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The phylogeny of purple bacteria: The beta subdivision
System. Appl. Microbiol. 5, 327-336 (1984) Department of Genetics and Development, 515 Morrill Hall, 505 South Goodwin Ave., University of Illinois, Urbana, Illinois 61801, U.S.A. • Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A. I Institut fur Allgemeine Botanik und Botanischer Garten, Universitat Hamburg, 2000 Hamburg, Federal Republic of Germany , Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, 8000 Munchen, Federal Republic of Germany
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The Phylogeny of Purple Bacteria: The Beta Subdivision C. R. WOESEl':', W. G. WEISBURG1, B. J. PASTER1, c. M. HAHNl, R. S. TANNER1, N. R. KRIEG 2 , H.-P. KOOPS 3 , H. HARMS3, and E. STACKEBRANDP
Received April 24, 1984
Summary The beta subdivision of the purple photosynthetic bacteria, defined by 16S ribosomal RNA (oligonucleotide) sequence comparisons comprises two main subclusters, one defined by Rhodopseudomonas gelatinosa, the other by Rhodospirillum tenue and its close relative Rhodocyclus purpureus. [All other characterized species of purple non-sulfur bacteria belong to the alpha subdivision of the purple bacteria.] Each of these subclusters contains a prepondenance of non-photosynthetic species, representing genera such as Spirillum, Aquaspirillum, Alcaligenes, Pseudomonas, Chromobacterium, Thiobacillus, and various ammonia and nitrite oxidizing bacteria. The evolutionary significance of this admixture of biochemical phenotypes is discussed.
Key words: Beta purple bacteria - Evolution - Phylogeny - rRNA catalog
Introduction
Given the simplicity of bacterial phenotypes (as we perceive them), reliable phylogenetic classification of bacteria is not possible using purely phenotypic criteria. However, bacterial genomes are more than complex enough to serve as the basis for a classification scheme. Therefore, sequence comparisons should be used as the primary basis for bacterial classification. The ribosomal RNAs are ancient, functionally constant, large, complex, and
* To whom requests for reprints should be sent.
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precisely defined molecules, and for these reasons serve as excellent molecular chronometers, by which genealogical relationships can be determined. Partial sequencing of 165 ribosomal RNAs - in terms of oligonucleotide catalogs - has been used to reveal the genealogical relationships among the 300 or so bacterial strains so far characterized in these terms (Fox et al., 1980; Woese et al., in preparation). The phylogenetic tree so produced appears to be well defined, except for the order of the earliest branchings in both bacterial kingdoms. [The order of branching once the common ancestor of a kingdom emerges seems to be relatively rapid; thus the major subdivisions appear all to stem from a common point in time.] The accepted taxonomy of the purple photosynthetic bacteria is incorrect in two important respects: It is based solely on photosynthetic genera, and it assumes that the purple non-sulfur genera (i.e. those with the prefix "Rhodo-") and the group of purple sulfur genera (such as Chromatium, Thiocapsa, etc.) represent the two sides of a primary phylogenetic division within the group (Pfennig and Truper, 1974). Regarding the first point, we now know that the true phylogenetic unit contains a number of genera of non-photosynthetic organisms, intermingled genealogically with the various photosynthetic genera (Fox et al., 1980). Regarding the second, there are (at least) three primary groups in this phylogenetic unit, not two; the purple non-sulfur bacteria split into two groups phylogenetically, each no closer to the other than to the purple sulfur group (Gibson et al., 1979). It is important that microbiology recognize the true phylogenetic relationships formally, by a taxonomy accurately reflecting them. Failure to do so in effect imposes a flawed, if not false, conceptual structure on the field. Because (1) the existing taxonomy represents vested interests, (2) it projects a (false) authority stemming from the use of the classical nomenclature and classification system borrowed from eucaryote taxonomy, (3) discarding the existing scheme would entail the discarding of a number of deeply held prejudices, and (4) an alternative formal scheme for classifying bacteria does not yet exist - a proper phylogenetic classification of bacteria will be some time in coming. In the meantime, in order to hasten the transition, it is important to emphasize the true genealogical relationships and stress their differences with the old taxonomy. In previous publications it has been shown that the purple photosynthetic bacteria and relatives are a distinct phylogenetic unit that comprises three major subdivisions, temporarily designated alpha, beta, and gamma. The first two cover the purple non-sulfur bacteria, the latter the purple sulfur bacteria (Gibson et al., 1979; Woese et al., 1982; Woese et al., 1984 a, b). The present communication covers the beta subdivision (Gibson et al., 1979; Fox et al., 1980; Woese et al., 1984b).
Materials and Methods The organisms used in this study are the following: Rhodospirillum tenue strain 3760 and Rhodopseudomonas gelatinosa strain TG-9 - reported previously by Gibson et aL (1979) ; Aquaspirillum gracile ATCC 19624, Aq. serpens ATCC 12638, and Spirillum volutans ATCC 19554 - reported by Woese et al. (1982); Pseudomonas acidovorans ATCC 15668, Ps. cepacia ATCC 17616, Ps. testosteroni ATCC 11996 and Comamonas terrigena ATCC 8461 - reported by Woese et al. (1984a). Reported for the first time are: Nitrosococcus mobilis Nc2 (Koops et aL, 1976), Nitrosomonas europeae Nm35 (from Dr. N. Walker), Nitrosovibrio tenuis Nv1 (Harms et aL, 1976), Nitrosolobus multiformis Nl7 (isolated
The Phylogeny of Purple Bacteria: The Beta Subdivision
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H.H.), Nitrosospira sp. Nsp1 (isolated H-PK), Aquaspirillum aquaticum ATCC 11330, Aq. dispar ATCC 27510, Aq. bengal ATCC 27641, Alcaligenes eutrophus DSM 531, AI. faecalis ATCC 8750, Chromobacterium violaceum ATCC 12472, C. lividum DSM 1522, Sphaerotilus natans ATCC 15291, Vitreoscilla stercoraria DSM (a gift of Prof. H. Reichenbach), Thiobacillus intermedius ATCC 15466 and T. denitri{icans ATCC 2364. [The Sab value between Aq. serpens and Aq. bengal is 0.93; this is high enough that the two should be considered strains of the same species. In all following discussion Aq. serpens will be taken to represent both strains.] Extraction and labelling of 16S rRNA and generation of the oligonucleotide catalogs followed either of two standard procedures (Uchida et al., 1974; Woese et al., 1976; Stackebrandt et al., 1982). The analysis of catalogs in terms of Sab values is described in Fox et al. (1977).
