System. Appl. Microbiol. 5, 337-351 (1984)

1 Department of Genetics and Development, University of Illinois, Urbana, Illinois 61801,U.S.A.

2 Department of Dairy Science, University of Illinois, Urbana, Illinois 61801, U.S.A.3 Lehrstuhl Hir Mikrobiologie, Technische Universitat Miinchen, 8000 Miinchen, Federal

Republic of Germany

The Phylogeny of the Spirochetes

B. J. PASTER1,2, E. STACKEBRANDT 3, R. B. HESPELL 2, C. M. HAHN1,and C. R. WOESE I, ':.

Received March 12, 1984

Summary

A number of species of Spirochaeta, Treponema, Leptospira and Borrelia have beencharacterized by the method of ribosomal RNA oligonucleotide cataloging, in order todetermine their phylogenetic relationships. The group so formed is phylogenetically verydeep, with a major division occurring between the leptospiras on the one side and theremaining spirochetes on the other. At a comparably deep level the greater group alsoencompasses the recently recognized group of obligate anaerobic halophiles. The maincluster within the greater group comprises three sublines: (1) that containing the bulk ofthe spirochetes and treponemes examined, (2) that represented by B. hermsii, and (3) thatrepresented by T. hyodysenteriae. The first of these in turn divides into two main sub­clusters, represented by S. halophila on the one hand and S. stenostrepta on the other.

Key words: Spirochete - Treponemes - Leptospira - 16S rRNA - Phylogeny

Introduction

The spirochetes are a group of bacteria that have in common a unique and com­plex morphology. They are helical and possess internal organelles of motility, theperiplasmic fibrils. Moreover, they are resistant to the antibiotic rifampin (whichinterferes with the transcription process in eubaeteria) (Stanton and Canale-Parola,1979) and, except for the leptospiras, possess ornithine as the diamino acid com­ponent of their cell wall peptidoglycan (Joseph et aI., 1973). Despite these basicsimilarities, there are differences among them regarding habitat, energy yieldingmetabolism, end products of glucose fermentation, and DNA composition (whichruns from less that 30 mol% G + C in some borrelias to 66 mol% G + C in somespirochetes) (Canale-Parola et aI., 1968; R. C.]ohnson, unpublished).

* To whom requests for reprints should be sent.

338 B.]. Paster, E. Stackebrandt, R.B. Hespell, C. M. Hahn, and C. R. Woese

At present the group is classified as one family consisting of five genera: (Smibert,1974) Spirochaeta - facultatively or obligately anaerobic free-living spirochetes;Treponema - host-associated pathogenic and non-pathogenic spirochetes; Borrelia- pathogenic spirochetes transmitted to birds and mammals via insects; Leptospira- aerobic, free-living and pathogenic spirochetes; and Cristispira -large spirochetesfound in molluscs.

The commonality of their complex morphological features and their resistanceto rifampin argue that they constitute a phylogenetically coherent grouping. How­ever, their diversity as regards metabolism suggests the opposite (Hespell and Ca­nale-Parola, 1971; Canale-Parola, 1977). This ostensible contradiction might bereconciled were the spirochetes a deeper phylogenetic grouping (and so more variedin phenotype) than their designation as a mere family suggests. In any case, there isconsiderable uncertainty regarding the phylogenetic relationships among the variousgroupings of spirochetes and no indication, whatever, as to their relationship toother eubacteria (Canale-Parola, 1977).

The technique of ribosomal RNA oligonucleotide cataloging has proven a veryeffective one for elucidating phylogenetic relationships among bacteria. The reasonsfor this are several: (1) Sequences are genotypic, not phenotypic properties of theorganism. The phase space of possible genotypes is enormous by comparison tothe space of corresponding phenotypes. Thus, (extensive) sequence similarity cannotreasonably represent evolutionary convergence. (2) The ribosomal RNA representsa very constant function, one that appears not to have changed significantly sincethe common ancestor of all eubacteria (Woese et al., 1983). Consequently, thesequence changes occurring in rRNAs during the course of evolution tend to beselectively neutral and so random - making the molecule an excellent chronometerof evolution. (3) The rRNA molecule presents a spectrum of sequence conservation,from positions that vary from one species to the next within the same genus, tothose that are invariant across an entire kingdom. Consequently, as a chronometerthe rRNA molecule will measure not only close evolutionary relationships, but verydistant ones as well. And (4) the molecule is large enough that occurrences of theoccasional local saltations in sequence, that make smaller molecules unreliablechronometers, affect so small a fraction of the sequence as to be negligible in impact(Woese,1982).

At present, over 300 eubacteria have been characterized by the rRNA oligo­nucleotide cataloging method, by the two laboratories active in the area (Fox et al.,1980; Stackebrandt and Woese, 1981). A number of major groupings of eubacteria,each a eubacterial "phylum", have been identified by this method (Fox et al., 1980;Woese et al., in preparation). Here we use the technique to test the validity of thepresent taxonomy of the spirochetes and to explore their relationships to othereubacteria.

