Vol. 171, No. 6

Pea Aphid Symbiont Relationships Established byAnalysis of 16S rRNAs

BRIAN M. UNTERMAN, PAUL BAUMANN,* AND DONALD L. McLEANt

Department of Microbiology, University of California, Davis, California 95616

Received 15 November 1988/Accepted 15 March 1989

The pea aphid (Acyrthosiphon pisum Harris) harbors two morphologically distinct procaryotic intracellularsymbionts. The genes for the 16S rRNA from these symbionts have been cloned and sequenced. Comparisonswith sequences of 16S rRNAs from selected procaryotes indicate that the two symbionts are evolutionarilydistinct from each other and are members of the 'y-3 subdivision of the class Proteobacteria. One of thesymbionts is a member of the family Enterobacteriaceae, while the other constitutes a lineage distinct from theseorganisms. Both symbionts appear to have only one copy of their rRNA operon.

The pea aphid (Acyrthosiphon pisum Harris), like many or

all plant sap-feeding members of the order Homoptera, isdependent for survival on obligate intracellular procaryoticsymbionts (4, 15). These endosymbionts are housed inspecialized cells (mycetocytes) which aggregate into organ-like structures (mycetomes) found in the hemocoel of theaphid. The pea aphid contains two endosymbionts desig-nated the primary (P) and secondary (S) symbionts (21). Theoval P symbiont is the predominant organism and is locatedin the mycetocytes. The rod-shaped S symbiont is associatedwith the sheath cells partially surrounding the mycetome.Both symbionts have a gram-negative cell wall and are

surrounded by a unit membrane derived from the hostcytoplasmic membrane (15).Although the function of the symbionts has not been

definitively established, their importance for the aphid isindicated by the elaborate host mechanisms developed toensure the infection of progeny (4, 15). In addition, treat-ment of the aphid with procaryote-specific antibiotics leadsto the decrease or elimination of the symbionts, with con-

comitant reduction in weight, fecundity, and longevity of theaphid (5). Evidence suggesting that the symbionts mayprovide the aphid with plant cell wall-degrading enzymes (8,10), essential nutrients (5), or a protein (symbionin) neces-

sary for nymphal development (17) has been presentedelsewhere.

Since the endosymbionts have not been cultivated outsidethe host aphid, speculation as to their identity has beenbased on their morphology and the moles percent guanine-plus-cytosine content of their DNAs (16, 18). It has beensuggested that the symbionts are related to the rickettsiasand chlamydias, two groups of obligate intracellular patho-gens, or to the mycoplasmas (15, 21). It has also beenproposed that the endosymbionts constitute an evolutionarystage which culminated in the formation of mitochondria (14,15).

In this study we establish the evolutionary affinities of theendosymbionts of the pea aphid by comparing the sequences

of their 16S rRNAs with those from selected procaryotes.This approach has been pioneered by C. R. Woese and his

* Corresponding author.t Present address: Office of the Dean, College of Agricultural and

Life Sciences, University of Vermont, Burlington, VT 05405.

collaborators (24, 29), who have applied it extensively tostudies of evolutionary relationship. It is particularly appli-cable to the study of unculturable procaryotes, since the 16SrRNA sequences are highly conserved and can be clonedand detected by hybridization with genes coding for rRNA(rDNA) probes from a different organism.

MATERIALS AND METHODS

Insect species. Apterous, parthenogenic pea aphids werereared on broad bean, Viciafaba L. cv. Windsor. Plants andaphids were maintained in a growth chamber at 27°C with a

16-h light and 8-h dark photophase. The aphids were re-

moved from plants and either they were immediately storedat -70°C or the mycetomes were dissected (21) and stored at-700C.

Bacterial strains. Escherichia coli C600 and C600 Hfl wereincluded in the XgtlO cloning system purchased fromPromega Biotec (Madison, Wis.). E. coli JM101 was pur-chased from Bethesda Research Laboratories, Inc., (Gaith-ersburg, Md.).