Results and Discussion Conventionally, analyses of rRNA catalog data have been in terms of binary association coefficients, or Sab values, which are used to construct phylogenetic trees by an unweighted pair group method (Fox et aI., 1977). The method is sufficient for the phylogenetic placement of most bacteria, but, in certain exceptional cases, it is inadequate (Woese et aI., 1980). However, catalog data can also be analysed by a "signature" method - i.e. in terms of groups of oligonucleotides that define and distinguish the various clusters (Woese et aI., 1980). [This is possible because the rRNA sequence exhibits a broad range in the degree of conservation of the various positions in the molecule. Consequently, particular positions (oligonucleotide sequences) can be characteristic of any given grouping of organisms.] Table 1. Oligonucleotide signature distinguishing the beta purple bacterial subdivision from the alpha and gamma subdivisions. Numbers show percent representation of the oligonucleotide in a group. "Other" refers to (percent) occurrence in eubacteria other than the purple group (Gibson et al., 1979; Fox et al., 1980). sequence
alpha beta
ganma
other
-----------------------------------------------CCCCCUG UUAAUCG AAUUUUG CCAACCCG AAAUCCCCG CUUCACACG AUUAAUUCG UCAUACAAUG UAAUACAAUG CYUUACACAUG AAAAACCUUACC ••
0 0 0 5 0 0 0 0 0 0 0
55 100 95 75 80 65 65 80 20 100 100
25 90 <10 10 20 0 0 0 0 0 0
0 <5 <5
<5
0 0 20 0 <5 0 <5
Table 1 is a collection of sequences that defines the beta subdivision of the purple bacteria and distinguishes if from the alpha and gamma subdivisions. Table 2 in turn is an oligonucleotide signature that defines the main subgroups within the beta subdivision. The first of these, beta-I, contains Rhodopseudomonas gelatinosa, Sphaerotilus natans, Aquaspirillum aquaticum, Aq. gracile, Pseudomonas acidovorans, Ps. testosteroni, and Comamonas terrigena, with Thiobacillus intermedius 22 Systematic and Applied Mictobiology, Vol. 5
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C. R. Woese et aI.
as a peripheral member. A second, beta-2, contains Rhodospirillum tenue, Chromobacterium lividum, C. violaceum, Alcaligenes faecalis, AI. eutrophus, Aq. dispar, Aq. serpens, Ps. cepacia and the gliding organism Vitreoscilla stercoraria. [It should be mentioned at this juncture that an incomplete catalog exists for the 165 rRNA of Rhodocyclus purpureus (Gibson et aI., unpublished). Although these data do not permit a meaningful Sab value to be calculated for this organism, they do permit a signature analysis, and this shows Rc. purpureus to be a specific relative of R. tenue (Woese et aI., unpublished).] The remaining species, from the three closely related genera Nitrosovibrio, Nitrosospira, and Nitrosolobus, and from Spirillum volutans, Nitrosococcus mobilis, and Nitrosomonas europeae, seem also to group with beta-2, but in a more peripheral way. Table 3 is a more extensive analysis of this last grouping. It lists all those sequences found in any catalog from this group (taking N. tenuis to represent itself and its two very close relatives) and in at least one other catalog from the beta subdivision Table 2. Oligonucleotide signature distinguishing among the subgroups of the beta purple bacteria. Abbreviations are these: Rg - Rps. gelatinosa, Sn - S. natans, Aa - Aq. aquatile, Pa - Ps. aeidovorans, Pt - Ps. testosteroni, Ct - C. terrigena, Ag - Aq. gracile, Ti - T. intermedius, Ad - Aq. dispar, As - Aq. serpens, Cv - C. violaeeum, Pc - Ps. eepaeia, Ae - AI. eutrophus, Af - AI. faeealis, Cl- C.lividum, Rt - R. tenue, Rp - Re. purpureus, Td - T. denitrifieans, Vs - V. stereoraria, Sv - Sp. volutans, Nt - N. tenuis, Nm - N. mobile, Ne N. europeae. sequence AAACUCAAAG RUUAAAACUCAAAG
R SAP P CAT
g n a a t t g i
d s v c e f 1 t P d s
A A CPA A C R R T V
S N N N v t me + + + +
+ + + + + + +
+
+ + + + + + + + + + +
UCAUACAAUG UAAUACAAUG
+ + + + + + + +
+ + + + + + + + + + +
UAAUACAUCG
+ + + + + + +
CAAUCCCCUG CAACUCCCUG CCCCCUG CUCCCUG
+ + + +
+
CUACACACG CUUCACACG
+ + + + + + + +
AAUUCCG AAUUCCACG AAUUCCAUG
+ + + + + + + +
+ +
+ + + + + + + + + + +
+ + + +
+ + + + + + + + ? + +
+ + + +
+ + + + + + + + ? + +
+ +
+
,.
?
+
+ + + + +
+ +
+
+ + + + + + +
+ + + + + + + + + + + • + + + +
+ + +
+ + + + +
+ + +
+
(A,U)UUCAUG CUUUUG •• ACUUUUG
+ + + + + + + + + + +
+7+
+ ',;+ + + + + + +
UCAACUG UCAACUAG UCUACUAG
+
+ + + + + + +
UUUAAUUCG AUUAAUUCG
CCCUUAUAG CCCUUAUG
+ +
+ + + +
+ + + + + + + +
+
+
+ +
The Phylogeny of Purple Bacteria: The Beta Subdivision
331
Table 3. Oligonucleotide signature showing the specific relationship among the catalogs of S. volutans, N. tenuis, N. mobile and N. europeae and a specific relationship of these to the beta-2 subcluster. All oligonucleotides that fit the following conditions are included: (1) they occur in at least two catalogs from the beta subdivision, one at least of which is in the group Sv - Nt - Nm - Ne, (2) They do not occur in catalogs from both the beta-1 and the beta-2 subclusters. Abbreviations are as in Table 2 caption. The abbreviations in the final two columns indicate which catalogs in the group contain the oligonucleotide in question. sequence
Sv Nt Nm Ne
beta-1
beta-2
--------------------------------------------------------------AAAAACCUUACCUACCUUUG +
+
0
0
Pt
CAACCCUUAUCAUU AG
+
0
0
0
0
Td
AAACUACAAG
0
+
0
+
0
0
UAAUACAAUG
+
+
+
+
0
0
CUAAUCUCAG CCAAUCUCAG
+
+
0
0 0
0
0
+
0 0
Cv Af
CACUUUAAUG
0
+
+
0
0
0
+ +
+ +
+
0 0
0 0 0
+ 0 0
+
+
0 0 +
0 0 +
0 0 0
Ad,Af Td Cv,Pc,Ae,Af,Rt,Rp
0 0 0
0 0 0
+ + +
+ +
0 0 0 0 0 all 0 0
Af,Td all Cv,Pc·,Ae,Af 0 0 0 Pc,Rp Cv,Pc,Af
CUUAACCUG CUUCACACG CUUACCAAG CUCAACUUG UUUCACCAG AUUAAUUCG AAACCCUG UCACACUG UCAACUAG
+
0 +
+ +
+ +
+
+
+ 0
0 +
AACACAG CCCCCUG AAUACCG AAUAACG CUCCCUG AAUUCCG AAUCCUG AUUCAUG
+ + +
0
AAACUG ACACUG ACUCCG ACUACG UAAAAG CUAAUG AUUAUG
+
0 0 0 +
0 0 0 0 0
+
+
0 0 0 0
0 0
+ + +
+
0 0 0 0
+
0 0
0 0 0
+
0
0 + 0 0 0
+ +
+
+
+ 0 0
0
Ad,As,Rt
0 Cv,Td 0 all Aa,Pa,Pt,Ct,Ag 0 0 Ae, Vs Cv,Af 0 0 all
0 0 0 0 0 0 0
Ad,Vs Cv,Td 0 Ae,Vs Cl,Rp, Vs Cl,Td 0
of the purple bacteria, but not occurring in both of the major subgroups of the beta subdivision. The table reinforces the specific relationship of these organisms to the beta-2 subgroup and also shows a phylogenetic coherence among the four species. Thus these four form a coherent subcluster peripheral to the beta-2 subgroup. [Note in Table 2 the second pair of sequences. The phylogenetic relationships suggested would require the transition UCAUACAAUG -+ UAAUACAAUG to have occurred but once. Any other clustering would require multiple origins of one or the other of these oligonucleotides.]
