Proc. NatL Acad. Sci. USA Vol. 78, No. 11, pp. 6854-6857, November 1981 Biochemistry

Amino acid sequences of bacterial cytochromes c' and c-556 (bacterial evolution/heme ligands/protein structure) R. P. AMBLER*, R. G. BARTSCHt, M. DANIEL*, M. D. KAMENtt, L. MCLELLAN*, T. E. MEYERt, AND J. VAN BEEUMEN§ *Department of Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, Scotland; tDepartment of Chemistry A-002, University of California at San Diego, La Jolla, California 92093; and §Laboratory for Microbiology and Microbial Genetics, University of Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium Contributed by Martin D. Kamen, August 20, 1981

ABSTRACT The cytochromes c' are electron transport pro- chrome c' was eluted together with nucleic acid with 0.5 M teins widely distributed in photosynthetic and aerobic bacteria. sodium chloride when the column was developed with a linear We report the amino acid sequences of the proteins from 12 dif- gradient offrom 0.1 to 0.6 M NaCl in 0.02 M Tris HCl, pH 7.3. ferent bacterial species, and we show by sequences that the cy- The cytochrome c' was then precipitated with ammonium sul- tochromes c-556 from 2 different bacteria are structurally related fate (70-100% saturation fraction) and subjected to gel filtration to the cytochromes c'. Unlike the mitochondrial cytochromes c, through Sephadex G-75. The cytochrome c' was then adsorbed the heme binding site in the cytochromes c' and c-556 is near the to hydroxylapatite and eluted with approximately 0.16 M phos- COOH terminus. The cytochromes c-556 probably have a methi- onine sixth heme ligand located near the NH2 terminus, whereas phate when a linear gradient offrom 0 to 0.2 M potassium phos- the cytochromes c' may be pentacoordinate. Quantitative com- phate, pH 7.0, in 0.2 M NaCl was used. Final purification was parison ofcytochrome c' and c-556 sequences indicates a relatively achieved by repetition of the hydroxylapatite and DEAE-cel- low 28% average identity. lulose chromatography, with yields of about 5 1Lmol/kg ofwet cells. The preparation and properties ofthe Agrobacterium tu- mefaciens cytochrome c-556 has been reported by Van Beeu- The cytochromes c' constitute a class ofelectron transport pro- men et al. (9). teins that are widely distributed in phototrophic and aerobic bacteria (1). The heme is covalently bound to cysteine residues, so the proteins are classed as cytochromes c although there is RESULTS AND DISCUSSION little further similarity in properties and structure to the well- The evidence for the amino acid sequences ofthe cytochromes characterized cytochrome c of mitochondria. Thus the cyto- c' fromAlcaligenes sp. National Collection ofIndustrial Bacteria chromes c' heme is in a high-spin environment, similar to that (NCIB) 11015 (10), Rhodospirillum rubrum (11), and Chro- in the globins, but is unreactive with most common heme li- matium vinosum (12) has already been published, and the se- gands, with the exception ofnitric oxide and carbon monoxide. quences of the Rhodopseudomonas gelatinosa and Rhodospi- The tertiary structure of one of the cytochromes c' has been rillum tenue (13) andAgrobacterium tumefaciens strain B2a (14) determined (2, 3), and it shows that there is no relationship proteins have been reported. The additional sequences shown between the overall folding patterns ofcytochromes c' and the in Fig. 1 have been determined by similar methods, and to sim- mitochondrial cytochrome c family (4). The cytochromes c' do ilar standards, as described by Ambler et aL (12). show a fortuitous or distant similarity in folding pattern to Esch- The sequences shown in Fig. 1 include those ofproteins from erichia coli cytochrome b-562, ferritin, and hemerythrin (5). We 9 of the 12 species of Rhodospirillaceae described by Pfennig have determined the amino acid sequences of cytochromes c' and Truper in the 8th edition ofBergey's Manual (16). We find from 10 species of phototrophic bacteria and from 2 very dif- that the protein is absent (or expressed only at very low levels) ferent denitrifying bacteria. There are some structural features from the other three species, Rhodopseudomonas viridis, Rho- common to all the proteins, but the sequences are so divergent dopseudomonas acidophila, and Rhodomicrobium vannielii (1), that choice ofalignments is equivocal. We have also determined and also from the recently described Rhodopseudomonas glob- the amino acid sequences of low-spin cytochromes c-556 from iformis (17). Cytochrome c' is present in most of the strains of a photosynthetic and from an aerobic bacterium, and we dis- Rhodopseudomonas palustris that we have examined, but is cover that, despite spectral differences, the proteins are struc- apparently absent from the neotype strain 2.1.6 (ATCC 17001), turally very similar to cytochromes c'. although no photosynthetic or respiratory anomalies have been noticed in this strain. Cytochrome c' is present in Azotobacter MATERIALS AND METHODS vinelandii (18), Alcaligenes faecalis (unpublished results), and General methods for the isolation of cytochrome c' from pho- Methylococcus capsulatus (unpublished results). tosynthetic bacteria have been described by Bartsch (6). The Rhodopseudomonas palustris synthesizes a low-spin cyto- cytochrome c' from the halophilic "Micrococcus" American chrome c with an a band absorption maximum at 556 nm (1). Type Culture Collection (ATCC) 12084 [which should be pro- Spectrally similar cytochromes c-556 have also been found in visionally assigned to the genus Paracoccus (7)] was described Rhodopseudonnnas sulphidophila (19) by Bartsch (1), and in as a hydroxylamine reductase by Kono and Taniguchi (8). We various strains of the nonphotosynthetic bacterium Agrobac- prepared a cell extract as described by Tedro et aL (7) and ad- terium tumefaciens (9). The sequences of two proteins of this sorbed the colored proteins onto DEAE-cellulose. The cyto- type have been determined (Fig. 1 and ref. 14), and they clearly resemble the cytochromes c' (20) in having the heme attach- The publication costs ofthis article were defrayed in part by page charge ment site close to the COOH terminus. payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 6854 Downloaded by guest on September 28, 2021 Biochemistry: Ambler et aL Proc. Natd Acad. Sci. USA 78 (1981) 6855

