DNA G+C content of V. spinosum (58-59 mol %) is at least 6 mol % lower than those reported for Prosthecomicrobium species (66-70 mol %) (Staley, 1981) and Ancalomicrobium adetum (70 mol %) (Staley, 1981). Stella humosa, on the other hand, showing the same DNA G+C content as V. spinosum, embrace flat bacteria (Hirsch and Schlesner, 1981). Phylogenetically, prosthecate (or appendaged) non-budding and budding eubacteria constitute a diverse collection of organisms. Although 165 rRNA analysis has shown members of Prosthecomicrobium, Ancalomicrobium, "Dichotomicrobium", Caulobacter, Hyphomicrobium (Fischer, 1986; Fischer, Roggentin and Stackebrandt, unpublished) and Stella (Fischer et aI., 1985) to be members of the alpha subdivision of purple photosynthetic bacteria and their non-phototrophic relatives (Woese et aI., 1984a, b), these genera did not cluster together to the exclusion of

non-protesthecate and non-budding relatives. Unusual mode of reproduction, i. e. budding, is now known to be of restricted significance in the definition of taxa above the species level (Rothe et aI., 1986; Stackebrandt et aI., 1986; Schlesner and Stackebrandt, 1986; Fischer, 1986). It was therefore interesting to see whether or not the differences used to distinguish the newly described prosthecate organism Verrucomicrobium spinosum from members of Prosthecomicrobium and Ancalomicrobium are valuable taxonomic properties. In order to determine the phylogenetic position of V. spinosum we have therefore analysed its 165 rRNA by oligonucleotide cataloguing (Stackebrandt et aI., 1985 a) and reverse transcriptase sequencing (Lane et aI., 1985). Materials and Methods Cells of Verrucomicrobium spinosum IFAM 1439 (IFAM = Institut fur Allgemeine Mikrobiologie, Kiel) (Schlesner, 1987) were grown in aerated 8-liter bottles in medium M13 (Schlesner, 1986) for 48 h at 30°C. For cell desintegration, 2 g (wet weight) of bacteria were suspended in 20 ml buffer (0.01 M MgCI 2, (l.S M NH4 Cl, 0.01 M Tris/HCI, pH 7.4) and 50 g of glass beads (0.1

58

W. Albrecht, A. Fischer,

J. Smida,

and E. Stackebrandt

mm) were added. The mixture was precooled in ice and then shaken in an MSK cell homogenizer (Braun, Melsungen, FRG) for 30 s. Ribosomes were isolated from these Iysates as described previously (Traub et al., 1971, Lee and Evans, 1971). Proteins were removed by phenolization, rRNA precipitated by 2V of ethanol and then stored at -20 c Isolation of 16S rRNA by sodium dodecylsulfate polyacrylamide slab gel electrophoresis and determination of RNase T1 resistant oligonucleotides fol· lowed described methods (Stackebrandt et al., 1985a). Bulk RNA was obtained from desintegrated cells by phenol-cresol-deproteinization, ethanol precipitation Uohnson, 1981) followed by sodium acetate homogenization (Kirby, 1968). Calculation of similarity coefficients (SAB) was done as described by Fox et al. (1977). A dendrogram of relationships was derived therefrom by average linkage clustering (among the merged groups). Sequencing of large fragments of 16S rRNA followed the protocol described by Lane et al. (1985). Six oligodeoxynucleotides complementary to highly conserved 16S rRNA sequences were used as primers for the reverse transcriptase reaction. Primer sequences of size 17 to 20 bases cover positions 357, 690, 797, 1052,1100 and 1510 (according to the E. coli numbering system (Woese et al., 1983). Of the resulting 6 cDNA stretches 4 were used for determination of phylogenetic relationships. Because of overlapping regions these 4 stretches could be linked together to yield 2 long fragments. These were aligned and compared to published 16S rRNA primary structures. Homology values were calculated using the Beckman Microgenic program. Nucleotides whose identity could not be determined unambigously (designated N) and their homologous counterparts in the reference sequences were omitted from the calculation. Gaps were treated half the weight of matched positions (McCaroll et al., 1983). Calculation of Knuc-values was done as indicated by Hori (1975). Construction of the phylogenetic tree followed the algorithm described by Fitch and Marguliash (1967) using the Felsenstein program (Phylip, version 2.9; IBM XT).

e.