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Table 4. Sab values (Fox et aI., 1977) for the members of the beta purple group with one another. The number assignments are as follows: 1 - Rps. gelatinosa, 2 - S. natans, 3 - Aq. aquatieum, 4 - Ps. aeidovorans, 5 - Ps. testosteroni, 6 - C. terrigena, 7 - Aq. gracile, 8 - Th. intermedius, 9 - Aq. dispar, 10 - Aq. serpens, 11 - Ch. violaeeum, 12 - Ps. eepacia, 13 - Al. eutrophus, 14 - AI. faeealis, 15 - Ch.lividum, 16 - R. tenue, 17 - Th. denitrifieans, 18 - V. stereoraria, 19 - Sp. volutans, 20 - N. tenuis, 21 - N. mobilis, and 22 - N. europeae. Re. purpureus is not included because a meaningful Sab cannot be calculated for an incomplete catalog. beta-1 123 4 5 678
beta-2 9 10 11 12 13 14 15 16 17 18
1 2 3 4 5 6 7 8
Rgel Snat Aaqu Cter Paei Ptes Agra Tint
78 63 62 68 61 67 64
-62 62 66 61 64 63
-89 80 84 78 53
-77 82 76 57
-79 -77 75 -57 58 56 --
9 10 11 12 13 14 15 16 17 18
Adis Aser Cvio Peep Aeut Afae Cliv Rten Tden Vste
51 51 52 57 56 48 60 60 53 44
57 52 50 60 60 48 60 63 57 46
47 52 47 48 51 46 53 56 48 45
49 56 49 46 51 49 52 55 51 48
48 52 48 50 51 48 55 53 49 45
49 53 46 46 48 47 50 49 49 44
46 51 49 52 52 43 52 54 47 43
52 51 51 51 54 49 57 58 50 48
71 59 57 57 57 61 57 60 51
62 54 59 57 62 59 60 58
-56 54 58 55 57 56 54
-71 58 63 61 50 52
-62 63 63 54 55
-57 52 56 56
-64 -59 55 -54 48 47 --
19 20 21 22
Svol Nten Nmob Neur
49 42 44 38
49 44 46 40
45 42 38 40
44 46 42 43
45 40 38 39
41 41 38 40
45 42 38 39
41 43 46 42
47 52 48 41
51 57 48 45
50 58 41 43
54 53 50 43
58 57 53 49
53 56 47 44
56 52 50 45
53 58 53 44
57 47 51 42
50 50 45 44
beta-2a 19 20 21 22
55 -5155 45 54 58 --
Table 4 shows the relationships within the beta subdivision as defined by numeric analysis - i.e. in terms of Sab values (Fox et aI., 1977). Perhaps the most remarkable feature of the present phylogeny is the extent to which it disagrees with the previous classifications of these organisms based upon phenotypic criteria. Every phenotypically defined genus in the present study is in disagreement with its true genealogy; the classically defined units are neither comprehensive nor coherent phylogenetically. The most extreme examples are the classical genera Rhodopseudomonas and Rhodospirillum. Neither Rps. gelatinosa nor R. tenue clusters with the other species in its genus (Truper and Pfennig, 1981). [The latter are included in the alpha subgroup of the purple bacteria (Gibson et aI., 1979; Woese et aI., 1984b).] However, the lack of coherence and/or comprehensiveness applies as well to Pseudomonas (Woese et aI., 1984a) (Ps. cepacia is not specifically related to the Ps. acidovorans cluster, and the latter also includes Aq. aquaticum), to Chromobacterium, to Aquaspirillum (Woese et aI., 1982) (only Aq. serpens, and perhaps Aq. dispar form a coherent unit), and to Alcaligenes. The nitrogen, sulfur and hydrogen oxidizing chemolithotrophs also lack coherence phylogenetically. [Nitrobacter species are found in the alpha subdivision, while some other sulfur oxidizing species join the gamma subdivision of the purple bacteria (Woese et aI., 1984b; Woese et aI., in preparation). The hydrogen oxidizing re-
The Phylogeny of Purple Bacteria: The Beta Subdivision
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Aq. serpens Aq. bengal
Ps. cepackJ
.9
.8
.7
.6
.5
.4
a - group
.3 Fig. 1. Schematic phylogenetic tree representing the relationships shown by Sab analysis as modified by signature analysis. The order of branching is that revealed by the combined analysis, and the branch points arc drawn in approximate agreement with the Sab analysis.
