Phylogenetic Relationships Among the Sulfate Respiring Bacteria, Myxobacteria and Purple Bacteria

System. App!. Microbiol. 6, 257-263 (1985)

Phylogenetic Relationships Among the Sulfate Respiring Bacteria, Myxobacteria and Purple Bacteria H. OYAIZU and C. R. WOESE 1 Department of Genetics and Development, 515 Morrill Hall, University of Illinois, Urbana, Illinois 61801, USA

Received April 9, 1985

Summary Ribosomal RNA sequence comparisons have been used to refine the phylogenetic relationships among the sulfate (and sulfur) respirers, myxobacteria and the purple bacteria. Several relationships originally suggested by signature analysis of rRNA oligonucleotide catalogs have been substantiated. They are (1) a specific relationship between the beta (represented by Pseudomonas testosteroni) and gamma (represented by Escherichia coli) subdivisions of the purple bacteria, to the exclusion of the alpha subdivision (represented by Agrobacterium tumefaciens), and (2) a specific relationship between the sulfate (and sulfur) respirers (represented by Desulfovibrio desulfuricans) and the myxobacteria (repesented by Myxococcus xanthus). At a deeper level the group of sulfate respirers, myxobacteria and bdellovibrios appears specifically related to the previously defined purple bacterial unit.

Key words: Desulfovibrio - Myxococcus - Purple bacteria - Phylogeny-16S ribosomal RNA

Introduction Partial sequencing of 16S ribosomal RNA, i. e. the method of oligonucleotide cataloging, has been used over the past decade to define the phylogeny of the bacteria (Fox et ai., 1980; Stackebrandt and Woese, 1981a; Woese, 1985; Woese et ai., 1985b). To date over 400 bacterial species have been characterized by the method. Ten major phylogenetic groups of eubacteria - each at least the equivalent phylogenetically of a eucaryotic Phylum or Divison - have been defined, as have the major subdivisions within many of them (Woese et ai., 1985b; Woese 1985). The ten groups, and their known subdivisions, are shown in Table 1. The definition of these high level bacterial taxonomic units puts bacterial phylogeny on a par with eucaryote phylogeny, in the sense that the major or primary divsions have now been identified in each case, but in both cases the relationships among the higher level units, their branching order relative to one another, remain uncertain. Cataloging studies reveal a number of unexpected relationships among eubacteria, for example, the specific clustering of the sulfate and sulfur respirers with the myxobacteria and bdellovibrios (Ludwig et ai., 1983a; Hespell et 1

To whom requests for reprints should be sent.

ai., 1984; Stackebrandt et ai., in preparation). The approach also vitiates much of the conventional wisdom concerning bacterial relationships. The purple photosynthetic bacteria, organisms that were historically considered to be phylogenetically separate from most if not all non-photosynthetic bacteria, and to divide naturally into two distinct subgroups on the basis of type of sulfur metabolism, are a prime example of this (Pfennig and Truper, 1974; Pfennig, 1977). The purple photosynthetic bacteria, far from being isolated, are thoroughly intermixed phylogenetically with non-photosynthetic genera, Escherichia, Aquaspirillum, Legionella and Pseudomonas, for example (Gibson et ai., 1979; Woese et ai., 1982; Ludwig et ai., 1983b; Fowler et ai., 1984; Stackebrandt et ai., 1984a; Woese et ai., 1984a; 1985a). Moreover, they divide naturally into three distinct groups, not two - the socalled alpha (purple non-sulfur), beta (purple non-sulfur) and gamma (purple sulfur) subdivisions (Woese et ai., 1984a, b; 1985a). None appears specifically related to one of the others to the exclusion of the third. The most recent taxonomy of the purple bacteria maintains the classical bipartite division, which is a de facto phylogenetic statement, regardless of whether the authors actually claim phylogenetic validity for their taxonomy

258

H.Oyaizu and C. R. Woese Phylum or Subdivision

Detailed description found in references

(1) Gram Positive Eubacteria (a) Clostridial (or low G+C) subdivision (b) Actinomycete (or high G+C) subdivision (2 ) Purple Bacteria and Relatives (a) Alpha subdivision (b) Beta subdivision (c) Gamma subdivision

1-2 3-5 6-7 1, 8 9 10 11

(3) Sulafte Respires and Relatives (a) Sulfate and sulfur respires (b) Myxobacteria (c) Bdellovibrios

2 12 13

(4) Spirochetes and Relatives (a) Spirochetes and Treponemes (b) Leptospiras (c) Obligate Anaerobic Halophiles

14

15-16

(5) Bacteroides-Flavobacteria -Cytophagas (a) Bacteroides (b) Flavobacteria-Cytophagas

17

(6) Cyanobacteria and Chloroplasts

1-2, 18-20

(7) Green Sulfur Bacteria

21

(8) Green Non-sulfur Bacteria

21

(9) Radioresistant Bacteria

1,2,22

(10) Plantomyces

(1 ) Fox et al., 1980 (2) Woese et al., 1985b (3) Fox et al., 1977 (4) Stackebrandt et al., 1983a (5) Woese et al., 1980 (6) Stackebrandt & Woese 1981b (7) Stackebrandt et al., 1983b (8) Gibson et al., 1979 (9) Woese et al., 1984a (10) Woese et al., 1984b (11) Woese et al., 1985a

Table 1. The ten eubacterial phyla

2,23 (12 ) (13 )

(14 ) (15 ) (16) (17) (1 8) (19 ) (20 ) (21 ) (22 ) (23 )

Ludwig et al., 1983a Hespell et al., 1984 Paster et al., 1984 Oren et al., 1984a Oren et al., 1984b Paster et al., 1985 Bonen & Doolittle, 1976 Bonen & Doolittle, 1978 Seewaldt & Stackebrandt, 1<).\ 2 Gibson et al., 1985 Brooks et al., 1980 Stackebrandt et al., 1984b

(Imhoff et aI., 1984). It is possible to partially reconcile the classical and the molecular view of purple bacterial phylogeny on the assumption that the two purple non-sulfur groups branched from each other after they split, as a unit, from the purple sulfur bacteria, and the cataloging methods is not sufficiently sensitive to pick up these early divisons if they occurred in rapid succession. . To settle questions of this nature, it is necessary to use complete sequencing of ribosomal RNAs. The approach not only yields more data than the older cataloging method, but it allows the use of ribosomal RNA secondary structural features as phylogenetic characters, and permits a more accurate determination of evolutionary rates. In the present communication we use full sequencing of ribosomal RNA to give additional evidence for the unexpected relationship among the sulfate and sulfur respirers, the myxobacteria and the bdellovibrios, and to further define the phylogeny of the purple bacteria.

Materials and Methods Bacterial strains. Several grams of frozen Desulfovibrio desulfuricans (ATCC 27774) and Myxococcus xanthus strain MD207 cells were kindly provided by Profs. R. B. Hespell and M. Dworkin, respectively. Cloning. Nucleic acids, RNA and DNA, were isolated from the frozen cell pellets by standard procedures (Marmur, 1961); Woese et al., 1976). The rRNA genes of D. desulfuricans and M. xanthus were initially cloned as Sau 3A partial restriction fragments in the Bam HI site of lambda L47.1 (Loenen and Brammar, 1980). Subcloning into phage M13 utilized the Eco RI site located at position 674 in 16S rRNA (E. coli numbering; Brosius et al., 1978), to give two fragments covering the entire 16S rRNA gene in both cases. Sequencing methods. The dideoxynucleotide chain termination method (Sanger et al., 1977) was used, templates being incorporated into the single stranded phage M13 genome (Messing, 1983). Synthesized strands were labeled by the inclusion of d(alpha-[ 35S]thio)ATP (Biggin et al., 1983). Two types of G sequenc-

Phylogenetic Relationship of Sulfate Respiring Bacteria ing reactions were routinely employed, one normal, the other in which dGTP was replaced by dITP (ddGTP being used to terminate chain growth) (Mills and Kramer, 1979). The usual M13 priming site (Messing, 1983) as well as specific priming sites within the ribosomal RNA genes, for which primers were synthesized (most at the University of Illinois DNA Synthesis Facility), were used. The rRNA-specific primers were designed for regions of the 16S rRNA molecule whose sequence tends to be common to most if not all eubacteria, and in some cases archaebacteria as well (Woese et al., 1983). Oligonucleotides of length 15-17 nucleotides were synthesized that prime in the forward (i.e. same sequence as the rRNA) and reverse directions, covering the following positions: (E. coli numbers; F = forward, R = reverse) 10F, 50F, 125R, 260F, 270R, 350F/R, 520F/R, 790F, 920R, 1l00F/R, 1240F/R, 1400F/R and 1540R. Eighty to 95 % of each gene sequence was determined in both the forward and the reverse direction.