Materials and Methods

The spirochetes characterized in this study came from the stock culture collection ofRBH, unless otherwise indicated. The organisms were cultivated on media similar to thatused previously in each case (indicated in the reference following the strain designation)with the exception that the phosphate content of the medium was lowered to allow forefficient incorporation of 32P (Woese et al., 1976). The strains examined included: Spiro­chaeta halophila RS1 (Greenberg and Canale-Parola, 1976), S. stenostrepta Zl (Canale-

The Phylogeny of the Spirochetes 339

Parola et aI., 1968; 1976), S. zuelzerae ATCC 19044 (Veldkamp, 1960), S. aurantia J1(Breznak and Canale-Parola, 1969), S.litoralis R1 (Hespell and Canale-Parola, 1970),S. isovalerica MA-2 (Harwood and Canale-Parola, 1981), rumen spirochete strains 6A,CA and PB (Paster and Canale-Parola, 1982), pectinolytic oral spirochete P5 (from E. Ca­nale-Parola; Paster and Canale-Parola, 1982), Treponema hyodysenteriae 94A 007 (fromT.Stanton; Hespell and Canale-Parola, 1971), T. succinifaciens 6091 (Cwyk and Canale­Parola, 1979) T. bryantii B2-5 (from M. P. Bryant; identical in catalog to T. bryantii RUS-1;Stanton and Canale-Parola, 1980), T. phagedensis Reiter (from]. B. Baseman; Hespell andCanale-Parola, 1971), T. denticola TDlO (Hespell and Canale-Parola, 1971), free livingspirochete strain N1R (from K.Stetter, unpublished), Borrelia hermsii B31 (ATCC 35209from R. C.]ohnson; Kelly, 1976), Leptospira illini 3055 (from M. Charon; Hanson et aI.,1974), L. patoc stt. Patoc (from M. Charon), and L. interrogans B16 (from C.D. Cox;Baseman and Cox, 1969) (Since the last two Leptospira species yielded nearly identicaloligonucleotide catalogs, in their subsequent discussion we will use only L. patoc to re­present both species). The catalogs of the anaerobic halophiles, Haloanaerobium praevalens(DSM 2228) and Halobacteroides halobius (ATCC 35273), are published separately (Orenet aI., 1984a, b).

Extraction of 16S rRNA and generation of the oligonucleotide catalogs followed eitherof two standard procedures (Uchida et aI., 1974; Woese et aI., 1976; Stackebrandt et aI.,1982). The analysis of catalogs in terms of Sab values is described in Fox et al. (1977).

Results and Discussion

As judged by rRNA sequence comparisons, the spirochetes would appear to bea particularly ancient group of eubacteria (Fox et aI., 1980). The group as a wholeforms one of about ten recognized major sublines of eubacteria, e.g., the Gram­positive bacteria, the purple photosynthetic bacteria and relatives (Gibson et aI.,1979), the cyanobacteria (Bonen et aI., 1978), etc. (Fox et aI., 1980; Woese et aI.,in preparation). Within the greater spirochete group, the main cluster (see below)alone is characterized by lowest Sab values in the 0.30 range - which makes it asdeep (ancient) as the cyanobacteria and almost as deep as the clostridia (Fox et aI.,1980). The depth of the leptospira cluster appears equally great. And the relationshipbetween these two clusters is sufficiently remote that the customary numeric analysisof the data (Fox et aI., 1977; 1980) does not bring it out. Moreover, at this samelevel, the greater spirochete group also appears to include a third subgroup oforganisms, the recently recognized obligate anaerobic halophiles, the Haloanaero­biaceae (Oren et aI., 1984 a, b) - a relationship again, that is not apparent from thestandard numeric analysis.

The Coherence of the Greater Spirochete Group

Table 1A shows the highest 30 or so Sab values (Fox et aI., 1977; 1980) for theleptospiras, for the anaerobic halophiles, and for B. hermsii and T. hyodysenteriaewith other eubacteria; while Table 1B shows their average Sab values with thevarious eubaeterial groups (with which they have the higher Sab values). While it isclear that B. hermsii and T. hyodysenteriae (the two spirochetes forming the deepestbranchings within the main spirochete cluster - see below) are a part of the maincluster of spirochetes, the relationship of all spirochetes to the leptospiras or to theanaerobic halophiles does not emerge from this analysis (nor does a specific rela­tionship between the last two groups). Note too, that 1. iilini and H. prevaelens

340 B.]. Paster, E. Stackebrandt, R.B.Hespell, C.M.Hahn, and C.R. Woese

Table 1. Sab values (Fox et aI., 1977) for the leptospiras, the anaerobic halophiles, B.hermsii and T. hyodysenteriae with other eubacteria. A. - thirty or so highest Sab valuesin each case. B. - average Sab values with those groups of eubacteria that yield the higheraverage values. Abbreviations: B-cyanobacterial group -14 examples (Bonen and Doolittle,1978; Fox et aI., 1980); D - sulfate reducing group - 16 examples (Stackebrandt et aI.,mss in preparation); G - Gram positive bacteria - 161 examples (Fox et aI., 1980; andunpublished catalogs); L - leptospira; H - anaerobic halophiles (Oren et aI., 1984a,b);R - purple bacteria - 103 examples (Gibson et aI., 1979; Fox et aI., 1980; and unpublished);S - spirochetes - 15 examples. The table is constructed from the calculations of Sab valuesby G. E. Fox, based upon his compilation of the published and unpublished rRNA catalogsfrom the laboratories of C.R. Woese and E.Stackebrandt. Numbers preceeding groupdesignations indicate the number of catalogs in the group having that Sab value.