presentatives (A. eutrophus and Paracoccus denitri{icans are members of the beta and alpha subdivisions, respectively.) The true phylogenetic relationships are at variance in another way with classical taxonomic prejudice. Metabolic patterns that we have traditionally kept well separated taxonomically are seen actually to be evolutionarily clustered - e. g. photosynthetic types with non-photosynthetic, aerobes with anaerobes, nitrogen oxidizing with nitrogen reducing species. The ancestral phenotype that gave rise to the greater purple bacterial unit was photosynthetic; there is no other reasonable way to account for the admixture of purple photosynthetic phenotypes within the group. At various times then, nonphotosynthetic offshoots of the photosynthetic phenotypes arose and produced the various "Gram-negative" groupings known so well to the microbiologist - some
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more, some less extensive, depending upon the evolutionary stage at which the transition took place. The deepest divisions in the greater purple bacterial group were not, however, along the classically defined lines (even as regards the photosynthetic types alone). As mentioned, the alpha and beta subdivisions of the (non-sulfur) purple bacteria are no closer to one another than either is to the gamma group (purple sulfur bacteria). This genotypically defined division within the purple non-sulfur bacteria is supported by phenotypic distinctions as well. Rps. gelatinosa and R. tenue, which define the two major subgroups of the beta unit, are unique in molecular phenotype. Their cytochromes c are of the small subunit type, unlike the cytochromes c of the other "Rhodo-" genera (and are removed in sequence from them as well- Dickerson, 1980). The structure of their photo-reaction centers is unique too (Clayton and Clayton, 1978), and the tubular morphology characteristic of their photomembranes distinguishes them from all other purple photosynthetic bacteria (Truper and Pfennig, 1981). The structure within the two major subclusters of the beta subdivision is of interest, if only because, again, it is a mix of classical genera. As Table 4 shows, within the beta-1 subcluster Rps. gelatinosa and S. natans form a specific unit; a Ps. acidovorans unit also exists, which includes Aq. aquaticum and peripherally Aq. gracile; and, T. intermedius is the sole representative of yet another subcluster. Structure within the beta-2 cluster is not clearcut. The two aquaspirillum species, Aq. serpens and Aq. dispar, are specifically related. R. tenue and Rc. purpureus form a specific couple (by signature analY3is). Although the unweighted pair group analysis (Table 4 and unpublished) suggests further structure, this is not strongly supported by signatures, and so is considered uncertain. An interesting biochemical connection would seem to be emerging from these phylogenetic studies with regard to nitrogen metabolism in the greater purple group. The nitrogen oxidizing bacteria appear specifically related to nitrogen reducing species. For example, species of Nitrobacter (oxidizing nitrite) are close, specific relative of Rps. palustris within the alpha-2 group - to the exclusion of Rps. viridis, Rhizobium species, and others (Seewaldt et ai., 1982; Woese et ai., 1984b). The ammonia oxidizing species encountered here split into two specific groups. The first includes the very closely related species Nitrosovibrio tenuis, Nitrosospira sp., and Nitrosolobus multiformis, the second Nitrosococcus mobilis and Nitrosomonas europaea. However, Nitrosococcus oceanus, distinct in its intracellular morphology (Watson and Remsen, 1970), is not a member of the beta subdivision, though it is included in the greater purple group (Stackebrandt et ai., unpublished). It is interesting that N. tenuis shows internal membrane structure reminiscent of the tubular membranes of the photosynthetic members of the beta subdivision. These relationships suggest that all the forms of nitrogen metabolism represented by various species are to some extent inherent in their common ancestor. In other words, organisms that oxidize nitrogen compounds and those that reduce them would seem to use a more or less common pathway. Evolutionary adjustments then cause the pathway to run in the oxidizing vs reducing direction, or select a segment of the pathway to be developed, e.g. ammonia or nitrite oxidation. The phylogenetic distribution of sulfur metabolizing species in the greater purple group suggests that the various forms of sulfur metabolism may have evolved in a similar fashion. The microbiologist is beginning to be faced with two ostensibly unconnected
The Phylogeny of Purple Bacteria: The Beta Subdivision
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problems that perhaps at base are intimately related. One is that the current (eroding) classification schemes for bacteria are totally inadequate; they are false in their deepest assumptions. The problem is not to revise them, but to replace the existing taxonomy by an entirely new classification scheme that uses new criteria for classification (phenotypic and genotypic) and reflects bacterial genealogical relationships. The other problem is that our prejudices as to the evolution of bacterial metabolism, the relationships among the various bacterial biochemistries, is in definite need of revision if we are ever to understand much about the origins of various intermediary metabolisms. It is in the framework of a true bacterial phylogeny that we will come to understand the evolution and full significance of bacterial metabolism. Therefore, in an important sense these two disparate problems are part and parcel of one another. Acknowledgements. CRW was supported by a grant from the National Science Foundation; ES was financed by the Gesellschaft fur Biotechnologische Forschung to support the German Collection of Microorganisms. HPK and HH were supported by the Deutsche Forschungsgemeinschaft.
References Clayton, R. J., Clayton, R. K.: Properties of photochemical reaction centers purified from Rhodopseudomonas gelatinosa. Biochim. Biophys. Acta 501, 470-477 (1978) Dickerson, R. E.: Evolution and gene transfer in purple photosynthetic bacteria. Nature 283, 210-212 (1980) Fox, G.E., Pechman, K.]., Woese, C.R.: Comparative cataloging of 16S ribosomal ribonucleic acid: molecular approach to procaryotic systematics. Int. J. system. Bact. 27, 44-57 (1977) Fox, G. E., Stackebrandt, E., Hespell, R. B., Gibson, J., Maniloff, J., Dyer, T. A., Wolfe, R.S., Balch, W.E., Tanner, R., Magrum, L.J., Zablen, L.B., Blakemore, R., Gupta, R., Bonen, L., Lewis, B.]., Stahl, D.A., Luehrsen, K.R., Chen, K.N., Woese, C.R.: The phylogeny of prokaryotes. Science 209, 457-463 (1980) Fowler, V.]., Ludwig, W., Stackebrandt, E.: Ribosomal ribonucleic acid cataloging in bacterial systematics: the phylogeny of Actinomadura. In: M. Goodfellow, D. E. Minnikin (eds.) The use of chemotaxonomic methods for bacteria. New York Academic Press (1984 in press) Gibson, ]., Stackebrandt, E., Zablen, L.B., Gupta, R., Woese, C.R.: A phylogenetic analysis of the purple photosynthetic bacteria. Curro Microbiol. 3, 59-64 (1979) Harms, H., Koops, H.-P., Wehrmann, H.: An ammonia-oxidizing bacterium Nitrosovibrio tenuis nov. gen. nov. sp. Arch. Microbiol. 108, 105-111 (1976) Koops, H.-P., Harms, H., Wehrmann, H.: Isolation of a moderate halophilic ammoniaoxidizing bacterium Nitrosococcus mobilis nov. sp. Arch. Microbiol. 107, 277-282 (1976) Seewaldt, E., Schleifer, K. H., Bock, E., Stackebrandt, E.: The close phylogenetic relationship of Nitrobacter and Rhodopseudomonas palustris. Arch. Microbiol. 131, 287-290 (1982) Stackebrandt, E., Seewaldt, E., Ludwig, W., Schleifer, K.-H., Huser, B.A.: The phylogenetic position of Methanothrix soehngenii elucidated by a modified technique of sequencing oligonucleotides from 16S rRNA. Zbl. Bakt. Hyg. I. Abt. Orig. C 3, 90-100 (1982) Truper, H. G., Pfennig, N.: Characterization and Identification of the anoxygenic phototrophic bacteria. pp. 299-312. In: The Prokaryotes eds. M.P.Starr, H.Stolp, H.G.
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Triiper, A. Balows, and H. G. Schlegel. Berlin - Heidelberg- New York. Springer-Verlag 1981 Watson, S. W., Remsen, C. C.: Cell envelope of Nitrocystis oceanus. J. Ultrastruct. Res. 33, 148-160 (1970) Uchida, T., Bonen, L., Schaup, H. W., Lewis, B.]., Zablen, L.B., Woese, C.R.: The use of ribonuclease U2 in RNA sequence determination. Some corrections in the catalog of oligomers produced by ribonuclease T1 digestion of Escherichia coli 16S ribosomal RNA. J. Molec. Evo!. 3, 63-77 (1974) Woese, C.R., Sogin, M., Stahl, D.A., Lewis, B.]., Bonen, L.: A comparison of the 16S ribosomal RNAs from mesophilic and thermophilic bacilli: Some modifications in the Sanger method for RNA sequencing. J. Molec. Evo!. 7, 197-213 (1976) Woese, C. R., Manilotf, 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. Curro Microbio!. 7, 127-132 (1982) 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 (1984a) Woese, C.R., Stackebrandt, E., Weisburg, W.G., Paster, B.J., Madigan, M. T., Fowler, V.]., Hahn, C.M., Blanz, P., Gupta, R., Fox, G.E.: The phylogeny of purple bacteria: The alpha subdivision. System. App!. Microbio!. (submitted 1984b) Dr. Carl R. Woese, Dept. of Genetics and Development, University of Illinois, 515 Morrill Hall, 505 South Goodwin Avenue, Urbana, Illinois 61801, U.S.A.