Results and Discussion 165 rRNA cataloguing

The 16S oligonucleotide catalogue of Verrucomicrobium spinosum is shown in Table 1. Oligonucleotides whose position could be located either in large blocks of V. spinosum 16S rRNA sequences obtained by the use of reverse transcriptase or, because of their conserved sequence, in the primary structures of other eubacteria (Weisburg et al., 1986) are indicated by prefix numbers, adopted from the E. coli numbering system (Woese et al., 1983). Table 2 shows the similarity coefficients (SAB values) calculated for V. spinosum and representatives of 460 eubacteria and 30 archaebacteria. The average value of 0.15 found for V. spinosum and eubacteria is significantly higher than that of 0.05 shared by the former species and archaebacteria. Since the most ancient lines of descent within the eubacterial kingdom branch off at an SAB value of 0.12 (5tackebrandt et al., 1986), V. spinosum has to be considered a eubacterium. This is in accord with the presence of meso-diaminopimelic acid in whole cell hydrolysates found so far exclusively in eubacteria. Fig. 1 is a dendrogram of relationship displaying the phylogenetic position of V. spinosum among representatives of a variety of eubacterial divisions. This prosthecate organism stands

Table 1. Oligonucleotide catalogue of the 16S rRNA from Verrucomicrobium spinosum Position

Oligonucleotide

AAACCG 917 AAAUUG 1374 AAUACG AAUCAG 753 ACACUG 704 AUAUCG 736 AUCCUG AUUCUG CAAACG 934 CACAAG 1226 CACACG CACUCG CAUCCG CCACCG 862 CUAACG 813 UAAACG 747 UAUCUG UCUUCG 1380 UUCCCG 160 AAACUUG 607 AAAUCCG 192 ACUAAAG + 1317 CAACUCG CAAUACG CAAUCCG + 370 CACAAUG 1464 CCCUAAG CUUUCUG 531 UAACACG 542 UCUCAAG + 870 UUAAACG + 715 AACACUAG + 559 AAUCACUG 675 AAUUCUCG + 792 AUACCCCG AUCAACUG + x

+

=

Posirion

Oligonucleotide

1109 CAACCCUG CAAUACCG 311 CCACACUG 1208 CCCUUACG CCUAUCAG UAACUCCG + 170 UAACACAG 1354 UACAUCAG + 338 ACACCUACG 363 AUAAUCUUG CAACCCCUG CAAUACACG + 267 CCCACCAAG 507 CUAACUCUG 955 CUUAAUUCG 1393 UACACACCG ACCCCUUAUG + 429 UAAACUCCUG 1232 UACUACAAUG 977 AACCUUACCUG 640 AUACUCCCAUG + AUUACAUACCG + 618 CUCAACCUACG + 1277 AAAUCCUCAAAAUG + AAUCAUUCACAAUG + 903 AUUAAAACUCAAAG 1193 AUUAAUACCUCAUG modified oligonucleotides * 1498 UAACAAG * + 1406 UCACAUCAUG 3' terminus 1531 AUCACCUCCUUUCX'OH

not determined modified nucleotide V. spinosum specific signature oligonucleotide, occurring in the 165 rRNA catalogue of this organism but in less than 1 % of all other eubacteria investigated (Woese et al., 1985)

isolated, showing no closer relationships to other prosthecate organisms, e. g. members of Prosthecomicrobium, Stella, Ancalomicrobium, Caulobacter, Hyphomicrobium (alpha subdivision of purple bacteria), or Prostecochloris aestuarii (Chlorobium division) (Gibson et al., 1986) and their respective relatives than to non-prosthecate strains. Average linkage clustering of SAB-values links V. spinosum at a deep level to the Planctomycetales division (Stackebrandt et al., 1986). Reverse transcriptase sequencing

In order to verify the phylogenetic posItIOn of V. spinosum, 2 eDNA fragments of its 16S rRNA were generated by using the reverse transcriptase sequencing ap-

Verrucomicrobium spinosum, an Ancient Line of Descent Table 2. Average SAB values calculated for Verrucomicrohium spinosum, representatives of the eubacterial divisions and archae bacteria Division Eubacteria Purple bacteria and relatives alpha subdivision beta subdivision gamma subdivision delta subdivision Gram-positive bacteria Clostridium subdivision Actinomycetes subdivision Spirochaetales Chloroflexus and relatives Chlorobium and relatives Cyanobacteria/Chloroplasts BacteroideslCytophaga DeinococcuslThermus Planctomycetales Chlamydia Archaebacteria