A. highest Sab values

Sab L. patoc L. illini H. halob. H. prevael. B. hermsii To hyodys.---403938 G 2S3136 G3534 2G33 G,R 2G R,2S32 3G 6G 5G31 4G,2R 8G 3G30 3G,S S 18G,D '(G 2R29 l1G,2R,D H 1G,3R,S R,2S28 L 4R,3S21 2G, R,2S,D26 G,S 2G,R,5S,D25 1G,2B24 G.B,S llG,B,3S23 G,1R 13G,B,R,S,D22 5G,B,5R,D21 10G,3B,8R,S

B. average Sab values

G 18.3 24.9 20.4 26.8 24.4 21.5Il 19.2 20.8 21.4 23.3 21.3 18.6R 18.3 24.4 16.7 20.4 23.0 22,3S 11.1 24.6 21.1 21.2 21.4 26.1D 1'{ .4 24.5 16.1 23.6 22.3 23.8

L 28 28 16.0 21.0 23.0 19.0H 16.5 20.5 29 29 21.5 19.0

have higher Sab values with a number of outgroup catalogs than they do with theirspecific relatives (L. patoc and H. halobius respectively). This leads in the lattercase to a failure to group the two halophiles with one another by the standardnumeric analysis (Fox et aI., 1977; Fox, unpublished calculation).

Failure of this method to cluster organisms that are actually related can occurwhen one or more catalogs from a group of such organisms lack a higher thannormal fraction of the highly conserved (i. e. ancestral) oligonucleotides commonto most eubacterial catalogs (Woese et aI., 1980; Woese et aI., in preparation) - over80% of which are found in any typical eubacterial catalog (Woese, 1984). Thenormal catalogs in the group will then receive a larger contribution to Sab values

The Phylogeny of the Spirochetes 341

from many outgroup catalogs than from these deficient catalogs within their ownphylogenetic unit. Unless this contribution from shared ancestral sequences iscompensated by a greater contribution from shared derived sequences, Sab valueswithin such a group can be less than some Sab values with outgroup catalogs.Table 2 lists some highly conserved, ancestral sequences. If all are present in acatalog, they would contribute about 180 bases (or about 0.33) to the Sab values.Since the catalogs of Bacillus species, for example, contain almost all of the sequenceson this list (Woese, 1984), Sab's with these species would tend to be relatively highdue merely to the shared ancestral contribution.

Table 2. Occurrence of high frequency, "ancestral" sequences in the catalogs of 1. patoc(Lp), 1. illini (Li), H. halobius (Hh), H. praevalens (Hp), S. halophila (Sh), S. stenostrepta(Ss), and T. hyodysenteriae (Th). "+" indicates presence of the oligonucleotide in a givencatalog, "0" its absence. Alternative bases in a sequence are indicated by "I" - e.g. (CIA)indicates either a C or an A at that position. The alternative occurrences are indicatedsimilarly, e.g. 01+. "Y" stands for a pyrimidine, "R" for purine. For each column thesum of bases in those sequences present is given.

Sequence Lp Li Hh Hp Sh Ss Th--------------------------------------------------------

CACAAG + + + + +UAAACG 0 0 + + + + +AAUACG + + + + + + +UUCCCG + + 0 + + + +AUCCUG + + + + + + 0UAAUCG + + + + + + +

CAAACAG 0 0 0 0 + +CAACUCG + + 0 + 0 0 0UAAUACG + + 0 + 0 0 +

CCACAYUG 0 + + 0 + 0 +AUACCCUG 0 0 0 + + +CUACAAUG + + + + + 0 +

UC( C/ A) UCAUG +/0 0 0/+ +/0 0/+ 0/+ +/0(G/C)CCCUUAYG 0 0 0/+ U/+ +/0 +/0 +/0

UACACACCG + + + + + + +CUACACACG 0 + + + + + +CHACCAAG + + 0 0 + +* 0ACUCCUACG 0 + + + + 0 +CUAACUACG + + 0 + 0 0 +CUAAUACCG + + 0 0 0 0 0UUUAAUUCG + + + + + + +

AAACUCAAAG + + + + + + +AACCUUACC(A/U)G 0 0 +/0 0 +/0 0/+ 0

no. of bases 65 78 65 74 83 70 78

One way to circumvent this problem and bring out the true phylogenetic relation­ships in such cases is through a "signature" or set of sequences that are shared bymembers of the group but found sparingly if at all in putative outgroups (Woeseet aI., 1980). Table 3 includes such a set of sequences for the greater spirochetegroup. Relatives of the first sequence in the table can be found in most eubaeterialcatalogs (G.E.Fox, unpublished compilation of the data from the laboratories ofC.R. Woese and E.Stackebrandt). In all such cases (except one), however, thesequence in question is ten, not twelve, nucleotides in length (i. e. UCACACYAYG).[The one exception is of length twelve, but ends uniquely, i.e.... CAAG.] Since

342 B.]. Paster, E. Stackebrandt, R.B. Hespell, C. M.Hahn, and C. R. Woese

Table 3. Oligonucleotide signature linking the members of the greater spirochete group.Abbreviations as in Table 2 caption. The modification of a nucleotide is signified by alower case letter. "Spiro" column refers to the 17 spirochetes and treponemes in this study."Other" refers to the approximately 300 other eubacterial catalogs (as compiled by G. E.Fox, see above). Numbers in columns are the number of cases in which the sequence inquestion occurs.

sequence Lp Li Hh Hp Spiro. Other Comments(11) ( 300)

-------------------------------------------------------------------UCACACYAYCYG + + + 16 0UCACACCAAUCG + 0 0

UUUUAAG 5 2UCUUAAG 11 1UCUUAAACAUG + + 0 0

AAUCUUG + + 0 13AAUCUUCCR •. + + 9 40AAUAUUCCR •• 8 0

AACUAACG + + 0

AAAAACCUUACC •. + + 5

AUACCCCG + + + 0 7

CCCUAAACG + + 0 36 (27 in R)