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The Phylogeny of Purple Bacteria: The Beta Subdivision C. R. WOESEl':', W. G. WEISBURG1, B. J. PASTER1, c. M. HAHNl, R. S. TANNER1, N. R. KRIEG 2 , H.-P. KOOPS 3 , H. HARMS3, and E. STACKEBRANDP
Received April 24, 1984
Summary The beta subdivision of the purple photosynthetic bacteria, defined by 16S ribosomal RNA (oligonucleotide) sequence comparisons comprises two main subclusters, one defined by Rhodopseudomonas gelatinosa, the other by Rhodospirillum tenue and its close relative Rhodocyclus purpureus. [All other characterized species of purple non-sulfur bacteria belong to the alpha subdivision of the purple bacteria.] Each of these subclusters contains a prepondenance of non-photosynthetic species, representing genera such as Spirillum, Aquaspirillum, Alcaligenes, Pseudomonas, Chromobacterium, Thiobacillus, and various ammonia and nitrite oxidizing bacteria. The evolutionary significance of this admixture of biochemical phenotypes is discussed.
Key words: Beta purple bacteria - Evolution - Phylogeny - rRNA catalog
Introduction
Given the simplicity of bacterial phenotypes (as we perceive them), reliable phylogenetic classification of bacteria is not possible using purely phenotypic criteria. However, bacterial genomes are more than complex enough to serve as the basis for a classification scheme. Therefore, sequence comparisons should be used as the primary basis for bacterial classification. The ribosomal RNAs are ancient, functionally constant, large, complex, and
* To whom requests for reprints should be sent.
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precisely defined molecules, and for these reasons serve as excellent molecular chronometers, by which genealogical relationships can be determined. Partial sequencing of 165 ribosomal RNAs - in terms of oligonucleotide catalogs - has been used to reveal the genealogical relationships among the 300 or so bacterial strains so far characterized in these terms (Fox et al., 1980; Woese et al., in preparation). The phylogenetic tree so produced appears to be well defined, except for the order of the earliest branchings in both bacterial kingdoms. [The order of branching once the common ancestor of a kingdom emerges seems to be relatively rapid; thus the major subdivisions appear all to stem from a common point in time.] The accepted taxonomy of the purple photosynthetic bacteria is incorrect in two important respects: It is based solely on photosynthetic genera, and it assumes that the purple non-sulfur genera (i.e. those with the prefix "Rhodo-") and the group of purple sulfur genera (such as Chromatium, Thiocapsa, etc.) represent the two sides of a primary phylogenetic division within the group (Pfennig and Truper, 1974). Regarding the first point, we now know that the true phylogenetic unit contains a number of genera of non-photosynthetic organisms, intermingled genealogically with the various photosynthetic genera (Fox et al., 1980). Regarding the second, there are (at least) three primary groups in this phylogenetic unit, not two; the purple non-sulfur bacteria split into two groups phylogenetically, each no closer to the other than to the purple sulfur group (Gibson et al., 1979). It is important that microbiology recognize the true phylogenetic relationships formally, by a taxonomy accurately reflecting them. Failure to do so in effect imposes a flawed, if not false, conceptual structure on the field. Because (1) the existing taxonomy represents vested interests, (2) it projects a (false) authority stemming from the use of the classical nomenclature and classification system borrowed from eucaryote taxonomy, (3) discarding the existing scheme would entail the discarding of a number of deeply held prejudices, and (4) an alternative formal scheme for classifying bacteria does not yet exist - a proper phylogenetic classification of bacteria will be some time in coming. In the meantime, in order to hasten the transition, it is important to emphasize the true genealogical relationships and stress their differences with the old taxonomy. In previous publications it has been shown that the purple photosynthetic bacteria and relatives are a distinct phylogenetic unit that comprises three major subdivisions, temporarily designated alpha, beta, and gamma. The first two cover the purple non-sulfur bacteria, the latter the purple sulfur bacteria (Gibson et al., 1979; Woese et al., 1982; Woese et al., 1984 a, b). The present communication covers the beta subdivision (Gibson et al., 1979; Fox et al., 1980; Woese et al., 1984b).
Materials and Methods The organisms used in this study are the following: Rhodospirillum tenue strain 3760 and Rhodopseudomonas gelatinosa strain TG-9 - reported previously by Gibson et aL (1979) ; Aquaspirillum gracile ATCC 19624, Aq. serpens ATCC 12638, and Spirillum volutans ATCC 19554 - reported by Woese et al. (1982); Pseudomonas acidovorans ATCC 15668, Ps. cepacia ATCC 17616, Ps. testosteroni ATCC 11996 and Comamonas terrigena ATCC 8461 - reported by Woese et al. (1984a). Reported for the first time are: Nitrosococcus mobilis Nc2 (Koops et aL, 1976), Nitrosomonas europeae Nm35 (from Dr. N. Walker), Nitrosovibrio tenuis Nv1 (Harms et aL, 1976), Nitrosolobus multiformis Nl7 (isolated
The Phylogeny of Purple Bacteria: The Beta Subdivision
329
H.H.), Nitrosospira sp. Nsp1 (isolated H-PK), Aquaspirillum aquaticum ATCC 11330, Aq. dispar ATCC 27510, Aq. bengal ATCC 27641, Alcaligenes eutrophus DSM 531, AI. faecalis ATCC 8750, Chromobacterium violaceum ATCC 12472, C. lividum DSM 1522, Sphaerotilus natans ATCC 15291, Vitreoscilla stercoraria DSM (a gift of Prof. H. Reichenbach), Thiobacillus intermedius ATCC 15466 and T. denitri{icans ATCC 2364. [The Sab value between Aq. serpens and Aq. bengal is 0.93; this is high enough that the two should be considered strains of the same species. In all following discussion Aq. serpens will be taken to represent both strains.] Extraction and labelling of 16S rRNA and generation of the oligonucleotide catalogs followed either of two standard procedures (Uchida et al., 1974; Woese et al., 1976; Stackebrandt et al., 1982). The analysis of catalogs in terms of Sab values is described in Fox et al. (1977).