Results and Discussion Fig. 1 shows the sequences of the 165 ribosomal RNA genes from Escherichia coli (Brosius et al., 1978) Pseudomonas testosteroni (Yang et al., 1985), Agrobacterium tumefaciens (Yang et al., 1985), - representing the gamma, beta and alpha purple bacteria respectively (Woese et al., 1984a, b; 1985a) - Desulfovibrio desulfuricans and Myxococcus xanthus, aligned with those from Bacillus subtilis (Green et al., 1985), Anacystis nidulans (Tomioka and Sugiura, 1983) and a representative archaebacterium Methanococcus vannielli (Jarsch and Bock, 1985). The lower left hand triangle of Table 2 shows the percent homology among the various pairs of sequences, calculated for all positions in the Fig. 1 alignment that are represented in all of the eubacterial sequences, a total of 1460 positions. That these small differences in percent

Table 2. Homology matrix for the sequence of Fig. 1 Lower left triangle: pre cent homology for the various pairs of sequences in the Fig. 1 alignment. Only positions in which every eubacterial sequences has representation are considered, a total of 1460 positions. Upper right triangle: same as lower left, except that all positions of invariant composition among the eubacterial seuquences have been eliminated from consideration, leaving 576 positions. Numbers in parentheses: homology calculated from 576 position set, except that all identities between any two eubacterial sequences that also have the same composition in the outgroup, archaebacterial sequence are not considered. In order to permit comparison of these to the previous homologies (listed above them in each case), the numbers have been normalized, however - i. e. multiplied by the ratio of the average values in this and the previous case.

1 Ec

1 Ec

259

homology are indeed significant is shown by the percentages in the upper right hand triangle of Table 2, which have been calculated as before except that all position in the alignment whose composition is invariant among the eubacterial sequences have been eliminated from consideration. [These have no phylogenetic significance among the eubacteria in any case.] Three pairs of sequences in Table 2 have their highest homology with one another, i. e. E. coli and P. testosteroni (55.0 percent - upper right hand triangle), D. desulfuricans and M. xanthus (53.8 percent) and B. subtilis and A. nidulans (52.3 percent). These homologies, however, are the sum of three contributions - from derived similarity (that evolved in a particular subline), from ancestral similarity (sequence present in the common ancestor of the entire group), and from convergence (multiple phylogenetically independent occurrences of the same composition in different sublines). Only compositional similarity of the first (derived) type represents true specific relationship. Notice that all the eubacterial sequences are not equidistant from the outgroup archaebacterial sequence. B. subtilis and A. nidulans are significantly closer to it than are the other eubacteria. This suggests these two eubacterial sequences to have retained proportionately more ancestral character than have the others. If so, each of the two will appear more closely related to the others than is actually the case, and their specific pairing with one another in Table 2 may result from this, not from true specific relationship. The archaebacterial, outgroup, sequence can be used to partially identify the ancestral component, and so, correct for it. Eliminating froin the pair-wise count in the analysis those positions in which composition common to a given pair of eubacterial sequences is also common to the archaebacterial sequence, should preferentially eliminate ancestral component from the talley. This has been done

2 Pt

3 At

55.0 (61)

48.6 (49) 46.2 (47)

4 5 Dd Mx 576 positions 50.9 (54) 46.0 (46) 52.4 (54)

50.5 (54) 45.8 (48) 50.0 (50) 53.8 (56)

6 Bs

7 An

47.0 (46) 44.2 (41) 47.9 (45) 51.6 (50) 50.7 (49)

44.6 (44) 43.2 (40) 44.6 (44) 47.7 (47) 47.2 (46) 52.3 (47)

2 Pt

82.3

3 At

79.7

78.8

4 Dd

80.6

79.7

8l.2

5 Mx

80.5

78.6

80.3

81.8

6 Bs

79.4

78.0

79.5

80.9

80.5

7 An

78.2

77.6

78.2

79.4

79.2

8l.2

8 Mv

60.5

61.8

61.6

61.7

6l.2

63.4

Mv

34.9 38.4 37.8 38.0 36.6 42.2 41.3

63.0

1460 positions Abbreviations: Ec - E. coli; Pt - P. testosteroni; At - A. tumefaciens; Dd - D. desulfuricans; Mx - M. xanthus; Bs - B. subtilis; An - A. nidulans; Mv - M. vanielli.

260

H. Oyaizu and C. R. Woese Eo Pt At Dd Hx 8s An MY

Ee Pt At Dd Hx

8. An My

•• AAAUUGAAGA .CGAACUAUAGA CUCAACUUGAGA • UGAACUGGAGA •• CAAUUGGAGA • UUUAUCGGAGA • CAAA,AUGGACA • •••••• AUUCC

UGUCUGGGAA. UACAUCGGAA. CGCGUGGGAA. CGCGUGGAUAA CACGUGGAUAA CACGUGGGUAA CGCGUGAGAA. CACGUGGUUAA

GUUUGAUCAU GUUUGAUCCU GUUUGAUooU GUUUGAUUCU GUUUGAUooU GUUUGAUCCu GUUUGAUa:U

GGCUCA.GAUU GGCUCAGAtru GGCUCAGAAC GGCUCAGAUU GGCUCAGAAC GGCUCAGGAC GGCUCAGGAU

GGUUGAUCCC GCCGGAGGCU

ACUGCCUGAU CGUGCCUAGU UCUACCGUGC UCUGCooUUA UCUGooOOAG CCooooUGUA UCUGCCUACA CUUAACCUCA

GGAGGGGGAU ACUGGOGGAU CCUGCOOMU UGAUCGGGAU OOCUCGGGAU AGACUGGGAU reACGGGGAC GGUGGAGCAU

GGCAGGC •• CUA GGCAOOC •• UUU GGCAGGC •• UUA GGGGUGC •• UUA GGCGUGC •• CUA GGGGUGC •• CUA GGCGUGC •• UUA GGGGUUCGACUA

GAACGCUGGC GAACGCUGGC GAACGClJGGC GAACGCUGGC GAACGClJGGC GAACGCUGGC GAACGCUGGC . ACUGCUAUU

MCUACUGGA AACU;,cUCGA AGCUOCOOGA AACAGUUGGA AACCAGUCGA AACUooGGGA AACACUUGGA AACCUUGGCA

AACOOUAGCU AAGAGUo\GCU AACUGGMUU AACGGCUGCU AAGAWGGCU AAooGGGGCU

ACACAUGCAA ACACAOOCAA ACACAOOCAA ACACAUGCAA ACACAUGCAA AUACAOOCAA ACACAOOCAA AGCCAUGCGA

AAUACCGCAU MUACCGCAU AAUACCGCAU AAUAooGGAU AAUACCGGAU AAUAooGGAU MCGAC~CU AAUACCCGAU AACUGAGGAU AAUUCUCCAU

GUCGAACGGU GUCGAACGGU GUCGAACGCC GUCGAACGCC GUCGAGCGCG GUCGAGCGGA GUCGAACGGG

AGCtnJGCUUC UUUGCUGACG •• UUCG •••• GAUGCUGAGG •• GCAA •••• •••••• GGGG •• uuec ••• G UCCUGAGUM •• GeM •••• CCCUUAGUAG •• CUUG ••• C UCCCCAUGUU •• UUCG •••• ••••• GAGeD •• uueG •••• •••••••• GC