Insect DNA isolation. A 2-g portion (wet weight) of frozeninsects was pulverized in dry ice with a Waring blender.Grinding in dry ice minimizes the degradation of DNA byinsect nucleases. The finely ground powder was allowed tosublime at -20°C overnight and then was suspended in 10 mlof 50 mM Tris-hydrochloride, pH 8.0-40 mM EDTA-10 mgof lysozyme per ml. After incubation for 5 to 10 min at roomtemperature, 2 volumes of a solution containing 2% sodiumdodecyl sulfate, 400 mM EDTA, and 100 mM Tris-hydro-chloride, pH 8.0, was added. The suspension was incubatedat 50°C for 15 to 60 min. The DNA was subsequently purifiedby CsCl centrifugation by the procedure of Weeks et al. (28).This procedure routinely yields 2 to 5 mg ofDNA ranging insize from 30 to 60 kilobase pairs (kb).

General procedures. Most of the methods used in thisinvestigation have been described by Maniatis et al. (19).These include restriction endonuclease digestions, agarosegel electrophoresis, and DNA minipreparations of A and M13bacteriophage. The procedure of Hanahan (12) was used totransform competent E. coli JM101 cells with M13 bacterio-phage DNA. Enzymes and substrates were purchased fromBoehringer Mannheim Biochemicals (Indianapolis, Ind.),New England BioLabs, Inc. (Beverly, Mass.), and Promega

2970

JOURNAL OF BACTERIOLOGY, June 1989, p. 2970-29740021-9193/89/062970-05$02.00/0Copyright © 1989, American Society for Microbiology

16S rRNA PEA APHID SYMBIONT RELATIONSHIPS 2971->a b c d

--7< C)

100--~ ~4b--

0O 0 -- ew

3.4- W4- -, I23

X.9- 20

.. 1-

0.33

N

_. '

FIG. 1. Restriction enzyme and Southern blot analyses of DNAisolated from mycetomes dissected out of pea aphids (lane a, 1 ,ug ofDNA) and from whole aphids (lanes b through e, 7 ,ug of DNA) byusing an E. coli 16S rDNA probe. The following enzyme(s) was

used: HindIll (lanes a and b), EcoRI (lane c), EcoRI and Hindlll(lane d), EcoRI and EcoRV (lane e). Arrows indicate the bandsderived from the S symbiont. P-s, P symbiont; S-s, S symbiont.Numbers under these headings designate the fragment sizes inkilobases.

Biotec. All enzymes were used according to the directionsprovided by the manufacturers.

Southern blot and hybridization analyses. Following re-striction digestion, the DNA was electrophoresed on 0.8%agarose gels and electrophoretically transferred to Zeta-Probe membrane (Bio-Rad Laboratories, Richmond, Calif.)by the procedures recommended by the manufacturer. Thehybridization probe was the 16S rRNA gene from E. coli.This probe was prepared by subcloning a 2.9-kb PstI-XbaIfragment from pkk3535, a plasmid containing the E. coli rrnBrRNA operon (3), into M13mpl9. The M13mpl9 subclonecontains all of the E. coli 16S rRNA gene and part of thetRNAGIU found in the spacer region. The probe was radio-actively labelled to _108 cpm./4Lg by using a nick translationkit and [35S]dATP (Amersham Corp., Arlington Heights, Ill.)according to the instructions of the manufacturer. Theimmobilized DNA was hybridized for 48 h as described ininstructions by Bio-Rad. The radioactive probe was used at106 cpm/ml of hybridization buffer. The hybridization bufferalso contained 10% dextran sulfate (Sigma Chemical Co., St.Louis, Mo.). AR X-ray film (Eastman Kodak Co., Roches-ter, N.Y.) was exposed to the membrane for 48 h anddeveloped as recommended by the manufacturer.

16S rRNA gene cloning. Identical restriction fragmentshybridizing with the 16S rDNA probe were obtained whenthe source of the DNA was the whole aphid (Fig. 1, lane a)or the mycetome preparation (Fig. 1, lane b). Since DNAobtained from the latter source was more degraded, onlyDNA isolated from the whole aphid was used in subsequentcloning experiments. The DNA fragments indicated in Fig. 2(fragments a, c, d, and e) were purified by 1% agarose gelelectrophoresis. The region containing the specific fragmentwas excised from the gel, and the DNA was electroeluted byusing an Elutrap (Schleicher & Schuell, Inc., Keene, N.H.)according to the recommendations of the manufacturer.Fragment b (Fig. 2) was amplified from 2 ,ug of total DNA

by the TaqI polymerase (New England BioLabs) chainreaction by using rDNA-specific oligonucleotide primers bythe procedure of Saiki et al. (25). The conditions for thermal