helix A H helix B-J helix C Hhelix D-j 12 16 58 70 76 82 125 (A) QQSKPEDLLKLRQGLMQTLKSQWVPIAGFAAGKADLPADAAQRAEMLAMVAKLA--PI-GWAKGTEA-LPNG-ETKPE-AFGSKSAEFLEGWKALATESTKLAAAAKAG-PDALKAQAAATGKVCKACHEEFKQD I~ ~~~~~~~~II1 ~ ~1 ~ ~~~ ~ ~~~1111PII1 (B) QQSKPEELLKLRQGLtuTLKSQWAPIAGFAAGKADLPADAAQRAENMVLVAKLA--PI-GWAKGTEA-LPNS-ETKAE-AFGARGAMESLAAKAGPDALKAQAAATGRVCKACHEEFKQD l I II 1111 11 (C) AEPEDAIHYRQSALSVMGWQMGPMGAMAQG zIDADEFATRANNAVAH-LPWEGFTEGTLQGDDHGVETDALADIGDDWEGFEERQETFKQEAATLAQMDDGEEFSALRRQVGAVGKSCKGCHDDFRAE l I 1111 11 (D) EPAKSEDLIKWRQSAYQVLHWNMDRLKANIDSPQYNKDDGIKAANTIAAIANSG-W GSLFAAGTETGKGWH-PTSVKPAFFTDGKKVGEVANKNEKVAATG-DAAAVKAQFGKVGQTCKACHDDFRRKDII (E) AGLSPEEQIETRQAGYEFFWNGKIKANLEG-EYNAAQVEANVAIANS G-G-ALYGPGTDKNVGDV-KTRVKPEFFQNMEDVGKIAREFVGAATAVAATG-EAEAVKTAFGDVGAACKSCHEKYRAK (F) QFQKPGDAIEYRQSAFTLIANHFGRVAAMA4G-KAPFDAKVAAENIALVSTLSK-LPLTAFGPGTDKGHG---TEAKPAVWSDAAGFAAA LDKMAVDLGKTG-DFAQIKAAVGETGGACKGCHDKFKEK (G) QFAKPEDAVKYRQSALTLMASHFGRMTPVvKG-QAPYDMAQIKANVEVLKTLSA-LPWAAFGPGTE-GGD---ARPEIWSDAASFKQKQQAFQDNIVKLSAAADAG-DLDKLRAAFGDVGASCKACHDAYRKKK I~~~~~II1 ~~ ~~ ~~~ ~ ~ ~~~~~1 11 11 11 (H)(C)~wew -w--Pw-aADAEHVVEARKGYFSLVALEFGPLAAMAKG-EMriD^AAAHASDLVTLTKYDPSDLYAPGTSADDVKG--TAAKAAIWQDADGFQAKGMLAFFEAVAALEPAAGAG--QKELAAAVGKVGTGCKSCHDDFRVKR s -^AA-vVsttY~w svn~n~r~nmr (I) ADTKEVLEAREAYFKSLGKSNKAMTGVAK-SFDAEAAKAEAAALEKIATD-VA-PLFPAGTSSTDLPG-QTEAKAAIWTNMADFG;AKGKMNDHJAGAEVLAAANA DATAGLQKLGLGTC.KAHLDDLlYKR!EJ (J) DGMETVKARQDYFKSLGGAIKALSGVAK--NYDAEAAKAEAKLEAILATD-IK-PLFAPGTSDADFPG-ESEAKASIWENMEDFGAKGQAMHEAGMELIAAANITG-EASAFGPALKKLGGTCKACHDDYRAEH l~~~~l 11 ~ ~ ~1 ~~1 ~ ~ ~~ ~ ~~~~~l 11 11 11 (K) ADPAAYVEYRKSVLSATSNYFKAIGITLKE-DLAVPNQTADHAKAIASIMET-LP-AAFPEGTAGIAK---TEAKAAIWKDFEAFKVASKKSQDAALELASAAET1( -DKAAIGAKLQALGGTCKACHKEFKAD ASPEAYVEYRKQALKASGDHMKALSAIVKG-QLPLNAEAAHEAIAAIMES-LP-AAFPEGTAGIAK---TEAKAVVWSKADEFKADAVKSADAAKALAQAATAG-DTA~MGKALAALGGTCKGCHETFRE (L) Ij11 l l l11 11 (M) AGEVEKREGMM-KQIGGAMGSLAAISKG~-ER.&FDADTVKAAVTTIGTNAKAFP-EQFPAGTETG---SAAAPAIWENFEDFKAKAAKLGTDADIVLANLPDL,-Q~AGvATAM-KTLGADCGTCHQTYRIKK I ~~~~~~~~~I~ ~ 1 11 11 (N) QQDLVDKTQKLMKDNGRNMMVLGAIAKG-EKPYDQAAVDAALKQFDETAKDLP-KLFPDSVKGLKPFDSKYSSSPKIWAERAKFDTEIADFAKAVDGAKGKIK---DVDTLKAAMQPIGKACGNCHENFRDKEG ll l l 11111 (0) QTDIAQKALKQGETKPAAlKG-ju'DAVQKLAAADSKLPALPASKTGD--AAPKWEKAKDDFALAAAAQTDVIQRKALKQMEATRIMMLG-EA~VQAWKSLAIADDKKLPALFPASKTGl)--AALPIWEDAKFDDFAKLAAATAQGTK -DASLKAIGGVGNCKCHDDRAKK