AACAG(;AAGA AACAGGUC •• 00 •••••••• AMGGGAC •• AAUAGGG ••• CAGGUGGGAG CUC •••••••

GUCUAUGGU •••••••••••

MCGUCGC ••••••••••••••••• M GA(;AUCUA ••••••••••••••••• CG ACGCCCUA ••••••••••••••••• CG ACGCUCAAAA ••••••• UGAACUUUUU AAGCCCACGGUU. UCUUCGGAGACUGA GGUUGUUUGAAC •• CGCAUGGUUCAAA G.UGCCGA••••••••••••••••• GA AAGAAAAGCAGUCUGGAACGAUUCUUU

GACCAAAGAG GGG.GACCUUC GAUGAMCCA CGG.GACCUUC GGGGAMGA ••••••••• UUU GAGGAAAGAU GGGCUCUGCUU GGGAAAAGGU GGCCUCOOUAU CAUAAAAGGU GGC ••••• UUC GGUGA.\ACA ••••••••• UUU UCUGAAAGC. • •• " ••• • AUA

AGUGGCGGAC AGUGGCGAAC AGUGGCAGAC AGUGGCGCAC AGCGGCGCAC AGCGGCGGAC AGUGGCGGAC CAUGCCGGAC

GGGC.CUCUUG GGGC.CUUGUG A••••••• UCG GCAUGCUAUCA ACAACCUAUCA G••• GCUACCA A••••••• tJGG U••••••• GeG

GGGUGACUAA GGGUGAGUAA GGGUGAGUAA GGGUGAGUM GGGUGCGUAA GGGUGAGUAA GGGUGAGUAA GGCUCAUUAA

CCADCGGAUG CUACUAGAGC GGGUAUGAUG CGUAAGGAUG CADUCAGAUG CUUACAGAOO CCUGUAGAUG CCCGAGGAUA

120

UGCCCAGAUG GGCUGAUGGC AGCC
GUAGGUGGGG GUUGGUGGGG GUUGGUGGGG GUUGGGOOCC GUUGGCGGGG GUUGGOOAGG GUUGGUGGGG CUUGGUGGGG

UAAC
CCUAGGCGAC CCAAGCCUGC CCAAGGCGAC CCAAGGCAUC CCAAGGCAAC CCAAGGCAAC CCAAGGCGAC CCAAGooUAC

GAUa:;CUAGC GAUCUGUAGC GAUooAUAGC GAUGGGUAGC GACGGGUAGC GAUGCGUAGC GAUCAGUAGC GAUCCAUACC

UGGUCUGAGA UGGUCUGAGA UGGUCOOAGA CGAUUUGAGA UGGUCOOAGA CGAooUGAGA UGGUCUGAGA GGCCUUGAGA

GGAUGACCAG GGACGACCAG GGAOOAUCAG GGAOOAUCGG GGACGAUCAG GGGUGAUCGG GGAUGAUCAG GACGGAGCCC

CCACACUGGA CCACACUGGG CCACAUUGGG CCACACUGGA CCACACUGGA CCACACUGGG CCACACUGGG GGAGAUGGGG

ACUGAGAGAC ACUGAGACAC ACOOAGACAC ACUGAAACAC ACUGAGACAC ACUGAGACAC ACUGAGACAC ACUGAGACAC

GGUCCAGACU GGCCCAGACU GGCCCAAACU GGUCCAGACU GGUCCACACU GGCCCAGACU GGCCCAGACU GGCCCCAGGC

CCUACGGGAG CCUACGGGAG CCUACGGGAG CCUACGGGAG CCUACGGGAG CCUACGGGAG CCUACGGGAG CCUACGGGGC

GCAGCAGUGG GCAGCAGUGG GCAGCAGUGG GCAGCAGUGG GCAGCACUGG GCAGCAGUAG GCAGCAGUGG GCAGCAGGCG

360

GGMUAUUCC GGAAUUUUGG GGAAUAtruGG GGAAUAUUGC GGAAUUUUCC GGAAUCUUCC GGAAUUUUCC CGAAACCUCC

ACAAUGGGCG ACAAUGGGCG ACAAUGGGCG GCAAUGGGCG GCAAUGGGCG GCAAUGGACG GCAAUGCCCG CCAAUGCACC

CAAGooUGAU AAAGooOOAU CAAGooOOAU AAAGCCUGAC AAAGOCUGAC AAAGUCUGAC CAAGreUCAC AAACUCCGAC

GCAGCCAUGC CCAGCAAUGC CCAGOCAUGC GCAGCGACGC GCAGCAACGC GGAGCAACGC GGAGCAACGC GGGGGGACCC

CGCGUGUAOO CGCGOOCAGG CGCGUGAGUG CGCGUGAGGG CGCGUGUGUG CGCGUGAGUG CGCCUGCCGC CMGUGCUCA

AAGAAGGooU AOOAAGGCCC AUGAAGGCCU AUGAAGGUUU AUGAAGGUCU AUGAAGGUUU AGGAAGGUUU UGC •••••••

UCGGGUUGUA UCGGGUUGUA UAGCGUUGUA UCGGAUCGUA UUGGAUUGUA UCGGAUCGUA UUGGACUGUA ACA ••••• GC

AAGUACUUUC AACUGCUUUU AAGCUCUUUC AACCUCUGUC AAGCA,CUUUC AAGCUCUGUU AACCCCUUUU AUGGGCUUUU

ACCGGGGAGG GUACGCAACG ACCGGAGAAG AGAAGGGAAG GACCGGGAAC GUUAGGGAAG CUCAGGCAAG AUCAAGU •••

!.AGG. GACOAA ACUUAAUACC AAAA.GCCUGG GGCUAAUAUC AUAA ••••••••••••••••• AAACUACGUUG UGCUAAU. CA AAAA. CCCCUU GGCUAAC . AU AACMGUACCG UUCGAAUAGC AAGA ••••••••••••••••• • ••• GUAMCA ••••••••••