CCUUUAUG + + 0 10

AUUAAG + + 15

CYAAUUACG + 14 0

AAUACCAAUG + 0

UACCUUUG + 0

ACUCUAAG + + 0 5

AAAUACG + + 0

AUACUAG + + 0 16

UAUCCCG + + 0 13 (11 in B)

UUAACG + + 0 12

AUUACG + + 0 0

cCcG + + + 0 0

this particular oligonucleotide changes in sequence only rarely, the fact that allmembers of the greater spirochete group possess this unique dodecamer versionof it, is likely to represent common ancestry rather than convergence. [No relativecan be found in one of the spirochete catalogs - ostensibly an experimental error.]This sequence alone constitutes a strong suggestion that the greater spirochetegroup as defined is phylogenetically coherent. Other sequences in Table 3 supportsuch a thesis: The second entry (family) comprises two obviously related sequences,

The Phylogeny of the Spirochetes 343

UYUUAAG - which covers all spirochete examples (except the borrelia) and isfound elsewhere among eubacteria only in three scattered examples - and UCU­UAAACAUG - which occurs only in the two leptospiras. The sequence AACUAACGoccurs only in the two leptospiras and B. hermsii. AAAAACCUUACC ... , a rarevariant of the near universal sequence ... GAACCUUACC. .. , is found in bothleptospiras and T. hyodysenteriae. And AUUAAG, an infrequently occuring hexam­er occurs in the leptospiras and in one spirochete. Coincidences among rare-occur­rence sequences such as these are deemed meaningful because the rRNA in a lineof descent is characterized not only by certain oligonucleotide sequences but by thepattern of allowable variation in these sequences (Woese et al., in preparation).

Additional sequences supporting the relationship of the anaerobic halophiles tothe spirochetes are few, but striking in quality. CYAAUUACG is a rare variant ofa highly conserved sequence CYAACUACG (Woese, 1984), which is confined solelyto the spirochetes and H. halobius. AAUACCAAUG occurs only in this halophileand one of the spirochetes among all the eubacterial catalogs. Also the modifiedoligonucleotide cCcG is found in one modification or another in all eubacterialcatalogs (Woese et al., unpublished); however, only in the three examples cited inTable 3 is the third C residue modified in this particular way (which gives the oligo­nucleotide a unique mobility on the primary electrophoretogram) (Uchida et al.,1974; Woese et al., unpublished). No comparable collection of rare (derived) se­quences can be assembled that would relate any of these major sublines of thegreater spirochete group to some eubacterial outgroup. Therefore, the data suggestthat the spirochetes, leptospiras, and anaerobic halophiles constitute a coherent andvery ancient phylogenetic unit: an eubacterial "phylum". The point is being furtherexplored through full sequencing of the appropriate 16S rRNA genes (unpublished).

The Leptospiras

While the Sab value between the two leptospira species (Table 1) is the highestone seen for L. patoc, this is not so for L. illini. The reason for this discrepancy isseen in Table 2. The table contains four sequences that do not contribute to theSab between the leptospiras - i. e. CCACAYUG, AUACCCUG, CUACACACG, andACUCCUACG - but would contribute to the Sab between L. illini and almost anyother eubacterial catalog - a total contribution of 0.06-0.07 to the Sab value. Inaddition, the L. illini catalog contains three more oligonucleotides that are notshared with the L. patoc catalog (i.e. UCCACG, UAACACG, and AUAAACCG)but that occur with high frequency in some of the major eubacterial groups, andwould further increase the Sab values with L. illini in these cases. By eliminatingsequences such as these (which have no phylogenetic significance) from considera­tion - as Table 3 does - the specific relationship between the two leptospiras appearsclearer and stronger.

The Anaerobic Halophiles

The specific relationship between H. halobius and H. prevaelens is, as mentionedabove, more difficult to detect than is that between the leptospiras. Table 2 containsseven sequences that can contribute to the Sab value between one or the other ofthese two organisms and a variety of outgroup catalogs, but do not contribute 'tothe Sab value between the two themselves - which readily accounts for the factsthat H. halobius has a higher Sab with S. halophila than with H. prevalens, and

344 B.J.Paster, E.Stackebrandt, R.B.Hespell, C.M.Hahn, and C.R. Woese

that this last has higher Sab values with a number of Gram positive species thanwith H. halobius. The signature that relates the two anaerobic halophiles to oneanother is included in Table 3 as well (Oren et al., 1984b).

The Spirochete-Treponeme Cluster

The main subgroup of the greater spirochete group comprises all species in thisstudy except T. hyodysenteriae and B. hermsii (and the leptospiras and anaerobichalophiles, of course). This large cluster subdivides into two subclusters, and variousfurther clusterings occur within these. Table 4 is a signature that defines the two

Table 4. Oligonucleotide signature defining and dividing the main spirochete-treponemecluster. Some abbreviations are defined in the Table 2 caption. The organisms are abbre­vited as follows: Sh - S. halophila, Sa - S. aurantia, Sl - S.litoralis, Si - S. isovalerica,SN - strain NIR, Ss - S. stenostrepta, Sz - S. zuelzerae, Td - T. denticola, Tp - T. phaga­densis, Ts - T. succinifaciens, Tb - T. bryantii, os - oral strain, strains 6A, CA, and PB(so indicated), Th - T. hyodysenteriae, and Bh - B. hermsii. "Other" refers to the 300 orso catalogs from other eubacteria; (see Table 3 caption). In the "comments" columnsequences are distinguished by whether they are presumably ancestral (An) or derived(Dr), and sequence variants, indicated by" in the table, are defined.

sequ~nce S S S S S SST T T T 0 6 CPT Bothhal iNs z d P s b s A A B h h er

comments

A.UCUUAAG + + + + + + + + + + + 1UUUUAAG + + + + + 2

CCACAYUG + + + + + + + 252 AnRCACAYUG + + + + + + + + 4 Dr

ACUCCAUG + + + + + + + 39 AnACUUCAUG + + + + + 4 Dr

AAUCUUCCR + + + + + • + +1. + + 41 AnAAUAUUCCR + + + + + .?+ + + 0 Dr

RAACCUUACCAG + + + + + 111 (GAAC .. )RAACCUUACCUG + + + • + + + + + + 51 (GAAC .. )

•• •RAAUC .•UCAcACCACCCG + + + + + 0UCAcACCAUCCG + 0UCACACCAUCCG + + + + + + + + + • 0 •.. CACUA..