FIG. 1. Amino acid sequences of bacterial cytochromes c' (A-L and 0) and c-556 (M and N). Residues are numbered from the R. molischianum sequence, for which the tertiary structure is known (2, 3), and the extent of the a helices in this protein are indicated (5). Insertions and deletions have been positioned so as to emphasize conserved residues of possible structural significance (see the text), which are indicated by vertical bars between the sequences. The sequences are from: (A)R. molischianum strain S (ATCC 14031), (B)R.fulvumstrain 1360 (ATCC 15798), (C)Paracoccus sp. (ATCC 12084), (D) R. tenue strain 3761, (E) Chromatium vinosum strain D (ATCC 17899), (F)Rhodopseudomonasgelatinosa strain 2.2.1 (ATCC 17011), (G)Alcaligenes sp. (NCIB 11015), (H)Rps. sphaeroides strain 2.4.1. (ATCC 17023), (I)Rps. capsukita strain SP7 (15), (J)Rhodopseudomonas sp. strain TJ12 (isolated from Tiuana river water by P. F. Weaver), (K) R. rubrum strain Si (ATCC 11170), (L) R. photometricum strain SP113, (M) Agrobacterium tumefaciens strain B2a, (N) Rps. palustris strain 2.1.37 (ATCC 17007) (cytochrome c-556), and (0) Rps. palustris strain 2.1.37. The sequence of the cytochrome c' from Rps. capsulata strain Ml10 (a carotenoid mutant of strain St. Louis, ATCC 23782) probably differs from that of strain SP7 (I) in 11 positions, but it is not separated by any insertion or deletion events. The one-letter notation used is that recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine.