Verrucomicrobium spinosum 0.16 0.13 0.15 0.18 0.17 0.18 0.15 0.16 0.14 0.15 0.12 0.14 0.16 0.14 0.05

proach. The length of the 2 fragments were 515 (285 and 230) nucleotides, corresponding to about 33.4 % of total. Difficulties encountered with the dideoxynucleotide-terminated sequencing technique using reverse transcriptase and synthetic oligonucleotide primers have been extensively dealt with by Lane et al. (1985). The major problem is the appearance of anomalous "masking" bands which are seen across all four lanes of the sequence ladder making it impossible to identify the proper nucleotide. As discussed by Lane et al. (1985) no single explanation can be given for this phenomenon. In our case these ambiguities were found predominantly in the 5' (primer-357) and 3' (primer-1510) regions of the molecule were they amounted to 12.5 and 5%, respectively, of total nucleoti des determined. These 2 fragments were consequently omitted from data analysis. 5ince the percentage of nonidentificable nucleotides was markedly increased in all eDNA fragments when 165 rRNA, purified by PAGE, electro elution and subsequent phenolization, was used as a template, fragmented rRNA is likely to be one major cause for the anomalies. Others may be the high degree of stabilized secondary structure known for the region between positions 120 and 350, and the concentration of modified residues between positions 1400 and 1410. Comparison of dendrograms and trees. It has been shown previously that phylogenetic trees constructed from partial 165 rRNA sequences had virtually identical topologies to those based on complete sequences (McCaroll et aI., 1983; Lane et aI., 1985). In the present case the phylogenetic tree is based on 2 sequences of a total of 515 nucleotides. These are aligned to the homologous regions from E. coli, Desulfovtbrio desulfuricans, Agrobacterium tumefaciens, Bacillus subtilis,

59

Anacystis nidulans, Bacteroides fragilis, and Chlamydia psittaci, listed comprehensively by Weisburg et al. (1985) (Table 3). Table 4 shows the percent homology (lower left triangle) and the Knuc-values (upper right triangle), Figs. 2a and 2b display a dendrogram of relationships and a phylogenetic tree, respectively, derived therefrom. As already detected in the oligonucleotide-based dendrogram (Fig. 1), V. spinosum is a deep branching organism within the eubacterial kingdom, sharing homology values with other eubacteria in the range of 73.5 to 77.1 %. Only 63.5% homology is found for the pair V. spinosum and the archaebacterium Halobacterium volcanii. The branching pattern of the reference organisms based on the analysis of 515 nucleotides only is very similar to those based on full 165 rRNA sequences (Oyaizu and Woese, 1985; Yang et aI., 1985; Weisburgetal.,1986) and to the dendrogram based on 5AB values. The only exception refers to Bacillus subtilis whose position changes with the number and selection of organisms included and with the method used for the determination of relationships. While in Fig. 1, B. subtilis, a representative of about 150 gram-positive eubacteria investigated, constitutes an individual line of descent, the same organism appears to be peripherally related to the division of purple bacteria (Fig.2b), while the same organisms even groups within the radiation of the latter organisms in Fig.2a. Interestingly, even phylogenetic trees based on full 165 rRNA sequences reflect the unsteadiness in the branching point of this Gram-positive eubacterium: it groups either with Anacystis nidulans (Weisburg et aI., 1986), or it branches off between the branching points separating A. nidulans and the purple bacteria (Yang et aI., 1985). The same problem is encountered with the topography of 5AB dendrograms generated on a very limited number of organisms only. It can be assumed that with additional complete 165 rRNA sequences of Gram-positive organisms available their position within the phylogenetic tree will be stabilized. At present, the remote relationship between V. spinosum and planctomycetes, as determined by 165 rRNA cataloguing, is neither supported by other molecular data, nor by shared phenotypic features. The primary structure of the 165 rRNA of Pirella staleyi, reported to be analysed (Weisburg et aI., 1986), has not yet been published. As discussed by these authors, Chlamydia psittaci, a remote relative of P. staleyi, has to be considered a fast evolving organism. However, this relationship is not expressed by standard tree construction algorithms, by which the two species cluster separately, but only by analysis of certain signature nucleotides (Weisburg et aI., 1986). The question whether or not rRNAs of V. spinosum and planctomycetes is (or has been) subjected to tachytelic evolution cannot yet be decided. Both groups of eubacteria are characterized by unique morphological features but can easily be differentiated by phenotypic characters. While V. spinosum exhibits a mesodiaminopimelic acid containing murein (Schlesner, 1987), murein is missing in planctomycetes and chlamidiae at all. In any case, the branching point of V. spinosum is sufficiently distinct, not only to support the validity of this new