CAACCCCUAUUG + + + 6CAACCCCUACYG + + + + + + + + + 2

B.ACUCCG + + + +

uAACACG + + + + + +

CAAUACG + + + +

(C,U)UAACCCG + + + +

IJCCCAUUAG + + +

ACAUC .••.. + + + +ACAUACA ... + + + + +

The Phylogeny of the Spirochetes 345

subclusters in question as well as some internal structure within the larger of thetwo. It will be noticed that the divisions are not along the lines of the genera Spiro­chaeta and Treponema as currently defined (Smibert, 1974).

The primary division of the group places S. halophila, S. aurantia, S. litoralis,S. isovalerica, and (peripherally) strain N1R into one cluster, and S. stenostrepta,S. zuelzerae, T. phagadensis, T. denticola, T. succinifaciens, T. bryant;i, the oralstrain, and strains CA, 6A, and PB into the other. Within the second (larger) ofthese clusters, the first four strains (S. stenostrepta and so on) separate, at a finerlevel, from the last six (T. succinifaciens et seq.). Further substructuring has notbeen attempted in Table 4, but Table 5 allows one to see all these relationships interms of Sab values (Fox et aI., 1977) as well as a specific relationship between thepairs S. stenostrepta-S. zuelzerae, T. denticola-T. phagadensis, T. bryanti-oral iso­late, and strain CA-strain 6A, and also the grouping of strains CA, 6A, and PBspecifically with one another.

Table 5. Sab values among the spirochetes and leptospiras (expressed as percentages) ­taken from the unpublished compilation and calculations of G.E.Fox (see above). Abbre­viations are self-evident.

1 S. halo2 S. auran3 S. litor4 S. isov5 str N1R

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19x

49 x45 49 x45 49 58 x38 38 40 37 x

6 S. steno7 S. zuel8 To dent9 To phag

10 To succ11 To bryan12 oral str13 str 6A14 str CA15 str PB

44 43 39 38 3931 30 34 34 3426 30 25 29 2533 35 31 33 2637 32 32 31 4235 34 31 28 3734 35 30 29 3341 37 30 31 3443 38 32 33 3537 36 30 29 30

x56 x42 48 x48 50 54 x54 42 26 37 x51 46 34 41 50 x43 40 33 34 42 47 x47 40 30 38 56 46 38 x53 45 35 42 55 50 42 73 x44 37 32"36 41 4040 49 59 x

16 B. herm 38 33 38 33 25 27 23 24 24 24 22 25 26 29 26 x

17 T. hyo 27 28 28 26 29 29 27 26 26 25 25 26 26 25 28 22

18 L. patoc 21 18 20 20 24 17 16 13 15 17 18 17 17 17 14 19 17 x19 L. illin 30 27 25 26 26 25 20 1'{ 18 27 23 25 28 26 23 27 21 28 x

Borrelia hermsii

While B. hermsii has its highest Sab values with certain spirochetes (e.g. S. halo­phila) - and so would cluster with them (see also Table 1B) - it has higher Sabvalues with many of the low G + C Gram positive eubacteria than it does with themajority of the spirochetes (Table 1), again for the reason discussed in the previoussections. Table 6 includes the collection of all oligonucleotides from the B. hermsiicatalog that occur at least once elsewhere (in the groups considered in the table)and are not widely distributed among the major eubacterial "phyla" (i.e. are notancestral characters). These sequences in the B. hermsii catalog are most often and

23 Systematic and Applied Microbiology, Vol. 5

346 B.J.Paster, E.Stackebrandt, R.B.Hespell, C.M.Hahn, and C.R. Woese

Table 6. Sequences found in the B. hermsii catalog that occur in at least one other catalogamong eubacteria, but are not widely distributed among the major eubacterial groups.Abbreviations: lp - leptospiras (2 examples); an - anaerobic halophiles (2 examples);sp - spirochetes (15 examples, i. e. excluding T. hyodysenteriae); G+ - Gram positivegroup (161 examples); cy - cyanobacteria (14 examples); pb - purple bacterial group (103examples); gb - green photosynthetic bacterial group (5 examples); db - cytophaga-flavobacter-bacteroides group (22 examples). Numbers are percent of organisms in a groupwhose catalogs contain the sequence in question.

sequence lp an sp G+ cy pb gb cfb2 2 15 161 14 103 5 22

--------------------------------------------------------------AAACUCG 50 a 54 6 29 8 a 0AUCACCG a 0 a a a 4 a aAUACCCG a a 27 0 a a 14 5UACAACG a a a 20 0 0 a aCAUAAAG a a 14 2 a 1 a aUUACCAG a a 7 3 a 10 a aAUUCCAG a 0 0 a 0 1 0 0UAUACAG a a 14 5 0 2 0 aUCAAUUG a 0 0 0 a 1 a aUUAAUUG 0 a a 1 0 0 0 0UUAAUCG 0 0 a a a 74 a 0