The amino acid sequences of13 different cytochromes c' and residues, which would also be capable of forming hydrogen of two homologous cytochromes c-556, shown in Fig. 1, are so bonds. From the R. molischianum structure, Thr-70 is also suf- divergent that alignment to demonstrate their common features ficiently near that it may hydrogen bond to one of the propio- requires many insertions and deletions, and there is a large sub- nates, and there is a threonine residue in 11 ofthe 15 sequences jective element in the choice of their locations. Weber et aL that can be matched to this position if the insertions and dele- (2, 3) have determined the tertiary structure ofone cytochrome tions shown in Fig. 1 are postulated. In three of the remaining c', and their study gives an indication as to which residues in sequences, there is a different hydroxyl-containing amino acid Rhodospirillum molischianum cytochrome c' are likely to be in this position, while the last one [that ofAlcaligenes sp. (12)] involved in the heme-polypeptide interactions. We have .may have a deletion in this region. Four residues after the highly aligned the sequences in Fig. 1 on the assumption that the es- conserved Arg-12 residue, the R. molischianum sequence con- sential features ofsuch interactions are likely to have been con- tains a methionine residue that is positioned over the heme face served despite the large amount of divergence that separates but does not bond to the iron (3). Methionine does not occur the sequences. The scheme used enables us to emphasize the frequently at this position in the cytochrome c' sequences, but conservation of residues that are likely to have functional im- is present in both the cytochromes c-556, and we suggest that portance and to give a preliminary quantitative estimate of the this residue is the probable contributor of the sixth ligand, similarities between the sequences (Fig. 2). However, the R. which has been identified as methionine by NMR spectroscopy molischianum sequence is one ofthe most divergent ofthe set, (unpublished results). All the cytochromes c' contain lysine as and so may differ from other of the cytochromes in features of the residue next after the first heme-binding cysteine residue, its tertiary structure. whereas in both cytochromes c-556 this position is occupied by In the alignment ofsequences that we have chosen, there are a glycine residue. No obvious structural role can yet be assigned only three absolutely invariant residues, the two cysteine res- to this lysine residue, so it is unlikely that its substitution by idues that we presume are covalently linked to the heme and glycine can be directly responsible for the difference in ligand the fifth ligand histidine residue. In all of the cytochromes c', binding properties between the high-spin and low-spin proteins. and in the Agrobacterium (but not the Rps. palustris) cyto- The alignment in Fig. 1 emphasizes the apparent conserva- chrome c-556, there is an arginine residue near the NH2 ter- tion of several aromatic residues in the sequences. In R. mol- minus, corresponding to Arg-12 in R. molischianumcytochrome ischianum cytochrome c', Trp-58, Phe-82, and Phe-125 are all c', in which it forms a salt bridge or hydrogen bond with one very near the heme (3). In 10 of the sequences there is a tryp- of the heme propionates (3). The heme propionate may also be tophan residue at the position aligning with Gly-76 in the R. hydrogen bonded to the sequentially adjacent Gln-13, which molischianum sequence, and which may be within Van der is conserved in 9 of the 15 sequences in Fig. 1. In the other Waals contact radius of the other three "conserved" aromatic sequences this position is occupied by lysine or glutamic acid rings. Downloaded by guest on September 28, 2021 6856 Biochemistry: Ambler et al Proc. Nad Acad. Sci. USA 78 (1981)

(A) 91 (0) (B)

26 (8) 26 (8) (C)

29 (5) 27 (5) 30 (6) (D)

20 (5) 20 (5) 27 (4) 48 (2) (E)

26 (7) 26 (7) 32 (3) 31 (4) 30 (2) (F)

28 (7) 29 (7) 36 (5) 31 (4) 27 (3) 48 (2) (G)

26 (8) 26 (8) 33 (4) 25 (61 25 (5) 39 (5) 38 (5) (H)