UUUGCUCAUU CCCGGGUCAU ••••••••• U GCAGCGUACU CCAACGGCUU GCGGUACCUU • ••••• AAGU

480

Ec GACGUUACCC

GCACAAGAAG UAAGAAUAAC GGAGAAGAAG UCAAAGGAAG GGAGMGAAG AACCAGAAAG GAOOAAUAAG GAGGAAUAAG

CACCGGCUAA. CACCGGCUAA. CCCCGGCUAA. CACCGGCUAA. CACCOOCUAA • CCACGGCUAA. CCUCOOCUAA. GGCUGGGCAAG

CUCCGUGCCA CUACGUGCCA CUUCGUGCCA CUCCGUGCCA CUCUGUGCCA CUACGUGCCA WCCGUGCCA UUCGCUGCCA

GCAGOCGCGG GCACCCGCCG GCAGooGCGG GCA.GCCGCGG GCAGCCGCGG GCAGCCGCGG GCAGCCGCOO GCAGCCGCGG

UAAUACGGAG UAAUACGUAG UAAUACGAAG UAAUACGGAG UAAUACAGAG UAAUACGUAG UAAUACGGCA UAAUACCCAC

GGUGCAAGCG GGUGCAAGCG GGGGCUAGCG GGUGCAAGCG GGUGCAAGCG GUGGCAAGCG GAGGCAAGCG GGCCCGAGUG

UUAAUCGGAA UUAAUCGGAA UUGUUCGGAA UUAAUCGGAA UUGUUCGGAA UUGUCCGGAA UUAUCCGCAA GUAGCCACUC

UUACUGGGCG UUACUGGGCG UUACUGGGCG UUACUGGGCG UUAUUGGGCG UUAUUGGGCG UUAUUGGGCG UUAUUGGGCC

UAAAGCGCAC UAA.\GCCUGC UAA.\GCGCAC UAAAGCGCAC UAAAGCGCGU UAAAGGGCUC UAAAGCGCCU UAAAGCGUCC

GCAGGCGGUU GCAGGCGGUU GUAGGCGGAU GUAGGCUGUA GUAGGCGGCG GCAGGCGGUU GCAGGCGGUU GUAGCCGGUC

UGUUAAGUCA UUGUAAGACA AUUUAAGUCA GUGUAAGUCA UGACAAGUCG UCUUAAGUCU AAUCAAGUCU CAGUAACUCC

600

CCCCGGGCUC CCCCGGGCUC CCCAGAGCUC CCCACGGCUC CCCUCAGCUC CCCCCGGCUC CGUGGGGCUC UCUCUGGCUU

AACCUGGGAA AACCUGGGAA AACUCUGGAA AAOCGUGGAA AACUGAGGAA AACCGGGGAG AACCUCAUAC AACCAGAGGA

CUGCAUCUGA CUGCCAUUGU CUGCCUUUGA CUGCCUUOOA CUGCGCCCGA GGUCAUUGGA AGGCAAUGGA CUGGCAGGGA

UACUGGCAAG GACUGCAAGG UACUGGGUAU UACUGCACAA AACUGUUGUG AACUGGGGAA AACOOAUUGA UACUGCUGGA

CUUGACUCUC CUAGAGOOCG CUUGAGUAUG CUUGAAUCCG CUUGAGUGCC CUOOAGUGCA CUAGAGUAOO CUUGGGACCG

GUAGAGGGGG GCAGAC.GGGG GAAGAGGUAA GGAGAGGGUG GGAgAGGGUG GAAGAGGAGA GUAGGGGUAG GGAGAGGACA

GUAGAAUUCC AUGGAAUUCC GUGGAAUUCC GCGGAAUUCC GCGGAAUUCC GUGGAAUUCC CGGGAAUUCC AGGGUACUCC

AGGUGUAGCG GCGUGUAGCA GAGUGUAGAG AGGUGUAGGA CCAAGUAGAG ACGUGUAGCG AGGUGUAGCG AGGGGUAGCG

GUGAAAUGCG GUGAAAUGCG GUGAAAUUCG GUGAAAUCCG GUGAAAUUCG GUGAAAUGCG GUGAAAUGCG< GUGAMUGUG

UAGAGAUCUG UAGAUAUGCG UAGAUAUUCG UAGAUAUCOO UAGAUAUGGG UAGAGAUGUG UAGAUAUCUG UUGAUCCUUG

GAGGAAUACC GAGGAACACC GAGGAACGCC GAGGAACAUC GAGGAACACC GAGGAACACC GAAGAACACC GAGGACCACC

720

GCGGCCCCCU GCAAUCCCCU GCGGCUUACU GCGGCCACCU GCGGCCACCU GCGACUCUCU GCGCGCUACU GCACUUGUCU

GGACGAAGAC GGGCCUGCAC GGUCCAUUAC GGACCGGUAU GGACGGUAAC CGUCUGUAAC GGGooAUAAC GGAACGGGUC

UGACGCUCAG UGACGCUCAU UGACGCUGAC UGACGCUGAG UGACGCUGAG UGACGCUGAG UGACGCUCAU CGACGGUGAG

GUGCGAAACC GCACGAAAGC GUGCGAAAGC GUGCGAMGC ACGCGAAAGC GAGCGAAAGC GGACGAAAGC GGACGAAAGC

GUGGGGAGCA GUGGGGAGCA GUGGGGAGCA GUGGGGAGCA GUGGGGAGCA GUGCGCACCG UAGGGGAGCG CAGGGGCGCG

AACAGGAUUA AACAGGAUUA AACAGGAUUA AACAGGAUUA AACAGGAUUA AACAGGAUUA AAAGGGAUUA AACCGGAUUA

GAUACCCUGG GAUACCCUGG GAUACCCUGG GAUACCCUGG GAUACCCUGG GAUACCCUGG GAUACCCCUG CAUACCCGGG

UAGUCCACGC UAGUCCACGC UAGUCCACGC UAGUCCACGC UACUCCACGC UAGUCCACCC UAGUCCUAGC UACUCCUcec

CGUAAACGAU CCUAAACGAU CGUAAACGAU UGUAAACGAU CGUAAACGAU CGUAAACGAU CGUAAACGAU CGUAAACUCU

GUCCACUUce GUCAACUGGU GAAUGUUAGC GGAUGCUAGA GAGAACUAGG GAGUGCUAAG GAACACUAGG GCGAACUAGG

AGGUUGUGCC UGUUGGGUCU CGUCGGGCAG UGUCGGGGA. UGUCGUGGGA UGUUAGGGGG UGUUGCGUGA UGUCACCUGG

840

Ec •• CUllGA •• GGCGU GGCUUCCGGA CCUAACGCGU UAAGUCGACC GCCUGGGGAG

UACGGCCGCA UACGGooGCA UACGGUCGCA UACGGUCGCA UACGGUCGCA UACCGUCGCA UACGCACGCA UACGGUCGCA

AGGUUAAAAC AGGUUGAAAC AGAUUAAAAC AGGCUGAAAC AGACUAAAAC AGACUGAAAC AGUUGGAAAC AGACUGMAC

UCAAAUGAAU UCAAAGGAAU UCAAAGGAAU UCAAAGAAAU UCAAAGGAAU UCAAAGGAAU UCAAAGGAAU UUAAAGGAAU

UGACGGGGGC UGACGGGGAC UGACGGGGGC UGACGGGGGe UCACGGGGGC UGACGGGGGC UGACGGGGGC UGGCGGGGGA

CCGC. ACAAGC CCGC. ACAAGC CCCC. ACAAGC ecce. ACMGC ccce. ACAAGC CCGC. ACAAGC CCGC. ACAAGC GCACCACAACG

GGUGGAGCAU GGUGGAUGAU GGUCGAGCAU GGUGGAGUAU GGUGGAGCAU GGUGGAGCAU GGUGGAGUAU GGUGGAGCCU

GUGGUUUAAU GUCGUUUAAU GUGGUUUAAU GUGGUUUAAU GUGGUUUAAU GUGGUpUAAU GUGGUUUMU GCGGUUUAAU

960

GCCUGGGGAG GCCOOGGGAG GCCUGGCGAG GCCUGGCAAG GCCUGGGGAG GCCUGGGGAG GCCUGGGGAG

CGGAAGUUUU •• AGGAACUUAC •• GGGUUUGCGCAG CGGAACCCUC.. CAGAAUCCUU.. CUGACAAUCC •• CCGAAUCUCU.. AUGAUGACGGCC

CAGAGAUGAG CAGAGAUCCU UGGAGACAUU CCGAAAAGGA CAGAGAUGAG UAGAGAUAGG UGGAAACGAG AGGUUGACGA

AAUGUGCC. UU UUGGUGCUCGA GUCCUUCAGUU GGGGUGCCCUU GGAGUGCCCGC ACGUCCCC.UU AGAGUGCC. UU ceU •• • •••••

CG. GGAACCGU AAGAGAACCUG AGGCUGGCCCC CGGGGAGCCGU AAGGGAA,CUGA CG. GGGGCAGA CG. GGAGCGGG UGCCUGAAGCG

GAGACAGGUG CACACAGGUG AGAACAGGUG GAGACAGGUG GAGACAGGUG GUGACAGCUG GAGACAGGUG CUGAGAGGUG