60

W. Albrecht, A. Fischer,

J. 5mida, and E. 5tackebrandt

Table 3. Partial sequences of Verrucomicrobium (Vs) 165 rRNA determined by reverse transcriptase. Nucleotides not readable in the sequencing gels are marked as "N" Ec Dd An

UACCCGC.AG UACCUUC.AA UACCUGA.GG UAUUAUA.UG UACCUAA.CC UACCAGG.UA

GGCUAACUCU GGCUAACUCC GGCUAACUCC GGCUAAUUCC GGCUAACUCC AG~AAAGCCAC GGCUAACUAC AAGAAGCACC GGCUAACUCC

GUGCCAGCAG GUGCCAGCAG GUGCCAGCAG GUGCCAGCAG GUGCCAGCAG GUGCCAGCAG GUGCCAGCAG

CCGCGGUAAU CCGCGGUAAU CCGCGGUAAU CCGCGGUAAU CCGCGGUAAU CCGCGGUAAU CUGCGGUAAU

ACGGAGGUCU ACGGAGGGUG ACGGAGGGUG ACGGGAGAGG ACGGAGGAUC ACGUAGGUGG ACGGAGGU.G

CAAGGCGUAGU CAAGC.GUUAA CAAGC.GUUAA CAAGC.GUUAU CGAGC.GUUAU CAAGC.GUUUU CUAGC.GUUAA

Vs

Bf Bs Cp

GUGGCGUGGA GCGGUUUGUU GCUGUAGUGU GCGGUUAAUC GUGGACUGGU GCGGUUUCUU GCGGAAAGGA

AAGUCGGAUG AAGUCAGAUG AAGUCAGGGG AAGUCUGUUG AAGUCAGUUG AAGUCUGAUG AAGUUAGAUG

UGAAAUCCGG UGAAAUCCCC UGAAAUCCCA UCAAAGCGUG UGAAAGUUUG UGAAAGCCCC UUAAAUCUUG

GGGCUCAACC GGGCUCAACC CGGCUCAACC GGGCUCAACC CGGCUCAACC CGGCUCAACC GGGCUCAACC

UCCGAAUUGC UGGGAACUGC GUGGAACUGC UCAUACAGGC GUAAAAUUGC GGGGAGGGUC CCAAGCCAGC

GUCCGAUACU AUCUGAUACU CUUUGAUACU AAUGGAAACU AGCUGAUACU AUUGGAAACU AUCUAAUACU

CCCAUGC.UG GGCAAGCUUG GCACAACUUG GAUUGACUAG GUCAGUCUUG GGGGAACUUG

Vs Ec Dd An Bf Bs Cp

GUAGCAGUGA GUAGCGGUGA GUAGGAGUGA GUAGCGGUGA GUf,GCGGUGA GUAGCGGUGA GUAGCGGUGA

AAUGCGUAGA AAUGCGUAGA AAUCCGUAGA AAUGCGUAGA AAUGCUUAGA AAUGCGUAGA AAUGCGUAGA

UAUCGAGAGG GAUCUGGAGG UAUCUGGAGG UAUCUGGAAG UAUCACGAAG GAUGUGGAGG UAUGUCGAAG

AACACUAGUG AAUACCGGUG AACAUCAGUG AACACCAGCG AACUCCGAUU AACACCAGUG AACACCAGUG

GCGAAGGCGA GCGAAGGCGG GCGAAGGCGG GCGAAAGCGC GCGAAGGCAG GCGAAGGCGA GCGAAGGCGC

V3 Ec Dd An Bf Bs Cp

CGCGUu.lac CGCGUUAAGU CGCGUUAAGC CGCGUUAAGU AGCAUUAAGU CGCAUUAAGC CGCGUUAAGU

GUGCCGCCUG CGACCGCCUG AUCCCGCCUG GUUCCGCCUG AUUCCACCUG ACUCCGCCUG AUGCCGCCUG

GGAAGUGCGG GGGAGUACGG GGGAGUACGG GGGAGUACGC GGGAGUACGC GGGAGUACGG AGGAGGACAC

UCGCGAGAUU CCGCAAGGUU UCGCAAGGCU ACGCAAGUUG CGGCAACGGU UCGCAAGACU UCGCAAGGGU

AAAACUCAAA AAAACUCAAA GAAACUCAAA GAAACUCAAA GAAACUCAAA GAAACUCAAA GAAACUCAAA

Vs