22 (7) 23 (7) 23 (5) 28 (2) 24 (3) 25 (5) 28 (5) 39 (5) (I)

20 (7) 22 (7) 24 (6) 27 (2) 29 (3) 23 (5) 26 (5) 36 (6) 69 (1) (J)

21 (7) 22 (7) 27 (3) 23 (4) 21 (2) 33 (2) 27 (4) 25 (5) 35 (3) 33 (4) (K)

22 (8) 21 (8) 28 (4) 24 (4) 24 (3) 38 (3) 36 (5) 33 (5) 34 (3) 32 (4) 57 (1) (L)

15 (8) 14 (8) 20 (7) 21 (5) 23 (5) 29 (4) 29 (5) 30 (5) 27 (5) 28 (5) 24 (5) 31 (5) (M)

20 (8) 20 (8) 19 (5) 20 (6) 18 (5) 24 (6) 22 (7) 24 (5) 19 (6) 18 (6) 18 (5) 23 (5) 30 (4) (N)

18 (8) 17 (8) 27 (6) 24 (6) 23 (5) 33 (5) 28 (7) 29 (5) 22 (6) 18 (6) 25 (4) 27 (4) 32 (4) 35 (1) (0)

FIG. 2. Amino acid sequence similarity matrix for the cytochromes c' and c-556 aligned as shown in Fig. 1. Results are expressed as percentage identity. The five values that are much higher than the average similarity of 28 ± 10% are shown with a wavy underline and are discussed in the text. The numbers in parentheses are the minimal number of insertion or deletion events separating pairs of sequences when they are aligned as in Fig. 1.

The sequences shown in Fig. 1 range in length between 125 Our studies ofcytochromes c have been criticized by Woese and 133 residues. In all ofthem the heme attachment site is very et al. (22), who argue that "for a macromolecule to be useful as close to the COOH terminus, and the five- to eight-residue an evolutionary clock, it must maintain function and tertiary COOH-terminal tails that extend beyond the fifth heme-ligand structure constancy over the group oforganisms being studied" histidine residue contain a high proportion ofcharged residues, and that cytochromes c vary in structure and function so much though the net charge ofthese regions varies from -4 through that their sequences do not give valid information about pro- 0 to +3. In all cases the third residue after the histidine is an karyotic evolution. We accept this to be a valid argument against aromatic residue (Phe-125 in the R. molischianum sequence) excessive phylogenetic speculation (and we have made no at- and the fourth residue is basic. tempt to deduce a "tree"), but we consider that it also applies None of the proteins contain cysteine except at the heme to the use of 16S ribosomal RNA nucleotide map comparisons binding site. The absence of this residue from nearly 2000 res- (23) (for which there is no evidence for tertiary structure, or for idue lengths of sequence suggests that its presence would cause the prevalence or paucity ofinsertions or deletions) and to other structural or functional difficulties for this class of protein. genetic systems that have been proposed. Our crucial results, In Fig. 2 we give an identity matrix for the sequences aligned which must be explained in any critique ofbacterial evolution, as in Fig. 1. The figure also shows the minimal number of in- are the anomalous similarities such as those mentioned above sertion or deletion events separating each pair of sequences in for cytochromes c' and that between Paracoccus denitrifwans this alignment. cytochrome c-550 (24) and Rps. capsulata cytochrome c2 (21), The high-spin and low-spin cytochromes from Rps. palustris and we predict that many more such anomalies will be found can be aligned with each other throughout their length with the as more macromolecular sequences are determined. for a four-residue insertion in necessity postulating only single We acknowledge generous support for this work by grants from the the cytochrome c-556 sequence. However, the number of se- Medical Research Council to R. P.A., the Department ofEnergy (AT03- quence identities with this alignment is only a little higher than 78ER-70293) and the National Institutes of Health (GM 18528) to the average 28 10% value in the matrix in Fig. 2. M.D. K., and the Nationaal Fonds voor Wetenschappelhjk Onderzoek There are only five pairs of sequences with identity values to J.V.B. much higher than the average of Fig. 2. Three pairs-R. rub- 1. Bartsch, R. G. (1978) in The Photosynthetic Bacteria, eds. Clay- rum and R. photometricum, R. molischianum and R. fulvum, ton, R. K. & Sistrom, W. R. (Plenum, New York), pp. 249-279. and Rps. capsulata and Rhodopseudomonas sp. strain TJ12-are 2. Weber, P. C., Bartsch, R. G., Cusanovich, M. A., Hamlin, R. also related in their 21 C., Howard, A., Jordan, S. R., Kamen, M. D., Meyer, T. E., closely cytochrome c2 sequences (ref. Weatherford, D. W., Xuong, N. H. & Salemme, F. R. (1980) and unpublished results), and we consider them to illustrate the Nature (London) 286, 302-304. case oforganisms just beginning to diverge from each other. The 3. Weber, P. C. (1979) Dissertation (Univ. ofArizona, Tucson, AZ). other two pairs are C. vinosum and R. tenue and Rps. gelatinosa 4. Dickerson, R. E. (1980) Sci. Am. 242 (3), 136-153. and Alcaligenes sp. strain NCIB 11015. As we have pointed out 5. Weber, P. C. & Salemme, F. R. (1980) Nature (London) 287, 82-84. elsewhere (13), these similarities are unexpected, they do not 6. Bartsch, R. G. (1971) Methods EnzymoL 23, 344-363. correlate with other properties of the organisms, and, in our 7. Tedro, S. M., Meyer, T. E. & Kamen, M. D. (1977) J. BioL opinion, are most simply explained by lateral gene transfer. Chem. 252, 7826-7833. Downloaded by guest on September 28, 2021 Biochemistry: Ambler et al Proc. NatL Acad. Sci. USA 78 (1981) 6857