CUGCAUGGCU CUGCAUGGCU CUGCAUGGCU CUGCAUGGCU CUGCAUGGCU GUGCAUGGUU GUGCAUGGCU GUGCAUGGCC

GUCGUCAGCU GUCGUCAGCU GUCGUCAGCU GUCGUCAGCU GUCGUCAGCU GUCCUCAGCU GUCGUCAGCU AUeGUCAGCU

CGUGUUGUGA CGUGUCGUGA CGUGUCGUGA CGUGUCGUGA CGUGUCGUGA CGUGUCGUGA CGUGUeGUGA CGUACCGCGA

1080

CCAGUGAUAA CCGGUGACAA CCGGUGAUAA CCCGGGUUAA CCGGUGUUAA CCGGUGACAA CCGGUGACAA CUAGCGCUAA

ACUG. GAGGAA ACCG.GAGGAA GCCGAGAGGAA CCGG.GAGGAA ACCG. GAooM ACCG. CAGGAA ACeG. GAGGAA GCUA.GAGGAA

GGUGGGGAUG GGUGGGGAUG GGUGGGGAUG GGUGGGGACG GGUGGGCAUG GGUGCGGAUG GGUGUGGACG GGACCGGGCA

ACGUCAAGUC ACGUCAAGUC ACGUCAAGUC ACGUCAAGUC ACGUCAAGUC ACCUCAAAUC ACGUCAAGUC ACGAUAGGUC

1200

Ec GGAUUAGCUA

Pt AGAUUAGGUA At GGAUUAGCUA Dd CCAUUAGCUU Hx CCAUCAGCUA Bs GCAUUAGCUA An UGAUUAGCUA My CGAUUAGGUA Ec Pt At Dd Hx

Bs An My

Pt GACGGUACCG At GACGGUAUCC Dd GACGGUAOCU Hx GACGGUACCG Bs GACGGVACCU An GACGGUACCU MY •••••• GCUU Ec GAUGUGAAAU

Pt GUGGUGAAAU At GGGGUGAAAU Del GGGGUGAAAU Mx OOUGUGAAAG Bs GAUGUGAAAG An GUUGUCAAAG My CUGUlRJAAAU Ec GGUGGCGAAG

Pt At Dd Mx

GAUGGCCAAC AQJGGCGAAG AGUGGCGAAG GGUGGCGAAG 8. AGUGGCGAAG An AGCGGCGAAA Hv UAUGGCGAAG

Pt At Dd Mx Bs An

••• UAAC •• UGACU ••• UAUA •• CUGUU ••• GUAU ••• UCUU C•• UUGA.CCCCCG U•• UUCC.GCCCCU A•• UCGA.CCCGCG M.y GCCUCGAGCCCAGG

Ec UCGAUGCAAC

CAGUAACGAA CGGUGGCGCA CGGUGUCGUA CGGUGCCGAA UAGUGCUGCA CAGUGCCGUA UGGUGCCGAA

GCUAACGCGU GCUAACGCAU GUUAACGCGU CCUAACGCAU GCUAAGGCAU GCCAACGCGU GGGAAGCCGU

GAAGUUGACC UAAACAUUCC UAAGCAUCCC UAAGUUCUCC UAAGCACUCC UAAGUGUUCC UAAGUUCGCC

GCGAAGAACC GCGAAAAACC GCGCAGAACC GCGAAGAACC GCGCAGAACC GCGAAGAACC GCGAAGAACC GCCGGGCAUC

UUACCUGGUC UUACCCACCU UUACCAGCUC UUACCUAGGU UUACCUGGUC UUACCAGGUC UUACCAGGGU UCACCAGGAG

UAAGUCCCGC UAAGUCCCGC UAAGUCCCGC UAACUCCCGC UAAGUCCCGC UAAGUCCCGC UAAGUCCeCC UAAGUCAGGU

AACGAGCGCA AACCAGCGCA AACGAGCCCA A.ACGAGCGCA AACGAGCGCA AACGAGCGCA AACGAGCGCA AAGCAGCGAG

CUCAUGGCCe CUCAUGGCCC AUCAUGGCCC CUCAUGGCCU AUCAUGCeCC AUCAUGCCCC My CGCAUGCCCC

UUACGACCAG UUAUAGGUGG UUACGGGCUG UUACGCCUAG UUAOOACCAG UUAUCACCUG UUACAUCCUG GAAUCUCCUG

GGCUACACAC GGCUACACAC GGCUACACAC GGCUACACAC GGCUACACAC GGCUACACAC GGCUACACAC GGCUACACGC

GUGCUACAAU GUCAUACAAU GUCCUACAAU GUACUACAAU GUGCUACAAU GUGCUACAAU GUAGUACAAU GGGCUACAAU

GGCGCAUACA GGCUGGUACA GGUGGUGACA GGCGCGCACA GGooGGUACA GGACAGAACA GCUCCGGACA GGCUAGGACA

AAGAGAACCJ;; AAGGGUUGCC GUGGGCAGCG AAGGGGAGCG GAGCGUVGCC AAGGGCAGCG GCGAGACGCG AUGGGCUGCU

ACCUCGCCAG AACCCCCGAG AGACAGCGAU AGACCGCGAG AACCCGCGAG MACCCCGAG AAGCCGCGAG ACCCUGAAAA

AGCAAGCGGA GGGGAGCUAA GUCGAGCUAA GUGCAGCCAA GGGGAGCUAA GUUAAGCCAA GUGAAGCAAA GGGACGCGAA

CCUCAUAAAG UCCCAUAAAG UCUCCAAAA. UCCCAAAAAA UCGCAUAAAA UCCCACAAAU UCUCCCAAAC UCUCCGAAAC

UGCGUCGUAG CCAGUCGUAG GCCAUCUCAG CGCGUCCCAG CCGGUCUCAG CUGUUCUCAG CGGGGCUCAG CUAGUCGUAG

UCCGGAUUGG UCCGGAUCGC UUCGGAUUGC UCCGGAUUGC UUCAGAUUGG UUCGGAUCGC UUCAGAUUGC UUCGGAUCGU

AGUCUGCAAC AGUCUGCAAC ACUCUGCAAC AGUCUGCAAC AGUCUGCAAC AGUCUGCAAC AGGCUGCAAC GGGCUGUAAC

1320

Pt At Dd Hx Bs An

Ec UCGACUCCAU Pt UCGACUGCGU At UCGAGUGCAU Dd UCGACUGCAU Kx UCGACUCCAU Bs UCGACUGCGU An UCGooooCAU MY UCGCCCACGU

GAAGUCGGAA GAAGUCGGAA GAACUUGGAA GAACUUGGAA GAAGGAGGAA GAAGCUGGAA GAAGGCGGAA GMGCUGGAA

UCCCUAGUAA UCGCUAGUAA UCCCUAGUAA UCGCUAGUAA UCGCUAGUAA UCGCUAGUAA UCGCUAGUAA UCCGllAGUM

UCGUGGAUCA UCGUGGAUCA UCGCAGAUCA UUCCAGAUCA UCGCAGAUCA UCGCGGAUCA UCGCAGGUCA UCGCAGUUCA

GAAUGCCACG GAAUGUCACG GCAOOCOOCG Gc.AUGCUCGG GCACGCUGCG GCAooooGCG GCAUACUGCG UAAUACUGCG

GUGMUACGU GUGAAUACGU GUGAAUACGU GUGAAUGCGU GUGAAUACGU GUGAAUACGU GUGMUACGU GUCMUGUGU

UCCCGGGCCU UCCCGGGUCU UooCGGGooU UCCCGGGCCU UCCCGGGooU UCCCGGGCCU UooCGGGCCU CCCUGCUCCU

UGUACACACC UGUACACACC UGUACACACC UGUACACACC UGUACACACC UGUACACACC UGUACACACC UGCACACACC

GCCCGUCACA GCCCGUCACA GCCCGUCACA GCCCGUCACA GCCCGUCACA GCCCGUCACA GCCCGUCACA GCCCGUCACA

CCAUGGGAGU CCAUGGGAGC CCAUGGGAGU CCACGAAAGU CCAUGGGAGU CCACGAGAGU CCAUGGAAGU CCACCCGAGU

GGGUUGCAAA GGGUCUCGCC UCGUUUUACC CGGUUUUACC CGAUUGCUCC UUGUAACACC UGGCCAUGCC UGGGUUCAGG

AGAAGUAGGU AGAAGUAGGU CGAAGGUAGU CGAAGCCGGU AGAAAUCADC CGAAGUCGGU CGAAGUCGUU UGAGGCCUUG

1440

Ec AGCUUAACe. U UCG. GGAGGGC GCUUACCACU

UUGUGAUUCA GCGGGGUUCG GUAGGGUCAG GUAGGGCCGA GAGUGGUCGG GUGGGACAGA GUAGXCUGA CUGGGCUCAG

UGACUGGGGU UGACUGGGGU CCACUGGGCU UGAUUGCCGU UAACUGGGGU UGAUUGGGGU UGACUGGGGU CGAGGCGCCU

GAAGUCGUAA GAAGUCGUAA GAAGUCGUAA GAAGUCGUAA GAAGUCGUAA GAAGUCGUAA GAAGUCGUAA GAAGUCGUAA