Results and Discussion Conventionally, analyses of rRNA catalog data have been in terms of binary association coefficients, or Sab values, which are used to construct phylogenetic trees by an unweighted pair group method (Fox et aI., 1977). The method is sufficient for the phylogenetic placement of most bacteria, but, in certain exceptional cases, it is inadequate (Woese et aI., 1980). However, catalog data can also be analysed by a "signature" method - i.e. in terms of groups of oligonucleotides that define and distinguish the various clusters (Woese et aI., 1980). [This is possible because the rRNA sequence exhibits a broad range in the degree of conservation of the various positions in the molecule. Consequently, particular positions (oligonucleotide sequences) can be characteristic of any given grouping of organisms.] Table 1. Oligonucleotide signature distinguishing the beta purple bacterial subdivision from the alpha and gamma subdivisions. Numbers show percent representation of the oligonucleotide in a group. "Other" refers to (percent) occurrence in eubacteria other than the purple group (Gibson et al., 1979; Fox et al., 1980). sequence
alpha beta
ganma
other
-----------------------------------------------CCCCCUG UUAAUCG AAUUUUG CCAACCCG AAAUCCCCG CUUCACACG AUUAAUUCG UCAUACAAUG UAAUACAAUG CYUUACACAUG AAAAACCUUACC ••
0 0 0 5 0 0 0 0 0 0 0
55 100 95 75 80 65 65 80 20 100 100
25 90 <10 10 20 0 0 0 0 0 0
0 <5 <5
<5
0 0 20 0 <5 0 <5
Table 1 is a collection of sequences that defines the beta subdivision of the purple bacteria and distinguishes if from the alpha and gamma subdivisions. Table 2 in turn is an oligonucleotide signature that defines the main subgroups within the beta subdivision. The first of these, beta-I, contains Rhodopseudomonas gelatinosa, Sphaerotilus natans, Aquaspirillum aquaticum, Aq. gracile, Pseudomonas acidovorans, Ps. testosteroni, and Comamonas terrigena, with Thiobacillus intermedius 22 Systematic and Applied Mictobiology, Vol. 5
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C. R. Woese et aI.
as a peripheral member. A second, beta-2, contains Rhodospirillum tenue, Chromobacterium lividum, C. violaceum, Alcaligenes faecalis, AI. eutrophus, Aq. dispar, Aq. serpens, Ps. cepacia and the gliding organism Vitreoscilla stercoraria. [It should be mentioned at this juncture that an incomplete catalog exists for the 165 rRNA of Rhodocyclus purpureus (Gibson et aI., unpublished). Although these data do not permit a meaningful Sab value to be calculated for this organism, they do permit a signature analysis, and this shows Rc. purpureus to be a specific relative of R. tenue (Woese et aI., unpublished).] The remaining species, from the three closely related genera Nitrosovibrio, Nitrosospira, and Nitrosolobus, and from Spirillum volutans, Nitrosococcus mobilis, and Nitrosomonas europeae, seem also to group with beta-2, but in a more peripheral way. Table 3 is a more extensive analysis of this last grouping. It lists all those sequences found in any catalog from this group (taking N. tenuis to represent itself and its two very close relatives) and in at least one other catalog from the beta subdivision Table 2. Oligonucleotide signature distinguishing among the subgroups of the beta purple bacteria. Abbreviations are these: Rg - Rps. gelatinosa, Sn - S. natans, Aa - Aq. aquatile, Pa - Ps. aeidovorans, Pt - Ps. testosteroni, Ct - C. terrigena, Ag - Aq. gracile, Ti - T. intermedius, Ad - Aq. dispar, As - Aq. serpens, Cv - C. violaeeum, Pc - Ps. eepaeia, Ae - AI. eutrophus, Af - AI. faeealis, Cl- C.lividum, Rt - R. tenue, Rp - Re. purpureus, Td - T. denitrifieans, Vs - V. stereoraria, Sv - Sp. volutans, Nt - N. tenuis, Nm - N. mobile, Ne N. europeae. sequence AAACUCAAAG RUUAAAACUCAAAG
R SAP P CAT
g n a a t t g i
d s v c e f 1 t P d s
A A CPA A C R R T V
S N N N v t me + + + +
+ + + + + + +
+
+ + + + + + + + + + +
UCAUACAAUG UAAUACAAUG
+ + + + + + + +
+ + + + + + + + + + +
UAAUACAUCG
+ + + + + + +
CAAUCCCCUG CAACUCCCUG CCCCCUG CUCCCUG
+ + + +
+
CUACACACG CUUCACACG
+ + + + + + + +
AAUUCCG AAUUCCACG AAUUCCAUG
+ + + + + + + +
+ +
+ + + + + + + + + + +
+ + + +
+ + + + + + + + ? + +
+ + + +
+ + + + + + + + ? + +
+ +
+
,.
?
+
+ + + + +
+ +
+
+ + + + + + +
+ + + + + + + + + + + • + + + +
+ + +
+ + + + +
+ + +
+
(A,U)UUCAUG CUUUUG •• ACUUUUG
+ + + + + + + + + + +
+7+
+ ',;+ + + + + + +
UCAACUG UCAACUAG UCUACUAG
+
+ + + + + + +
UUUAAUUCG AUUAAUUCG
CCCUUAUAG CCCUUAUG
+ +
+ + + +
+ + + + + + + +
+
+
+ +
The Phylogeny of Purple Bacteria: The Beta Subdivision
331
Table 3. Oligonucleotide signature showing the specific relationship among the catalogs of S. volutans, N. tenuis, N. mobile and N. europeae and a specific relationship of these to the beta-2 subcluster. All oligonucleotides that fit the following conditions are included: (1) they occur in at least two catalogs from the beta subdivision, one at least of which is in the group Sv - Nt - Nm - Ne, (2) They do not occur in catalogs from both the beta-1 and the beta-2 subclusters. Abbreviations are as in Table 2 caption. The abbreviations in the final two columns indicate which catalogs in the group contain the oligonucleotide in question. sequence
Sv Nt Nm Ne
beta-1
beta-2
--------------------------------------------------------------AAAAACCUUACCUACCUUUG +
+
0
0
Pt
CAACCCUUAUCAUU AG
+
0
0
0
0
Td
AAACUACAAG
0
+
0
+
0
0
UAAUACAAUG
+
+
+
+
0
0
CUAAUCUCAG CCAAUCUCAG
+
+
0
0 0
0
0
+
0 0
Cv Af
CACUUUAAUG
0
+
+
0
0
0
+ +
+ +
+
0 0
0 0 0
+ 0 0
+
+
0 0 +
0 0 +
0 0 0
Ad,Af Td Cv,Pc,Ae,Af,Rt,Rp
0 0 0
0 0 0
+ + +
+ +
0 0 0 0 0 all 0 0
Af,Td all Cv,Pc·,Ae,Af 0 0 0 Pc,Rp Cv,Pc,Af
CUUAACCUG CUUCACACG CUUACCAAG CUCAACUUG UUUCACCAG AUUAAUUCG AAACCCUG UCACACUG UCAACUAG
+
0 +
+ +
+ +
+
+
+ 0
0 +
AACACAG CCCCCUG AAUACCG AAUAACG CUCCCUG AAUUCCG AAUCCUG AUUCAUG
+ + +
0
AAACUG ACACUG ACUCCG ACUACG UAAAAG CUAAUG AUUAUG
+
0 0 0 +
0 0 0 0 0
+
+
0 0 0 0
0 0
+ + +
+
0 0 0 0
+
0 0
0 0 0
+
0
0 + 0 0 0
+ +
+
+
+ 0 0
0
Ad,As,Rt
0 Cv,Td 0 all Aa,Pa,Pt,Ct,Ag 0 0 Ae, Vs Cv,Af 0 0 all
0 0 0 0 0 0 0
Ad,Vs Cv,Td 0 Ae,Vs Cl,Rp, Vs Cl,Td 0
of the purple bacteria, but not occurring in both of the major subgroups of the beta subdivision. The table reinforces the specific relationship of these organisms to the beta-2 subgroup and also shows a phylogenetic coherence among the four species. Thus these four form a coherent subcluster peripheral to the beta-2 subgroup. [Note in Table 2 the second pair of sequences. The phylogenetic relationships suggested would require the transition UCAUACAAUG -+ UAAUACAAUG to have occurred but once. Any other clustering would require multiple origins of one or the other of these oligonucleotides.]