GCAACGCGAA GCAACGCGAA GCAACGGGAA GCAACGCGAA GAUACGCGAG GCAACGCGAA GCAACGCGAA

GAACCUUACC GAACCUUACC GAACCUUACC GAACCUUACC GAACCUUACC GAACCUUACC GAACCUUACC

UGGGCUUGAC UGGUCUUGAC UAGGUUUGAC AGGGUUUGAC CGGGCUUAAA AGGUCUUGAC UGGGCUUGAC

NU.GCACUG.U AU.CCACGGAA AU.CCACGGAA AU. CCCCCGAA UUGCAGUGGAA AUCCUCU .GAC AU.GUAU.UUG

UCGUCAGCUC UCGUCAGCUC UCGUCAGCUC UCGUCAGCUC UCGUCAGCUC UCGUCAGCUC UCGUCAGCUC

GUGUCGUGAG GUGUUGUGAA GUGUCGUGAG GUGUCGUGAG GUGCCGUGAG GUGUCGUGAG GUGCCGUGAG

AUGUUGGGUN AUGUUGGGUU AUGUUGGGUU AUGUUGGGUU GUGUCGGCUU AUGUUGGGUU GUGUUGGGUU

AAN AAG 1094 AAG AAG AAG AAG AAG

VS

Df B3 Cp

Ec Dd An

Ec Dd An

Bf B3 Cp Vs

Ec Dd An

Br Bs Cp

GUGUCGcr:!~c

GilGAAGAGAC AAGAAGCACC AGGAAGCACC AAUAAGCCUC AA'JAAGGAUC

UCGGAAUCAC UCGGAAUUAC UCGGAAUUAC CCGGAAUUAU CCGGAUUUAU CCGGAAUUAU UCGGAUUUAU

UGGGCGUAAA UGGGCGUAAA UGGGCGUAAA UGGGCGUAAA UGGGUUUAAA UGGGCGUAAA UGGGCGUAAA

AUCUUU~UAG

AGAACUGGAG AGUCUCGUAG AAUCCGGGAG AGUAUGGUAG AGUACAGUAG AGUGCAGAAG AGGGUAGACG

GGGGAUCUGG AGGGGGGUAG AGGGUGGCGG GGGUAGCGGG UGGUGGGCGG AGGAGAGUGG GAGAAAAGGG

GAUCCUGCAG CCCCCUGGAC CCACCUGGAC GCUACUGGGC CUCACUGGAC CUCUCUGGUC UUUUCUAAUU

AGUAUCUGAC GAAGACUGAC CGCUAUUGAC CAUAACUGAC UGCAACUGAC UGUAACUGAC UACACCUGAC

ACUGAUGCAC GCUCAGGUGC GCUGAGGUGC GCUCAUGGAC ACUGAUGCUC GCUGAGGAGC GCUAAGGCGC

GAAGG--GAAAG--CAAAG--GAAAG--GAAAG--GAAAG--GAAAG---

GAAAUUGAC. UGAAUUGACG GAAAUUGACG GGAAUUGACG GGAAUUGACG GGAAUUGACG AGAAUUGACG

GGGGACCGCA GGGGCCCGCA GGGGCCCGCA GGGGCCCGCA GGGGCCCGCA GGGGCCCGCA GGGGCCCGCA

CAAGCGGUGG CAAGCGGUGG CAAGCGGUGG CAAGCGGUGG CAAGCGGAGG CAAGCGGUGG CAAGCAGUGG

AGUAUGUGGC AGCAUGUGGU AGUAUGUGGU AGUAUGUGGU AACAUGUGGU AGCAUGUGGU AGCAUGUGGU

GUCGUCGGUG GUUUUCAGAG CCCUCCCGAA .UCUCUUGGA .UGAUGUGGA .UAUCCUAGA .ACCGCGGCA

AAAGCCGGCU AUGAGAAUGU AAGGAGGGGU AACGAGAGAG AACAUGUCAG GAUAGGAC.G GAAAUGUC.G

A.GUGUAGCA G.CCUUCGGG GCCCUUCGGG UGCCUUC.GG UG.AGCA.AU UC.CCCU.UC UU.UUCC.GC

AUAGCGC.UNN A.ACCG •• UGA GAGCCG .. UGA GAGCGG •• GGA CACCGC •. UGU GGGGGCAGAGU AAGGACAGAUA

GGGUGCGUNG GCGCACGCAG GCGCACGUAG GCGCCUGCAG GGGAGCGUAG GGGCUCGCAG GGGCGUGUAG AAUUCUCGGU AAUUCCAGGU .HUUCCAGGU AAUUCCAGGU AAUUCGUGGU AAUUCCACGU AAUUCCACGU