CAAGGUAACC CAAGGUAGCC CAAGGUAGoo CAAGGUAGCC CAAGGUAGCC CAAGGUAGCC CAAGGUAGCC CAAGGUAGCC

GUAGCGGAAC GUAUCGGAAG GUA(X;GGAAC GUAGGGGAAC GUAGGGGAAC GUAUCGGAAG GUACCGGAAG GUAGCGGAAC

CUGCGGUUGG GUGCGGCUGG CUGCGGCUGG CUGCGGCUGG COOCGGCUGG GUGCGGCUGG GUGUGCCUGG CUGCGGCUGG

AUCACCUCCUUA • • AUCACCUCCUUUCU AUCACCUCCUUUCU AUCACCUCCUUU •• AUCACCUCCUUUCU AUCACCUCCUUUCU AUCACCUCCUUU • • AUCACCUCC •• • ••

Pt At Dd Mx Bs

UCGAUGCAAC UCGAAGCAAC UCGAUGCAAC UCGACCCAAC UCGAAGCAAC An UCGAUGCAAC My UGGAUUCAAC Ec AAUGOUGGGU

Pt GAUGUUGGGU At GAIJGUUGGGU Dd GAUGUUGGGU Hx GAUGUUGGGU Bs GAUGUUGGGU An GAUGUUGGGU M.y GGCGUCCUGU Ec AUCAUGGCCC

Pt At Dd Hx Bs An

AGCCUAACC.G GCGCUAACC.G GAGCCAACCAG UC ••••••• AC GAGGUAACCUU ACCCUAACCGU Hv Gec ••••••••

UAA.GGAGGGC CAA.GCAGGCA CAAUGGAGGCA CAA •••••• CA UU .AGGAGCCA UCGCGGAOOGG UUU •••• GGCU

GCUUACCACG GCUAACCACG GCCGUCUACG GGUGCUCAAG GCCGCCGAAG GGCGCCCAAG AGGGUCCAAC

UUGACAUCCA UUGAGAUGeC UUGA.GAUUCG UUGACAUCCA UVGACAUCCU UUCACAUCCU UUGACAUCCC CGA •.• CAGC ACCCUUAUCC ACCCUUGCCA ACCCUCecec ACCCCUAUGG ACCCUCGCCU ACCCUUGAUC ACCCACGUUU ACCCGUGCCC

UUUGUUGCCA UUACUUGCUA UUAGUUGCCA AUAGUUGCCA. UUAGUUGCC. UUAGUUGCCA UUAGUUGCCA UAUGUUGCGA

GCGG ••• UCCGG ••• CCGGGAACUCA.AAGGAGACUG • CAU •••• UCAGU ••• UGAGCACOCU. AAUGGGACUG GCAU •••• UUAGU ••• UCGGCACUCU .AAGGGGACUG GCAA •••• GUAAUG. UUGGGCACUCU .AUUCAGACUG • AC ••••• GCAA •••• GUGGAUCUCU .AGAGGGACUG GCA •••• UUCAGU ••• UGGGCACUCU .AAGGUGACUG UCA •••• UUCAGU ••• UGGGCACUeU .AGAGAAACUC CUACUUUCUCCGGAAGGUAAGCACUCAUAGGGGACCG

1542

Fig. 1. Alignment of 165 ribosomal RNA sequences. Alignment is by procedures described previously (Woese et aI., 1983; Yang et aI., 1985). Abbreviations of species names are as in Table 2 caption. Lower case letters in the sequence denote uncertainty in the determination.

Phylogenetic Relationship of Sulfate Respiring Bacteria

for the numbers in parantheses in the upper triangle of Table 2. [Note however, that these are not the actual percentages, but numbers normalized to facilitate comparision to their counterparts, above them.] With this correction the specific relationship between B. subtilis and A. nidulans disappears (as suspected), but the relationship between E. coli and P. testosteroni, is notably enhanced, so would seem to be a genuine relationship. In spite of the fact that the P. testosteroni sequence exhibits a generally low homology with all other eubacterial sequences (Table 2), the E. coli sequence is closest of all to it. This specific relationship - which becomes more prominent in the upper triangle of Table 2 - is also seen in the oligonucleotide catalogs of the beta and gamma purple bacteria in general and in the secondary structure of the molecule. For example, of the nine oligonucleotides composing the signature that defines the purple bacterial "phylum" (Woese et al., 1984a), three are confined solely to the beta and gamma subdivisions; UUAAUCG (vacinity of positions 555 in E. coli 16S rRNA) is found in 100 % and 90 % of catalogs respectively from the beta and gamma subdivisions, but occurs elsewhere only three times, among more than 300 eubacterial 16S rRNA catalogs (Woese et al., 1984a). Similarly, CCCCCUG (vacinity pos. 740) is found in 54 % and 31 % respectively of the beta and gamma subdivision catalogs, but nowhere else (Woese et al., 1984a). And, CUYACCAqUG]UUG (pos. 1465) has only three occurrences outside of the beta (46 % of catalogs) and gamma (27 %) subdivisions (none in alpha). [Y = pyrimidine; bases in brackets are alternatives to one another). Table 3. Secondary structure for the 195-220 region of the 16S ribosomal RNA Sequences are those of Fig. 1 plus Heliobacterium chlorum (Woese et al., 1985c) and Mycoplasma capricolum (lwami et al., 1984). Boxed sequences indicate pairing in the region (Woese et al., 1983). Numbering is according to the E. coli sequence (Brosius et al., 1978). sequence from

helix

200

210

I

220

@ill

UUCG

I IGG • ccucuul G

AM

IGCAGGG I GA. @II

UUCG

JGG • CCUUGul G

AM

@!l •••

.Q.. desulfuricans

AM

~.~

AM

IGAUGGC I cuc @£I IGGUGGC I CUC @§]

!.~

AM I GGUGGcl •••

!!..~

AM ~ •••••• "

!. coli

AM !GAkGIGA.

!_

testosteroni

!.

tumefaciens

~.

capricolum

!.~

~.

vannielii

... ..

AM

IGMCC ~ ••••

AM

AM

.. ..

UUUA

..

••. [Qf]

G

CUUG

~u IGCUAUC I A

UAUA

@AIGCUAUcl A

UUCG GCM

..