8. Kono, M. & Taniguchi, S. (1960) Biochim. Biophys. Acta 43, 17. Pfennig, N. (1974) Arch. Microbiol 100, 197-206. 419-430. 18. Yamanaka, T. & Imai, S. (1972) Biochem. Biophys. Res. Commun. 9. Van Beeumen, J., Van den Branden, C., Tempst, P. & De Ley, 46, 150-154. J. (1980) Eur. J. Biochem. 107, 475-483. 19. Hansen, T. A. & Veldkamp, H. (1973) Arch. MicrobioL 92, 10. Ambler, R. P. (1973) Biochem. J. 135, 751-758. 45-58. 11. Meyer, T. E., Ambler, R. P., Bartsch, R. G. & Kamen, M. D. 20. Van Beeumen, J. (1980) Ptotides Biot Fluids Proc. Colloq. 28, (1975)J. Biol Chern. 250, 8416-8421. 61-68. 12. Ambler, R. P., Daniel, M., Meyer, T. E., Bartsch, R. G. & Ka- 21. Ambler, R. P., Daniel, M., Hermoso, J., Meyer, T. E., Bartsch, men, M. D. (1979) Biochem.J. 177, 819-823. R. G. & Kamen, M. D. (1979) Nature (London) 278, 659-660. 13. Ambler, R. P., Meyer, T. E. & Kamen, M. D. (1979) Nature 22. Woese, C. R., Gibson, J. & Fox, G. E. (1980) Nature (London) (London) 278, 661-662. 283, 212-214. 14. Van Beeumen, J., Tempst, P., Stevens, P., Bral, K., Van 23. Fox, G. E., Stackebrandt, E., Hespell, R. B., Gibson, J., Man- Damme, J. & De Ley, J. (1980) Protides BioL Fluids Proc. Colloq. iloff, J., Dyer, T. A., Wolfe, R. S., Balch, W. E., Tanner, R. S., 28, 69-74. Magrum, L. J., Zablen, L. B., Blakemore, R., Gupta, R., Bonen, 15. Weaver, P. F., Wall, J. D. & Gest, H. (1975) Arch. MicrobioL L., Lewis, B. J., Stahl, D. A., Luehrsen, K. N., Chen, K. N. & 105, 207-216. Woese, C. R. (1980) Science 209, 457-463. 16. Pfennig, N. & Truper, H. G. (1974) in Bergey's Manual of De- 24. Ambler, R. P., Meyer, T. E., Kamen, M. D., Schichman, S. A. terminative Bacteriology, eds. Buchanan, R. E. & Gibbons, N. & Sawyer, L. (1981)J. Mol Biol 147, 351-356. E. (Williams & Wilkins, Baltimore), 8th Ed., pp. 24-64. Downloaded by guest on September 28, 2021