AACUAACG 100 a a a a 0 0 0CACACUUG a 0 7 a a a a aUCACACUG o 50 a 4 4 10 0 0ACUUCAUG a a 27 1 a 1 a aUAUAUCAG 0 a a a a a a 5

AAUCUUCCG o 50 40 15 a a a 0CUAAUUACG a 50 73 a a a a a

ACAUAUACAG 0 a 7 a 0 0 0 a

CAACCCUUAUUG 0 a a 7 a a a 0UCAc ACCACCCG 50 50 27 a a a 0 a

most highly represented in the main spirochete group, which encompasses elevenof them. The eleven collectively show no pronounced phylogenetic bias within themain spirochete cluster, however, and they are widely distributed among the spiro­chete species. Only one of the 16 spirochete catalogs, that of T. hyodysenteriae(significantly - see below), contains none of them, and eleven contain two or moreof them. B. hermsii can thus be considered to represent a deep branching in theline of descent that gave rise to the main spirochete cluster.

T. hyodysenteriae

T. hyodysenteriae is the only species of Spirochaeta or Treponema examinedthat is excluded from the main spirochete-treponeme group. This could reflecteither true phylogenetic distance or a "fast clock" in that line of descent (Woese,1984). The latter explanation does not appear correct, in that Tables 1 and 5 showT. hyodysenteriae to be no more distant from outgroup species such as the lepto­spiras than are the other spirochetes and treponemes. Table 7 is a collection ofsequences supporting the thesis that T. hyodysenteriae is phylogenetically outsideof the main spirochete-treponeme cluster. Three of the four sequences listed therein

The Phylogeny of the Spirochetes 347

Table 7. Sequences indicating that T. hyodysenteriae is outside of the main spirochete-tremponeme cluster. Abbreviations as defined in previous table captions.

sequence T.hyo B.herm Lepto Halo Spiro comment::>

._---------------------------------------------------------AAAAACCUUACC •. 1 0 2 0 0

AACCUUACC .• 0 1 0 2 9 AnAAACCUUACC .. 0 0 0 0 5

uAAUACG 1 1 2 1· 0 An-UAAUACG; ·-not moduAACACG 0 0 0 0 6

CUAACUACG 1 0 2 2 AnCYAAUUACG 0 1 0 13

RCUAAUACCR 1 1 2 1 0 An-CUAAUACCGRUAAUACCG 0 0 0 0 12

50

30

20

Fig. 1. Phylogenetic tree for the greater spirochete group. Branching points are in accordwith Sab values and the signatures discussed above. [Sab values are given as percentages.]

348 B.J.Paster, E.Stackebrandt, R.B.HespelI, C.M.Hahn, and C.R.Woese

- i.e. uAAUACG, CUAACUACG, and (R)CUAAUACCG - are ancestral typesfound in the T. hyodysenteriae catalog but existing as derived versions only in themain cluster of the spirochetes (italics mark the position that is altered in thespirochete examples). [Table 2 shows the T. hyodysenteriae catalog as well not tolack a disproportionate number of the highly conserved sequences that are possessedby other spirochete and treponeme catalogs - which would be a symptom of arapidly evolving line of descent (Woese, 1984).] While none of this directly provesour case, it makes unlikely the alternative that the deep branching of T. hyodysente­riae results from a "fast clock" in its particular line of descent.

Fig. 1 is a schematic representation of the relationships deduced here in tree form.The order of branching accurately reflects the branchings deduced from the varioustables above.

The phylogeny depicted in the figure is consistent with much of the current pheno­typic information on these organisms. Unlike the other spirochetes studied, theleptospiras are aerobic organisms that catabolize long-chain fatty acids for energyand carbon, and have one or both ends of the cells hooked. The Leptospira speciesserovars illini and interrogans have G + C contents of their DNA's of 53 mol%and 37 mol%, respectively. On the basis of the many and varied phenotypic differ­ences between them Hovind-Hougen (1979) has suggested that the genus Leptospirabe excluded from the family Spirochaetaceae and instead be placed in a new familyLeptospiraceae. This family would have two genera: Leptospira and Leptonema,with L. interrogans and L. illini, respectively, as type species. The present resultsare certainly in agreement with such a classification.

By oligonucleotide analysis, a cluster of spirochetes can be defined and could beconsidered as the genus Spirochaeta. The members of this cluster include S. halo­phila, S. aurantia, S. litoralis, S. isovalerica, and spirochete strain N1R, but wouldexclude S. stenostrepta and S. zuelzerae. The first five spirochetes have, whereexamined, common phenotypic traits: two periplasmic flagella per cell; high G + Ccontent of their DNA's (50.5 mol% to 55.8 mol%); fermentative metabolism ofsugars to similar end products (C02, H2, ethanol, acetate); a clostridial-type ofpyruvate clastic reaction; presence of ferredoxins and/or rubredoxins (see Canale­Parola, 1976; Harwood and Canale-Parola, 1983); and, except for S. aurantia, a saltrequirement for growth (Smibert, 1974). [However, free-living pigmented, faculta­tively anaerobic marine spirochetes have been isolated (C.Harwood, personalcommunication) which may be related to or are salt tolerant strains of S. aurantia.]