332
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Table 4. Sab values (Fox et aI., 1977) for the members of the beta purple group with one another. The number assignments are as follows: 1 - Rps. gelatinosa, 2 - S. natans, 3 - Aq. aquatieum, 4 - Ps. aeidovorans, 5 - Ps. testosteroni, 6 - C. terrigena, 7 - Aq. gracile, 8 - Th. intermedius, 9 - Aq. dispar, 10 - Aq. serpens, 11 - Ch. violaeeum, 12 - Ps. eepacia, 13 - Al. eutrophus, 14 - AI. faeealis, 15 - Ch.lividum, 16 - R. tenue, 17 - Th. denitrifieans, 18 - V. stereoraria, 19 - Sp. volutans, 20 - N. tenuis, 21 - N. mobilis, and 22 - N. europeae. Re. purpureus is not included because a meaningful Sab cannot be calculated for an incomplete catalog. beta-1 123 4 5 678
beta-2 9 10 11 12 13 14 15 16 17 18
1 2 3 4 5 6 7 8
Rgel Snat Aaqu Cter Paei Ptes Agra Tint
78 63 62 68 61 67 64
-62 62 66 61 64 63
-89 80 84 78 53
-77 82 76 57
-79 -77 75 -57 58 56 --
9 10 11 12 13 14 15 16 17 18
Adis Aser Cvio Peep Aeut Afae Cliv Rten Tden Vste
51 51 52 57 56 48 60 60 53 44
57 52 50 60 60 48 60 63 57 46
47 52 47 48 51 46 53 56 48 45
49 56 49 46 51 49 52 55 51 48
48 52 48 50 51 48 55 53 49 45
49 53 46 46 48 47 50 49 49 44
46 51 49 52 52 43 52 54 47 43
52 51 51 51 54 49 57 58 50 48
71 59 57 57 57 61 57 60 51
62 54 59 57 62 59 60 58
-56 54 58 55 57 56 54
-71 58 63 61 50 52
-62 63 63 54 55
-57 52 56 56
-64 -59 55 -54 48 47 --
19 20 21 22
Svol Nten Nmob Neur
49 42 44 38
49 44 46 40
45 42 38 40
44 46 42 43
45 40 38 39
41 41 38 40
45 42 38 39
41 43 46 42
47 52 48 41
51 57 48 45
50 58 41 43
54 53 50 43
58 57 53 49
53 56 47 44
56 52 50 45
53 58 53 44
57 47 51 42
50 50 45 44
beta-2a 19 20 21 22
55 -5155 45 54 58 --
Table 4 shows the relationships within the beta subdivision as defined by numeric analysis - i.e. in terms of Sab values (Fox et aI., 1977). Perhaps the most remarkable feature of the present phylogeny is the extent to which it disagrees with the previous classifications of these organisms based upon phenotypic criteria. Every phenotypically defined genus in the present study is in disagreement with its true genealogy; the classically defined units are neither comprehensive nor coherent phylogenetically. The most extreme examples are the classical genera Rhodopseudomonas and Rhodospirillum. Neither Rps. gelatinosa nor R. tenue clusters with the other species in its genus (Truper and Pfennig, 1981). [The latter are included in the alpha subgroup of the purple bacteria (Gibson et aI., 1979; Woese et aI., 1984b).] However, the lack of coherence and/or comprehensiveness applies as well to Pseudomonas (Woese et aI., 1984a) (Ps. cepacia is not specifically related to the Ps. acidovorans cluster, and the latter also includes Aq. aquaticum), to Chromobacterium, to Aquaspirillum (Woese et aI., 1982) (only Aq. serpens, and perhaps Aq. dispar form a coherent unit), and to Alcaligenes. The nitrogen, sulfur and hydrogen oxidizing chemolithotrophs also lack coherence phylogenetically. [Nitrobacter species are found in the alpha subdivision, while some other sulfur oxidizing species join the gamma subdivision of the purple bacteria (Woese et aI., 1984b; Woese et aI., in preparation). The hydrogen oxidizing re-
The Phylogeny of Purple Bacteria: The Beta Subdivision
333
Aq. serpens Aq. bengal
Ps. cepackJ
.9
.8
.7
.6
.5
.4
a - group
.3 Fig. 1. Schematic phylogenetic tree representing the relationships shown by Sab analysis as modified by signature analysis. The order of branching is that revealed by the combined analysis, and the branch points arc drawn in approximate agreement with the Sab analysis.
presentatives (A. eutrophus and Paracoccus denitri{icans are members of the beta and alpha subdivisions, respectively.) The true phylogenetic relationships are at variance in another way with classical taxonomic prejudice. Metabolic patterns that we have traditionally kept well separated taxonomically are seen actually to be evolutionarily clustered - e. g. photosynthetic types with non-photosynthetic, aerobes with anaerobes, nitrogen oxidizing with nitrogen reducing species. The ancestral phenotype that gave rise to the greater purple bacterial unit was photosynthetic; there is no other reasonable way to account for the admixture of purple photosynthetic phenotypes within the group. At various times then, nonphotosynthetic offshoots of the photosynthetic phenotypes arose and produced the various "Gram-negative" groupings known so well to the microbiologist - some
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more, some less extensive, depending upon the evolutionary stage at which the transition took place. The deepest divisions in the greater purple bacterial group were not, however, along the classically defined lines (even as regards the photosynthetic types alone). As mentioned, the alpha and beta subdivisions of the (non-sulfur) purple bacteria are no closer to one another than either is to the gamma group (purple sulfur bacteria). This genotypically defined division within the purple non-sulfur bacteria is supported by phenotypic distinctions as well. Rps. gelatinosa and R. tenue, which define the two major subgroups of the beta unit, are unique in molecular phenotype. Their cytochromes c are of the small subunit type, unlike the cytochromes c of the other "Rhodo-" genera (and are removed in sequence from them as well- Dickerson, 1980). The structure of their photo-reaction centers is unique too (Clayton and Clayton, 1978), and the tubular morphology characteristic of their photomembranes distinguishes them from all other purple photosynthetic bacteria (Truper and Pfennig, 1981). The structure within the two major subclusters of the beta subdivision is of interest, if only because, again, it is a mix of classical genera. As Table 4 shows, within the beta-1 subcluster Rps. gelatinosa and S. natans form a specific unit; a Ps. acidovorans unit also exists, which includes Aq. aquaticum and peripherally Aq. gracile; and, T. intermedius is the sole representative of yet another subcluster. Structure within the beta-2 cluster is not clearcut. The two aquaspirillum species, Aq. serpens and Aq. dispar, are specifically related. R. tenue and Rc. purpureus form a specific couple (by signature analY3is). Although the unweighted pair group analysis (Table 4 and unpublished) suggests further structure, this is not strongly supported by signatures, and so is considered uncertain. An interesting biochemical connection would seem to be emerging from these phylogenetic studies with regard to nitrogen