.1 GCUAccl ..lmJ

A G

GUUU

IGGUUCI A

@!l •••

UUUA

••• @ill

G

@ill •••

AUAU

... @

G

261

Although very few -helices in 16S rRNA show differences in general structure among the eubacteria, two of the three composite helices in the region from position 143-220 do so (Gutell et al., 1985). Table 3 shows the versions of the helix 198-207/212-219 (Woese et al., 1983) found in various eubacterial16S rRNAs. The form common to E. coli and P. testosteroni appears both representative of, and unique to, the beta and gamma subdivisions. The first is suggested by the fact that the oligonucleotide ACCUUCG, see Table 3, occurs in 29 % of catalogs from the gamma subdivision (representing at least four phylogenetic ally independent occurrences if it is not the ancestral version for the group) and 13 % of beta catalogs (at least two independent occurrences) (unpublished analysis). [This oligonucleotide is found elsewhere among the eubacteria in only 3 catalogs out of more than 300 (unpublished analysis) - and in these cases does not necesarily occur in the structure under consideration]. The most common form of this structure - ostensibly the ancestral form because it is seen in the archaebacteria as well (Gutell et al., 1985) - is the short version occurring in the alpha subdivision representative, A. tumefaciens. The structure can be tracked in this case by the oligonucleotide AUUUAUCG, which occurs in 78 % of alpha subdivision catalogs and elsewhere among the eubacteria about 15 times, scattered among five of the "phyla"; it is not found in catalogs from either the beta or the gamma subdivision (Woese et al., 1984a). The specific relationship seen in Table 2 between sulfate and sulfur respirers (represented by D. desulfuricans) and the myxobacteria (represented by M. xanthus) was previously detected by oligonucleotide signature (Hespell et al., 1984; Stackebrandt et al., in preparation; Woese et al., 1985b). The relationship is also evident in several common secondary structure features (i. e. in molecular phenotype). The form of the 198-207/212-219 helix found in these two organisms - see Table 3 - appears both general for, and unique to this group: The sequence CCUCUG (see Table 3) identifies this particular version of the structure in oligonucleotide catalogs. It occurs in one of the three bdellovibrio catalogs, three of ten catalogs from the sulfate and sulfur respirers, and three of five myxobacterial catalogs - but only five more times among the remaining eubacterial catalogs (Ludwig et al., 1983a; Stackebrandt et al., in preparation; unpublished analysis). Neither the three-base bulge that covers positions 204-205 in the sequences of D. desulfuricans and M . xanthus (Table 3) nor the single bulge after position 213, has been encountered in any other sequences to date. A second feature unique to these two sequences (with one exception) is the CIA juxtaposition replacing a normal base pair at positions 256/270 (Woese et al., 1983; unpublished analysis). In several other respects, however, the secondary structure of the two differs, indicating the two organisms not to be especially close to one another. A specific relationship between the alpha subdivision and the beta-gamma couple that excludes the sulfate respiring-myxobacteria-bdellovibrio group can not be deduced from Table 2. However, one secondary structure feature not found in the sulfate respirer or the myxobac-

262

H.Oyaizu and C. R. Woese

terium, is common to the three previously recognized subdivisions of the purple bacteria. The 184-1861191-193 helix defined for E. coli comprises a stalk of only three base pairs (Woese et al., 1983; Gutell et al., 1985). As Figure 1 shows, the alpha and beta subdivision representatives also have this form. A much more extended form characterizes all other eubacteria and archaebacteria sequenced, with one exception (unpublished results). However, this single (almost) unique secondary structural chracteristic cannot be taken as proof of a specific relationship. The data of Table 2 actually suggest that the sulfate reducers and their relatives are a part of the purple bacterial "phylum"; they can be considered its delta subdivision. This is not clearly seen in the lower triangle of Table 2 (where D. desulfuricans and M. xanthus have relatively high homologies with B. subtilis), but is evident from the numbers in parentheses in the upper one. While the exact interrelationships among the sulfate respirers and relatives (delta purple bacteria), the alpha purple bacteria, and the beta-gamma couple must be considered unresolved, there can no longer be any doubt that the beta and gamma subdivisions of the purple bacteria are specifically related to one another to the exclusion of the other two subdivisions. Therefore, one genus of purple non-sulfur bacteria, Rhodocyclus (Imhoff et al., 1984), is related to the purple sulfur bacteria, to the exclusion of all other purple non-sulfur genera - i. e. Rhodospirillum, Rhodomicrobium, and Rhodopseudomonas. The old notion that the purple bacteria fall naturally into two groups, the purple sulfurs vs. the purple non-sulfurs, must now be discarded. Acknowledgements. This work was supported by grants from NASA and the National Science Foundation. HO was supported in part by a fellowship from the Toyobo Biotechnology Foundation.

References Biggin, M. D., Gibson, T. J., Hong, G. F.: Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc. nat. Acad. Sci (Wash.) 80, 3963-3965 (1983) Bonen, L., Doolittle, W. F.: Partial sequences of 16S rRNA and the phylogeny of blue-green algae and chloroplasts. Nature 261, 669-673 (1976) Bonen, L., Doolittle, W. F.: Ribosomal RNA homologies and the evolution of the filamentous blue-green bacteria. ]. Molec. Evo!. 1, 283-291 (1978) Brooks, B. W., Murray, R. G. E., Johnson, j. L., Stackebrandt, E., Woese, C. R., Fox, G. E.: Red-pigmented micrococci: a basis for taxonomy. Int. ]. system. Bact. 30, 627-646 (1980) Brosius, J., Palmer, j. L., Kennedy, j. P., Noller, H. F.: Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc. nat. Acad. Sci. (Wash.) 75, 4801-4805 (1978) Fox, G. E., Peckman, K. j., Woese, C. R.: Comparative cataloging of 16S ribosomal ribonucleic acid: molecular approach to prokaryotic systematics. Int.]' system. Bact. 27, 44-57 (1977) Fox, G. E., Stackebrandt, E., Hespell, R. B .• Gibson,]., Maniloff, j., Dyer, T. A., Wolfe, R. S., Balch, W. E., Tanner, R., Mag-