On the other hand, spirochete species S. stenostrepta and S. zuelzerae currentlyin that genus, by oligonucleotide analysis belong to a larger cluster of spirochetesdominated by rumen spirochetes and treponeme strains - see Fig. 1. This largercluster is somewhat heterogeneous with respect to the types of organism (and theirphenotypic traits) encompassed, but can be subdivided into lines defined both byRNA cataloging and G + C content of the DNA's. The highest G + C (56 to 60mol%) subline is represented by species S. stenostrepta and S. zuelzerae, the inter­mediate G + C (41 to 46 mol%) (Canale-Parola et al., 1968) by rumen spirochetestrains 6A, CA, and PB (Paster and Canale-Parola, 1982), and the lowest G + C(35 to 38 mol%) by the remaining members of this cluster. However, this last sublineis further divided into two groups by RNA cataloging, which subclustering is inturn, supported by some differences in phenotypic traits. T. bryantii and T. suc­cinifaciens are carbohydrate fermenters and grow on rather simple media (Cwzkand Canale-Parola, 1979; Stanton and Canale-Parola, 1979), whereas T. denticola

The Phylogeny of the Spirochetes 349

and T. phagedensis can ferment amino acids and require complex, serum-containingmedia for growth (Hespell and Canale-Parola, 1971). Whether this large cluster ofthree different G + C ranges should be considered as a single genus "Treponema"is for now a moot point.

Using DNA-rRNA hybridization methods, T. hyodysenteriae strains have beenshown to be highly related to one another, but not to T. phagedensis or T. pallidum(Miao et al., 1978). By RNA cataloging, T. hyodysenteriae is a distinct spirochetalevolutionary line and only related to the other spirochete groups at a deep level(Fig. 1, Table 4). The data clearly indicate that T. hyodysenteriae should not beconsidered as part of the genus Treponema. We feel that this organism may repre­sent an entirely new undesignated spirochaetal genus, with the caveat that its rela­tionship to Cristispira remains unknown.

Currently, the borrelia are defined as loosely-coiled, microaerophilic spirochetesthat are pathogenic for mammals and birds, being transmitted by insect vectors.Recent studies have shown these organisms to have a low DNA G + C content(27-31 mol%) and a high degree of DNA-DNA homology (59-100%, by hybridiza­tion studies) amongst themselves, but little if any DNA homology with the trepo­nemes and leptospiras (R. C.]ohnson, personal communication). These findings,along with the present study, clearly indicate that the borrelia are a distinct branchof spirochetes.

In summary, RNA cataloging has shown the spirochetes to be a phylogeneticallycoherent group of organisms that are of ancient origin, one of about ten majorsublines ("phyla") of eubacteria. However, the phylogenetically derived evolutionarylines of spirochetes are not entirely consistent with the current determinative taxo­nomic schemes found in Bergey's Manual (Smibert, 1974). Phenotypic taxonomiccriteria such as gross cell morphologies, numbers of periplasmic flagella, free-livingvs host association, G + C content of DNA, or pathways of energy metabolism(when used individually) do not provide a phylogenetically valid taxonomy forspirochetes.

Acknowledgements

We thank G.E.Fox for calculating Sab values and preparing the compilation of oligo­nucleotide catalogs we used to construct the tables herein. CRW is supported by a grant,DEB-8107061, from the National Science Foundation. ES was financed by the Gesellschaft£tir Biotechnologische Foschung to support the German Collection of Microorganisms.BJP was supported in part by a grant from the University of Illinois Agricultural Experi­ment Station (ILLU 35-362).

References

Azuma, 1., Taniyama, T., Yamamura, Y., Yanagihara, Y., Hattori, Y., Yasuda, S., Mi­fuchi, 1.: Chemical studies on the cell walls of Leptospira biflexa strain Urawa andTreponema pallidum strain Reiter. Jap. J. Microbiol. 19,45-51 (1975)

Baseman, ]. B., Cox, C. D.: Intermediate energy metabolism of Leptospira. J. Bact. 97,992-1000 (1969)

Breznak, ]. A., Canale-Parola, E.: Spirochaeta aurantia, a pigmented, facultatively an­aerobic spirochete. J. Bact. 97, 386-395 (1969)

350 B.J.Paster, E.Stackebrandt, R.B.Hespell, C.M.Hahn, and C.R.Woese

Bonen, I., Doolittle, W. F.: Ribosomal RNA homologies and the evolution of the fila­mentous blue-green bacteria. J. Molec. Evo!. 10, 283-291 (1978)

Canale-Parola, E.: Physiology and evolution of spirochetes. Bact. Rev. 41, 181-204 (1977)Canale-Parola, E., Udris, Z., Mandel, M.: The classification of free-living spirochetes.

Arch. Mikrobiol. 63, 385-397 (1968)Canale-Parola, E., Holt, S. Coo Udris. Z.: Isolation of free-living, anaerobic spirochetes.

Arch. Mikrobiol. 59. 41-48 (1976)Cwyk, W. M.,Canale-Parola, E.: Treponema succinifaciens sp. nov., an anaerobic spiro­

chete from the swine intestine. Arch. Microbiol. 122 231-239 (1979)Fox, G.E., Pechman. K.]., Woese, C.R.: Comparative cataloging of 165 ribosomal ribo­

nucleic acid: molecular approach to procaryotic systematics. Int. J. 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., Magrum, I.]., Zablen, I. B., Blakemore, R., Gupta, R.,Bonen, I., Lewis, B.]., Stahl, D.A., Luehrsen, K.R., Chen, K.N., Woese, C.R.: Thephylogeny of prokaryotes. Science 209, 457-463 (1980)

Gibson, ]., Stackebrandt, E., Zablen, I. B., Gupta, R., Woese, C. R.: A phylogenetic analysisof the purple photosynthetic bacteris. Curr. Microbiol. 3, 59-64 (1979)