P-symbiontEH V EE,. I I..lr I I 9J

V H EI

a V cS-symbiont b

E VH E H E,_ I 1 kb

d e

FIG. 2. Restriction map of the rDNAs of the P and S symbiontsof the pea aphid, based in part on the results shown in Fig. 1. Thethick bar designates the 16S rDNA. E, EcoRI; H, HindlIl; V,EcoRV. A through e, fragments which were cloned and sequenced(Fig. 3). Sizes (in kilobases) were as follows: a, 1.9; b, 0.33; c, 3.4;d, 2.3; e, 2.3.

cycling were as follows: denaturation, 1 min at 95°C; an-nealling, 2 min at 55°C; and polymerization, 5 min at 70°C. Atotal of 25 cycles were completed to yield 0.3 to 0.4 j.wg of thefragment. The amplified DNA was digested with EcoRI torelease the 330-base-pair fragment (Fig. 2).

Gel-purified fragments, a, d, and e were ligated directlyinto the EcoRI-cut and phosphatase-treated AgtlO (PromegaBiotec). Fragment c was first ligated to phosphorylatedEcoRI linkers (New England BioLabs), since the EcoRVrestriction enzyme produces a blunt end. Free linkers wereremoved by passage through a G-50 (Pharmacia, Uppsala,Sweden) spun column (19). The DNA was packaged in vitroby using the Lambda DNA Packagene System (PromegaBiotec) and plated on the indicator strain, E. coli C600 Hfl.Preparations routinely had 8.0 x 105 to 5.0 x 106 PFU/ml;-95% of the phage had recombinant DNA inserts of theappropriate size.The amplified fragment (fragment b, Fig. 2) was cloned

directly into M13mpl8 by the procedure of Messing (22). Inaddition, AgtlO clones of fragments, a, c, d, and e were allsubcloned into M13mp18 and M13mpl9 by the same proce-dure.

Detection of 16S rRNA recombinant clones in XgtlO. Ap-proximately 2.0 x 103 to 1.0 x 104 X bacteriophage particleswere plated on the host bacterial strain E. coli C600. Afterincubation for 7 to 8 h at 37°C, the petri plates were placedat 4°C for 15 min to harden the top agarose. The plates werenext overlaid with nitrocellulose filters (Schleicher &Schuell) which, after a brief incubation at room temperature,were carefully removed. Hybridization with the rDNA probewas performed as described above.DNA sequencing. Several approaches were used to obtain

the DNA sequence. These included the IBI Cyclone System(International Biotechnologies, Inc., New Haven, Conn.) togenerate a series of overlapping deletions, 16S rRNA uni-versal primers (Boehringer Mannheim), and primers synthe-sized according to specifications. In all cases the dideoxy-nucleotide method was used with [35S]dATP and theprocedures of Sanger et al. (26). Reaction mixtures preparedwith a 35S sequencing reagent kit (Pharmacia) were electro-phoresed on 6% polyacrylamide gels (Boehringer MannheimBiochemicals) and fixed in 10% acetic acid. Sequencing gelswere subsequently dried for 1 h at 80°C and autoradio-graphed for 18 h at room temperature with AR X-ray film(Eastman Kodak). Both strands were sequenced at leastonce.

Determination of evolutionary distance and tree construc-tion. The nucleotide sequences of the P and S symbiontswere aligned to other 16S rRNA sequences (6). The evolu-tionary distance was estimated for each pair of sequences bythe procedure of Distel et al. (7). By this method, the average

VOL. 171, 1989

2972 UNTERMAN ET AL.

TABLE 1. 16S rRNA sequence similarities in and evolutionary distances between pea aphid symbionts and selected free-living bacteria'

y Esche- Proteus monas grium Rocha- Myxo- Desulfo- Chla- Myco- Methano-Organism richia .. .- limaea coccus I mydia Ll plasma coccusbiont biont vulgaris testos- tumefa- esu- . subtiliscoli teroni ciens quintana xanthus furicans psittaci capricolum vannielli