rum, L., Zablen, L. B., Blakemore, R., Gupta, R., Bonen, L., Lewis, B. j., Stahl, D. A., Luehrsen, K. R., Chen, K. N., Woese, C. R.: The phylogeny of prokaryotes. Science 209, 457-463 (1980) Fowler, V. J., Pfennig, N., Schubert, W., Stackebrandt, E.: Towards a phylogeny of phototrophic purple sulfur bacteria 16S rRNA oligonucleotide cataloging of eleven species of Chromatiaceae. Arch. Microbio!. 139, 382-387 (1984) Gibson, J., Stackebrandt, E., Zablen, L. B., Gupta, R., Woese, C. R.: A phylogenetic analysis of the purple photosynthetic bacteria. Curro Microbio!. 3, 59-64 (1979) Gibson, j., Stackebrandt, E., Woese, C. R.: The phylogeny of the green photosynthetic bacteria: lack of a close relationship between Chlorobium and Chloroflexus. System. App!. Microbio!' 6, 152-156 (1985) Green, C. J., Stewart, G. c., Hollis, M. A., Void, B. S., Bott, K. F.: Nucleotide sequence of Bacillus subtilis ribosomal RNA operon, rrnB. Gene, in press (1985) Gutell, R. R., Weiser, B., Woese, C. R., Noller, H. F.: Comparative anatomy of 16S-like ribosomal RNA. Progr. Nucleic Acid. Res. Molec. Bio!. in press (1985) Hespell, R. B., Paster, B. j., Macke, T., Woese, C. R.: The origin and phylogeny of the bdellovibrios. System. App!. Microbio!. 5, 196-203 (1984) Jarsch, M., Bock, A.: The sequence of the 16S ribosomal RNA gene from Methanococcus vannielii. System. App!. Microbio!. 6, 54-59 (1985) Imhoff, j., Truper, H. G., Pfennig, N.: Rearrangement of the species and genera of the phototrophic "purple nonsulfur bacteria". Int. ]. system. Bact. 34, 340-343 (1984) Iwami, M., Muto, A., Yamao, F., Osawa: Nucleotide sequence of the rrnB 16S ribosomal RNA gene from Mycoplasma caprico/um. Molec. Gen. Genet. 196,317-322 (1984) Loenen, W. A. M., Brammar, W. J.: A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene 20, 249-259 (1980) Ludwig, W., Schleifer, K.-H., Reichenbach, H., Stackebrandt, E.: A phylogenetic analysis of the myxobacteria Myxococcus fulvus, Stigmatella aurantiaca, Cystobacter fuscus, Sorangium cellulosum and Nannocystis exedens. Arch. Microbio!. 135, 58-62 (1983a) Ludwig, W., Stackebrandt, E.: A phylogenetic analysis of Legionella. Arch. Microbio!. 135,45-50 (1983b) Marmur, j.: A procedure for the isolation of deoxyribonucleic acid from micro-organisms.]' Molec. Bio!. 3, 208-218 (1961) Messing, j.: New M13 vectors for cloning. Meth. Enzymo!. 101, 20-78 (1983) Mills, D. R., Kramer, F. R.: Structure-independent nucleotide sequence analysis. Proc. nat. Acad. Sci. (Wash.) 76, 2232-2235 (1979) Oren, A., Weisburg, W. G., Kessel, M., Woese, C. R.: Halobacteroides halobius gen. nov., sp. nov., a moderately halophilic anaerobic bacterium from the bottom sediments of the Dead Sea. System. App!. Microbio!. 5, 58-70 (1984a) Oren, A., Paster, B. j., Woese, C. R.: Haloanaerobiaceae: A new family of moderately halophilic, obligatory anaerobic bacteria. System. App!. Microbio!. 5, 71-80 (1984b) Pfennig, N.: Phototrophic green and purple bacteria: A comparative, systematic survey. Ann. Rev. Microbio!. 31, 275-290 (1977) Pfennig, N., Truper, H. G.: The phototrophic bacteria. In: R. E. Buchanan, N. E. Gibbons, (eds.), Bergey's manual of determinative bacterilogy, 8th ed. Baltimore, Williams and Wilkins (1974) Paster, B. J., Stackebrandt, E., Hespell, R. B., Hahn, C. M., Woese, C. R.: The phylogeny of the spirochetes. System. App!. Microbio!. 5, 337-351 (1984)

Phylogenetic Relationship of Sulfate Respiring Bacteria

Paster, B. j., Ludwig, W., Weisburg, W. G., Stackebrandt, E., Hespell, R. B., Hahn, C. M., Reichenbach, H., Stetter, K. 0., Woese, C. R.: A phylogenetic grouping of the bacteroides, cytophagas and certain flavobacteria. System. Appl. MicrobioI. 6, 34-42 (1985) Sanger, F., Nicklen, S., Coulson, A. R.: DNA sequencing with chain-terminating inhibitors. Proc. nat. Acad. Sci. (Wash.) 74, 5463-5467 (1977) Seewaldt, E., Stackebrandt, E.: Partial sequence of 16S ribosomal RNA and the phylogeny of Prochloron Nature 295,618-620 (1982) Stackebrandt, E., Woese, C. R.: The evolution of prokaryotes. In: Molecular and Cellular Aspects of Microbial Evolution, eds. M. j. Carlile, j. R. Collins, pp. 1-31. B. E. B. Moseley - Cambridge University Press (1981a) Stackebrandt, E., Woese, C. R.: Towards a phylogeny of the actinomycetes and related organisms. Curr. Microbiol. 5, 197-202 (1981b) Stackebrandt, E., Fowler, V. ]., Woese, C. R.: A phylogenetic analysis of the lactobacilli, Pediococcus pentosaceus and Leuconostoc mesenteroides. System. Appl. Microbiol. 4, 326-337 (1983a) Stackebrandt, E., Kroppenstedt, R. M., Fowler, V. j.: A phylogenetic analysis of the family Dermatophilaceae. ]. gen. Microbiol. 129, 1831-1838 (1983b) Stackebrandt, E., Fowler, V. j., Schubert, W., Imhoff, j. F.: Towards a phylogeny of phototrophic purple bacteria - the genus Ectothiorhodospira. Arch. Microbiol. 137, 366-370 (1984a)

Stackebrandt, E., Ludwig, W., Schubert, W., Klinik, F., Schlesner, H., Roggentin, T., Hirsch, P.: Molecular genetic evidence for early evolutionary origin of budding peptidoglycan-less eubacteria. Nature 307, 735-737 (1984b) Tomioka, N., Sugiura, M.: The complete nucleotide sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans, Molec. Gen. Genet. 191,46-50 (1983) Woese, C. R.: Why study evolutionary relationships among bacteria? In: The evolution of prokaryotes, K. H. Schleifer and E. Stackebrandt (eds.), p. 1-30. London, Academic Press (1985)

263

Woese, C. R., Sogin, M., Stahl, D. A., Lewis, B. j. Bonen, L.: A comparison of the 16S ribosomal from mesophilic and thermophilic bacilli: Some modifications in the Sanger method for RNA sequencing. ]. Molec. Evol. 7, 197-213 (1976) Woese, C. R., Maniloff, j., Zablen, L. B.: Phylogenetic analysis of the mycoplasmas. Proc. nat. Acad. Sci. (Wash.) 77, 494-498 (1980) Woese, C. R., Blanz, P., Hespell, R. B. Hahn, C. M.: Phylogenetic relationships among various helical bacteria. Curr. Microbiol. 7, 127-132 (1982) Woese, C. R., Gutell, R., Gupta, R., Nolier, H. R.: Detailed analysis of the higher-order structure of 16S-like ribosomal Ribonucleic acids. Microbiol. Rev. 47, 621-669 (1983) Woese, C. R., Stackebrandt, E., Weisburg, W. G., Paster, B. j.,

Madigan, M. R. Fowler, V. j. Hahn, C. M., Blanz, P., Gupta, R., Nealson, K. H., Fox, G. E.: The phylogeny of purple bacteria: The alpha subidvision System. Appl. Microbiol. 5, 315-326 (1984a)

Woese, C. R., Weisburg, W. G., Paster, B. j., Hahn, C. M., Tanner, R. S., Krieg, N. R. Koops, H.-P., Harms, H., Stackebrandt, E.: The phylogeny of purple bacteria: The beta subdivision. System. Appl. Microbiol. 5, 327-336 (1984b)

Woese, C. R., Weisburg, W. G., Hahn, C. M., Paster, B., Zablen, L. B., Lewis, B. j. Macke, T. ]., Ludwig, W., Stackebrandt, E.: The phylogeny of purple bacteria: the gamma subdivision. System. Appl. Microbiol. 6, 25-33 (1985a)

Woese, C. R., Stackebrandt, E., Macke, T. j., Fox, G. E.: A phylogenetic definition of the major eubacterial taxa. System. Appl. Microbiol. 6, 143-151 (1985b) Woese, C. R., Debrunner- Vossbrinck, B. A., Oyaizu, H.: Are Gram positive bacteria of photosynthetic ancestry? Science, 229, 762-765 (1985c)

Yang, D., Oyaizu, Y. Oyaizu, H., Olsen, G. j., Woese, C. R.: Mitochondiral Origins. Proc. nat. Acad. Sci. (Wash.) 82, 4443-4447 (1985)

Dr. Carl R. Woese, Dept. of Genetics and Development, University of Illinois, 515 Morrill Hall, 505 South Goodwin Ave., Urbana, Illinois 61801, U.S.A.