metabolism in the greater purple group. The nitrogen oxidizing bacteria appear specifically related to nitrogen reducing species. For example, species of Nitrobacter (oxidizing nitrite) are close, specific relative of Rps. palustris within the alpha-2 group - to the exclusion of Rps. viridis, Rhizobium species, and others (Seewaldt et ai., 1982; Woese et ai., 1984b). The ammonia oxidizing species encountered here split into two specific groups. The first includes the very closely related species Nitrosovibrio tenuis, Nitrosospira sp., and Nitrosolobus multiformis, the second Nitrosococcus mobilis and Nitrosomonas europaea. However, Nitrosococcus oceanus, distinct in its intracellular morphology (Watson and Remsen, 1970), is not a member of the beta subdivision, though it is included in the greater purple group (Stackebrandt et ai., unpublished). It is interesting that N. tenuis shows internal membrane structure reminiscent of the tubular membranes of the photosynthetic members of the beta subdivision. These relationships suggest that all the forms of nitrogen metabolism represented by various species are to some extent inherent in their common ancestor. In other words, organisms that oxidize nitrogen compounds and those that reduce them would seem to use a more or less common pathway. Evolutionary adjustments then cause the pathway to run in the oxidizing vs reducing direction, or select a segment of the pathway to be developed, e.g. ammonia or nitrite oxidation. The phylogenetic distribution of sulfur metabolizing species in the greater purple group suggests that the various forms of sulfur metabolism may have evolved in a similar fashion. The microbiologist is beginning to be faced with two ostensibly unconnected
The Phylogeny of Purple Bacteria: The Beta Subdivision
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problems that perhaps at base are intimately related. One is that the current (eroding) classification schemes for bacteria are totally inadequate; they are false in their deepest assumptions. The problem is not to revise them, but to replace the existing taxonomy by an entirely new classification scheme that uses new criteria for classification (phenotypic and genotypic) and reflects bacterial genealogical relationships. The other problem is that our prejudices as to the evolution of bacterial metabolism, the relationships among the various bacterial biochemistries, is in definite need of revision if we are ever to understand much about the origins of various intermediary metabolisms. It is in the framework of a true bacterial phylogeny that we will come to understand the evolution and full significance of bacterial metabolism. Therefore, in an important sense these two disparate problems are part and parcel of one another. Acknowledgements. CRW was supported by a grant from the National Science Foundation; ES was financed by the Gesellschaft fur Biotechnologische Forschung to support the German Collection of Microorganisms. HPK and HH were supported by the Deutsche Forschungsgemeinschaft.
References Clayton, R. J., Clayton, R. K.: Properties of photochemical reaction centers purified from Rhodopseudomonas gelatinosa. Biochim. Biophys. Acta 501, 470-477 (1978) Dickerson, R. E.: Evolution and gene transfer in purple photosynthetic bacteria. Nature 283, 210-212 (1980) Fox, G.E., Pechman, K.]., Woese, C.R.: Comparative cataloging of 16S ribosomal ribonucleic acid: molecular approach to procaryotic systematics. Int. J. system. Bact. 27, 44-57 (1977) Fox, G. E., Stackebrandt, E., Hespell, R. B., Gibson, J., Maniloff, J., Dyer, T. A., Wolfe, R.S., Balch, W.E., Tanner, R., Magrum, L.J., Zablen, L.B., Blakemore, R., Gupta, R., Bonen, L., Lewis, B.]., Stahl, D.A., Luehrsen, K.R., Chen, K.N., Woese, C.R.: The phylogeny of prokaryotes. Science 209, 457-463 (1980) Fowler, V.]., Ludwig, W., Stackebrandt, E.: Ribosomal ribonucleic acid cataloging in bacterial systematics: the phylogeny of Actinomadura. In: M. Goodfellow, D. E. Minnikin (eds.) The use of chemotaxonomic methods for bacteria. New York Academic Press (1984 in press) Gibson, ]., Stackebrandt, E., Zablen, L.B., Gupta, R., Woese, C.R.: A phylogenetic analysis of the purple photosynthetic bacteria. Curro Microbiol. 3, 59-64 (1979) Harms, H., Koops, H.-P., Wehrmann, H.: An ammonia-oxidizing bacterium Nitrosovibrio tenuis nov. gen. nov. sp. Arch. Microbiol. 108, 105-111 (1976) Koops, H.-P., Harms, H., Wehrmann, H.: Isolation of a moderate halophilic ammoniaoxidizing bacterium Nitrosococcus mobilis nov. sp. Arch. Microbiol. 107, 277-282 (1976) Seewaldt, E., Schleifer, K. H., Bock, E., Stackebrandt, E.: The close phylogenetic relationship of Nitrobacter and Rhodopseudomonas palustris. Arch. Microbiol. 131, 287-290 (1982) Stackebrandt, E., Seewaldt, E., Ludwig, W., Schleifer, K.-H., Huser, B.A.: The phylogenetic position of Methanothrix soehngenii elucidated by a modified technique of sequencing oligonucleotides from 16S rRNA. Zbl. Bakt. Hyg. I. Abt. Orig. C 3, 90-100 (1982) Truper, H. G., Pfennig, N.: Characterization and Identification of the anoxygenic phototrophic bacteria. pp. 299-312. In: The Prokaryotes eds. M.P.Starr, H.Stolp, H.G.
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Triiper, A. Balows, and H. G. Schlegel. Berlin - Heidelberg- New York. Springer-Verlag 1981 Watson, S. W., Remsen, C. C.: Cell envelope of Nitrocystis oceanus. J. Ultrastruct. Res. 33, 148-160 (1970) Uchida, T., Bonen, L., Schaup, H. W., Lewis, B.]., Zablen, L.B., Woese, C.R.: The use of ribonuclease U2 in RNA sequence determination. Some corrections in the catalog of oligomers produced by ribonuclease T1 digestion of Escherichia coli 16S ribosomal RNA. J. Molec. Evo!. 3, 63-77 (1974) Woese, C.R., Sogin, M., Stahl, D.A., Lewis, B.]., Bonen, L.: A comparison of the 16S ribosomal RNAs from mesophilic and thermophilic bacilli: Some modifications in the Sanger method for RNA sequencing. J. Molec. Evo!. 7, 197-213 (1976) Woese, C. R., Manilotf, 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. Curro Microbio!. 7, 127-132 (1982) 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 (1984a) Woese, C.R., Stackebrandt, E., Weisburg, W.G., Paster, B.J., Madigan, M. T., Fowler, V.]., Hahn, C.M., Blanz, P., Gupta, R., Fox, G.E.: The phylogeny of purple bacteria: The alpha subdivision. System. App!. Microbio!. (submitted 1984b) Dr. Carl R. Woese, Dept. of Genetics and Development, University of Illinois, 515 Morrill Hall, 505 South Goodwin Avenue, Urbana, Illinois 61801, U.S.A.