Greenberg, E. P., Canale-Parola, E.: Spirochaeta halophila, sp. N., a facultative anaerobefrom a high salinity pond. Arch. Microbiol. 110, 185-194 (1976)

Hanson, I. E., Tripathy, D. N., Evans, I. B., Alexander, A. D.: An unusual leptospira,serotype illini (a new serotype). Int. J. system. Bact. 24,355-357 (1974)

Harwood, C. S., Canale-Parola, E.: Branched-chain amino acid fermentation by a marinespirochete: Strategy for starvation survival. J. Bact. 148, 109-116 (1981)

Harwood, C. S. and Canale-Parola, E.: Spirochaeta isovalerica sp. nov., a marine anaerobethat forms branched-chain fatty acids as fermentation products. Int. J. system. Bact. 33,573-579 (1983)

Hespell, R. B., Canale-Parola, E.: Spirochaeta litoralis sp. N., a strictly anaerobic marinespirochete. Arch. Mikrobiol. 74, 1-18 (1970)

Hespell, R. B., Canale-Parola, E.: Amino acid and glucose fermentation by Treponemadenticola. Arch. Mikrobiol. 78, 234-251 (1971)

Hovind-Hougen, K.: Leptospiraceae, a new family to include Leptospira Noguchi 1917and Leptonema gen. nov. Int. J. system. Bact. 29,245-251 (1979)

Joseph, R., Holt, S. C., Canale-Parola, E.: Peptidoglycan of free-living anaerobic spiro­chetes. J. Bact. 115,436-435 (1973)

Kelly, R. T.: Cultivation and physiology of relapsing fever borreliae. In: R. C.]ohnson(ed.), The Biology of Parasitic Spirochetes, pp. 87-94. New York, Academic Press 1976

Miao, R. M., Fieldsteel, A. H., Harris, D. I.: Genetics of Treponema: Characterizationof Treponema hyodysenteriae and its relationship to Treponema pallidum. Infect. Im­mun. 22, 736-739 (1978)

Oren, A., Weisburg, W. G., Kessel, M., Woese, C.R.: Halobacteroides halobius gen nov.,sp. nov., a moderately halophilic obligatory anaerobic bacterium from the bottomsediments of the Dead Sea. System. Appl. Microbiol. 4,58-70 (1984a)

Oren, A., Paster, B.]., Woese, C.R.: Haloanaerobiaceae: A new family of moderatelyhalophilic, obligatory anaerobic bacteria. System. Appl. Microbiol. 4, 71-80 (1984b)

Paster, B.]., Canale-Parola, E.: Physiological diversity of rumen spirochetes. App!. En­viron. Microbiol. 43, 686-693 (1982)

Smibert, R. M.: The Spirochetes. In: Bergey's manual of determinative bacteriology (R. E.Buchanan, N. E. Gibbons, eds), 8th ed. Baltimore, Williams and Wilkins 1974

Stackebrandt, E., C. R. Woese: The evolution of prokaryotes. In: Molecular and CellularAspects of Microbial Evolution. Eds. M.]. Carlile, J. R. Collins, B. E. B. Moseley, pp. 1-31.Cambridge University Press 1981

Stackebrandt, E., Seewaldt, E., Ludwig, W., Schleifer, K.-H., Huser, B. A.: The phylo­genetic position of Methanothrix soehngenii Elucidated by a modified technique of

The Phylogeny of the Spirochetes 351

sequencing oligonucleotides from 16S rRNA. Zbl. Bakt. Hyg., I. Abt. Orig. C 3, 90-100(1982)

Stanton, T. B., Canale-Parola, E.: Enumeration and selective isolation of rumen spiro­chetes. Appl. Environ. Microbiol. 38, 965-973 (1979)

Stanton, T. B., Canale-Parola, E.: Treponema bryantii sp. nov., a rumen spirochete thatinteracts with cellulolytic bacteria. Arch. Mikrobiol. 127, 145-156 (1980)

Uchida, T., Bonen, 1., Schaup, H. W., Lewis, B. ]., Zablen, L. B., Woese, CR.: The use ofribonuclease U2 in RNA sequence determination. Some corrections in the catalog ofoligomers produced by ribonuclease T1 digestion of Escherichia coli 16S ribosomalRNA. J. Molec. Evol. 3, 63-77 (1974)

Veldkamp, H.: Isolation and characteristics of Treponema zuelzerae nov. spec., an an­aerobic free-living spirochete. Antonie v. Leeuwenhoek 26, 103-125 (1960)

Woese, C. R.: Archaebacteria and cellular origins: An overview. Zbl. Bakt. Hyg., I. Abt.Orig. C 3,1-17 (1982)

Woese, C.R.: Molecular perspectives on the relationship between tempo and mode inevolution. Science (submitted) 1984

Woese, CR., Sogin, M., Stahl, D.A., Lewis, B.]., Bonen, L.: A comparison of the 16Sribosomal RNAs from mesophilic and thermophilic bacilli: Some modifications in theSanger method for RNA sequencing. J. Molec. Evol. 7, 197-213 (1976)

Woese, C.R., Maniloff, ]., Zablen, L.B.: Phylogenetic analysis of the mycoplasmas. Proc.nat. Acad. Sci. (Wash.) 77,494-498 (1980)

Woese, CR., Gutell, R.R., Gupta, R., Noller, H.R.: A detailed analysis of the higherorder structure of 16S-like ribosomal RNAs. Microbiol. Rev. 47,621-669 (1983)

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