UUAAUUCGAU UUAAUUCGAU UUAAUUCGAU UUAAUUCGAU UUAAUUCGAU UUAAUUCGAA UUAAUU.GGU

GCACAGGUGC G.ACAGGUGC G.ACAGGUGC G.ACAGGUGG G •• AAGGUGC G.ACAGGUGG C.ACAGGUGC

584

684

965

UGCAUGGCUG UGCAUGGCUG 1061 UGCAUGGCUG UGCAUGGCUG UGCAUGGUUG UGCAUGGUUG UGCAUGGCUG

Reference sequences are taken from Weisburg et al. (1986): Ec - Escherichia coli, Dd - Desulfovibrio desulfuricans, An - Anacystis nidulans, Bf - Bacteroides fragilis, Bs - Bacillus subtilis, Cp - Clamydia psittaci . . = gap; --- = not sequenced. Vs Vs Ec At Dd An Bf Cp Bs

77.1 74.7 76.7 75.1 74.7 74.2 73.5

Ec 0.273 83.9 86.9 82.1 77.6 78.6 83.4

At 0.309 0.181 81.7 78.0 74.4 77.7 81.5

Dd 0.279 0.144 0.210 80.3 75.0 76.8 82.6

An

0.303 0.205 0.260 0.229 75.8 77.3 81.6

Bf 0.309 0.266 0.313 0.304 0.292 76.2 75.3

Bs

Cp 0.316 0.252 0.265 0.278 0.270 0.286 77.1

0.327 0.188 0.212 0.198 0.211 0.300 0.273

Table 4. Sequence comparison of long 165 rRNA stretches. Lower left triangle: Percent homology values calculated on the basis of 515 bases. Gaps were assigned half the weight assigned to a missmatch

Upper right triangle: Knuc-values derived from the homology values (Hori, 1975; McCaroll et aI., 1983) Ds = Verrucomicrobium spinosum, Ec = Escherichia coli, At = Agrobacterium tumefaciens, Dd = Desulfovibrio desulfuricans, An = Anacystis nidulans, Bf = Bacteroides fragilis, Cp = Chlamydia psittaci, Bs = Bacillus subtilis

genus (Schlesner, 1987) but to give this species the status of a separate eubacterial division as also done for the planctomycetes and for C. psittaci. The existence of a prosthecate organism phylogenetically unrelated to other eubacteria with similar morphology is another example for the convergent evolution of an ap-

parently valuable taxonomic marker. Thus the presence of prosthecates adds a new item to the list of characters of doubtful taxonomic significance for the definition of higher taxa as shown before for gliding motility (Reichenbach et al., 1986) anoxygenic photosynthesis (Woese et al., 1984a, b), budding reproduction modus (Rothe et al.,

Verrucomicrobium spinosum, an Ancient Line of Descent

61

,.------------l Agrobact.rium tunutJb.cums I lot-... dM.ion _ _ _ _ _ _ _ _ _ _ _~I~E~s~cluJ~nc~ . ~h~~'=C;O;li~~I----~ L.. l¥'-...dlwion doS'Ulfuricans I '------------------------l GDosulfovibrio - lWtiUw,,",

rl

Fig. 1. Dendrogram of relationships, showing the phylogenetic position of V. spinosum among various eubacterial divisions. This partial dendrogram is extracted from a comprehensive tree based on the analysis of more than 500 prokaryotes analysed by the 16S rRNA cataloguing approach (Fox, Stackebrandt, Woese, unpublished).

a

EscluJrich~

1---------------------- Bacillus subtilis 1---------------------- Anacllstis n.idulans 1---------------------- Chlam1idia psutaoi ' - - - - - - - - - - - - - - - - - - - - - - - Bac:t"roid.s fragilis

r------------------------ Pinlla

stalelli

' - - - - - - - - - -- - - - - - - - - - - - - - - V• .,.,....,omic:TObium spin.osum 0.2

0 ', 4

0'.8

C:Qli

DeS'UlfollibriD desulfuricans Bacillus subtilis Agrobacterium tumoJb.cwns

An
Bacteroides fragil is Varrucomicrobium spino sum

70

eo

10

1001

Komolou

...

Fig. 2. Dendrogram and phylogenetic tree based on sequence comparison of two long stretches of 16S rRNAs (Table 3). a. Dendrogram of relationships, based on average linkage clustering (among the merged groups) of homology values (Table 4, lower left triangle). b. Unrooted phylogenetic tree constructed from the evolutionary distances (Table 4, upper right triangle) with the treeing algorithm of Fitch and Margoliash (1967). The distance measure (bar) corresponds to 0.05 mutational events per sequence position.

1986), cell shape (Stackebrandt arid Woese, 1981; Woese et aI., 1984c), Gram-staining behaviour (Brooks et aI., 1981; Stackebrandt et aI., 1985b), or the presence of endospores (Stackebrandt et aI., 1987). Acknowledgement. This work was supported by the Deutsche Forschungsgemeinschaft and by the Gesellschaft fUr Biotechnologische Forschung (GBF) for performing research of relevance for the German Collection of Microorganisms (DSM). We thank H. Schlesner for providing us with a strain of V. spinosum and C. R. Woese and G. E. Fox for the gift of DNA primers.

References Brooks, B. W., Murray, R. G. E., Johnson, j. L., Stackebrandt, E., Woese, C. R., Fox, G. E.: A study of the red-pigmented micrococci as a basis for taxonomy. Int. ]. System. Bact. 30, 627-646 (1980) Fischer, A.: Phylogenetische Untersuchungen an 24 Stammen knospender und prosthekater Bakterien. Ph. D. Thesis, Technical University, Munich FRG (1986)

Fischer, A., Roggentin, T., Schlesner, H., Stackebrandt, E.: 16S ribosomal RNA oligonucleotide cataloguing and the phylogenetic position of Stella humosa. System. Appl. Microbiol. 6, 43-47 (1985) Fitch, W. M., and Marguliash, E.: Construction of phylogenetic trees. Science 155, 279-284 (1967) Fox, G. E., Stackebrandt, E.: The application of 16S rRNA cataloguing and 5S rRNA sequencing in microbial systematic&. In: Methods in Microbiology Vol. 19, R. Colwell (Ed.). (ip press) Fox, G. E., Pechman, K. R., Woese, C. R.: Comparative cataloguing of 16S ribosomal ribonucleic acid: molecular approach to prokaryotic systematics. Int. J. System. Bact. 29, 44-57 (1977) Gibson, j., Ludwig, W., Stackebrandt, E., Woese, C. R.: The phylogeny of the green photosynthetic bacteria: absence of a close relationship between Chlorobium and Chloroflexus. System. Appl. Microbiol. 6, 152-156 (1985) Hirsch, P., Schlesner, H.: The genus Stella, p. 461-465. In: The Prokaryotes. A handbook of habitats, isolation and identification of bacteria, M. Starr, H. Stolp, H. G. Triiper, A. Balows, H. G. Schlegel (eds.). Berlin, Springer-Verlag 1981 Hori, H.: Evolution of 5S RNA. J. Mol. Evol. 7, 75-86 (1975) Johnson, j. L.: Genetic characterization. In: Manual of methods

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J. Smida,

and E. Stackebrandt

for general microbiology, pp.450-475, P. Gerhard, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. Briggs Phillips (eds.), Washington, American Society for Microbiology 1981 Kirby, K. S.: Isolation of nucleic acids with pheno solvents, p. 87-99. In: Methods in Enzymology XII B. L. Grossman, K. Moldave (eds.). New York-London, Academic Press 1968 Lane, D. j., Pace, B., Olsen, G. j., Stahl, D. A., Sogin, M. L., Pace, N. R.: Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Nat!. Acad. Sci. USA 82, 6955-6959 (1985) Lee, S. G., Evans, W. R.: Hybrid ribosome formation from Escherichia coli and chloroplast ribosome subunits. Science 173, 264-271 (1971) McCaroll, R., Olsen, G., Stahl, D., Woese, C. R., Sogin, M.: Nucleotide sequence of the Dictyostelium discoideum smallsubunit ribosomal ribonucleic acid inferred from gene sequence: Evolutionary implications. Biochemistry 22, 5858-5868 (1983) Oyaizu, H., Woese, C. R.: Phylogenetic relationships among the sulfate respiring bacteria, myxobacteria and purple bacteria. System. App!. Microbio!. 6, 257-263 (1985) Reichenbach, H., Ludwig, W., Stackebrandt, E.: Lack of relationship between gliding cyanobacteria and filamentous heterotrophic eubacteria: comparison of 16S rRNA catalogues of Spirulina, Saprospira, Vitreoscilla, Leucothrix, and Herpetosiphon. Arch. Microbio!. 145,391-395 (1986) Rothe, B., Fischer, A., Hirsch, P., Sittig, M., Stackebrandt, E.: The phylogenetic position of the budding bacteria Blastobacter aggregatus and Gemmobacter aquatilis gen. nov., sp. nov. Arch. Microbio!. 147, 92-99 (1986) Schlesner, H.: Pirella marina sp. nov., a budding, peptidoglycanless bacterium from brackish water. System. App!. Microbio!. 8, 177-180 (1986) Schlesner, H.: Verrucomicrobium spinosum gen. nov., sp. nov.: a fimbriated prosthecate bacterium. System. App!. Microbio!. (in press) Schiesner, H., Stackebrandt, E.: Assignment of the genera Planctomyces and Pirella to a new family Planctomycetateae fam. nov. and description of the order Planctomycetales ord. nov. System. App!. Microbio!. 8,174-176 (1986) Stackebrandt, E., Woese, C. R.: Towards a phylogeny of actionomycetes and related organisms. Curr. Microbio!. 5, 131-136 (1986) Stackebrandt, E., Ludwig, W., Fox, G. E.: 16S rRNA oligonucleotide cataloguing, pp. 75-107. In: Methods in Microbiology, Vo!' 18, G. Gottschalk (Ed.). Orlando, Academic Press, 1985a

Stackebrandt, E., Pohla, H., Kroppenstedt, R. M., Hippe, H., Woese, C. R.: 16S rRNA analysis of Sporomusa, Selenomonas, and Megasphaera: on the phylogenetic origin of Gram-positive eubacteria. Arch. Microbio!. 143,270-276 (1985b) Stackebrandt, E., Fischer, A., Hirsch, P., Roggentin, T., Schlesner, H.: The phylogeny of an ancient group of budding peptidoglycan-less eubacteria: the genera Planctomyces and Pirella. Endocyt. C. Res. 3, 29-40 (1986) Stackebrandt, E., Ludwig, W., Weizenegger, M., Dorn, S., Fox, G. E., Woese, C. R., Schleifer, K. H.: Comparative 16S rRNA oligonucleotide analysis and murein types of roundsporeforming bacilli and non-sporeforming relatives. J. Gen. Microbio!. (in press) Staley, j. M.: The genera Prosthecomicrobium and Ancalomicrobium. In: The Prokaryotes. A handbook of habitats, isolation and identification of bacteria, M. Starr, H. Stolp, H. G. Truper, A. Balows, H. G. Schlegel (eds.), pp. 456-460. Berlin, Springer-Verlag 1981 Traub, P., Mizu:;,hima, S., Lowry, C. V., Nomura, M.: Reconstitution of ribosomes from subunit subribosomal components, p. 391-407. In: Methods in Enzymology XX, part C, L. Grossman, K. Moldave (eds.). London-New York, Academic Press 1971 Weisburg, W. G., Hatch, T. P., Woese, C. R.: Eubacterial origin of chlamydiae. J. Bact. 167, 570-574 (1986) Woese, C. R., Gutell, R., Gupta, R., Noller, H. F.: Detailed analysis of the higher-order structure of 16S-like ribosomal ribonucleic acids. Microbio!. Rev. 47, 621-669 (1983) Woese, C. R., Stackebrandt, E., Weisburg, W. G., Paster, B. j., Madigan, M. T., Blanz, P., Gupta, R., Fowler, V. j., Hahn, C. M., Fox, G. E.: The phylogeny of purple bacteria: The alpha subdivision. System. App!. Microbio!. 5, 315-326 (1984a) Woese, C. R., Weisburg, W. G., Paster, B. j., Hahn, C. M., Knoops, H. P., Harms, H., Stackebrandt, E.: The phylogeny of purple bacteria: the beta subdivision. System. App!. Microbio!. 5, 327-336 (1984b) Woese, C. R., Blanz, P., Hahn, C. M.: What isn't a pseudomonad: the importance of nomenclature in bacterial classification. System. App!. Microbio!. 5, 179-195 (1984c) Woese, C. R., Stackebrandt, E., Macke, T. j., Fox, G. E.: A definition of the major eubacterial taxa. System. App!. Microbio!' 6, 143-151 (1985) Yang, D., Oyaizu, Y., Oyaizu, H., Olsen, G. j., Woese, C. R.: Mitochondrial origins. Proc. Nat!' Acad. Sci. USA 82, 4443-4447 (1985)

Prof. Dr. E. Stackebrandt, Institut fur Allgemeine Mikrobiologie, Christian-Albrechts-Universitat, Olshausenstr. 40 D-2300 Kiel, FRG