P symbiont 0.082 0.098 0.099 0.203 0.268 0.276 0.214 0.224 0.284 0.249 0.273 0.559Escherichia coli 0.899 0.034 0.045 0.179 0.242 0.253 0.198 0.198 0.272 0.223 0.253 0.533S symbiont 0.881 0.948 0.045 0.189 0.239 0.252 0.206 0.209 0.289 0.229 0.261 0.548Proteus vulgaris 0.876 0.933 0.925 0.185 0.241 0.250 0.203 0.223 0.280 0.227 0.260 0.552Pseudomonas tes- 0.784 0.810 0.802 0.804 0.275 0.280 0.221 0.233 0.287 0.235 0.246 0.510

tosteroniAgrobacterium 0.734 0.751 0.752 0.754 0.739 0.059 0.248 0.257 0.330 0.256 0.286 0.528

tumefaciensRochalimaea quin- 0.727 0.745 0.747 0.748 0.733 0.928 0.260 0.272 0.337 0.262 0.311 0.534

tanaMyxococcus xan- 0.768 0.780 0.773 0.778 0.769 0.747 0.740 0.195 0.265 0.191 0.233 0.529

thusDesulfovibrio desul- 0.759 0.779 0.774 0.766 0.768 0.753 0.739 0.789 0.253 0.195 0.254 0.533furicans

Chlamydia psittaci 0.722 0.734 0.723 0.732 0.733 0.694 0.693 0.730 0.737 0.264 0.258 0.536Bacillus subtilis 0.742 0.761 0.761 0.758 0.754 0.747 0.748 0.778 0.775 0.730 0.182 0.495Mycoplasma capri- 0.736 0.741 0.743 0.742 0.754 0.727 0.710 0.747 0.738 0.735 0.794 0.497colum

Methanococcus 0.577 0.786 0.583 0.585 0.604 0.594 0.590 0.589 0.590 0.590 0.605 0.604vannielii

a Lower left-hand triangle, Sequence similarity based on published full 16S rRNA sequences (6); upper right-hand triangle, corrected evolutionary distance (7).

number of nucleotide substitutions per sequence position isestimated as -(3/4) x ln[(4S-1)13], where S is the observedfractional sequence similarity (24). The fractional sequencesimilarity was defined as MI[M + U + (G12)], where M is thenumber of positions with identical residues, U is the numberof positions with different residues, and G is the number ofpositions in which one of the compared sequences had aalignment gap.The phylogenetic tree was constructed by using the dis-

tance matrix method of Fitch and Margoliash (11).

RESULTS AND DISCUSSION

Restriction map of the 16S rDNA. Restriction digests of peaaphid DNA probed with 16S rDNA from E. coli are pre-sented in Fig. 1. HindIII-cut DNA obtained from the wholeaphid gave an intense band at 10 kb and a band of lesserintensity at 2 kb (Fig. 1, land b). Since the P symbiont greatlyoutnumbers the S symbiont (21), this result suggests that the10-kb fragment is derived from the numerically predominantP symbiont and the 2-kb fragment is derived from thenumerically less common S symbiont. The same 10- and 2-kbfragments are observed in the HindIII-digested DNA ob-tained from the dissected mycetomes. In this preparation,the intensity of the 10-kb band is less than that of the 2-kbband. This observation is explained by the facts that the Psymbiont is osmotically fragile (15, 21) and that during thedissection of the mycetome and its transient storage on iceprior to freezing, some P symbiont cells lyse and their DNAis degraded by nucleases. This degradation, which leads to adecrease of the 10-kb band (lane a), is minimized in theprocedure in which whole aphids are frozen and ground priorto DNA purification (lane b).

Restriction enzyme analyses of the DNA obtained fromthe dissected mycetome and the whole aphid gave identicalresults, indicating that all of the DNA which was detected byhybridization with the 16S rDNA probe was associated withthe mycetomes. The results obtained with EcoRI, EcoRV,and Hindlll (Fig. 1; additional digests not shown) allow

arrangement of the fragments to give the restriction mapspresented in Fig. 2. These results together with additionalrestriction digests with BamHI, Bcll, BglII, DraI, PstI,PvuII, and Scal (results not shown) are consistent with thepresence of only 1 copy of the 16S rRNA gene per genome ofthe P and S symbiont (1).

Sequence of the 16S rRNA and phylogenetic analysis. Thesequences of the 16S rRNA genes from the P symbiont, Ssymbiont, and E. coli are presented in Fig. 3. The P and Ssymbionts differ from each other in 188 base changes.Between E. coli and the P and S symbionts, there are 158 and81 base changes, respectively. The sequences of the 16SrRNAs of the P and S symbionts could readily be aligned togive the secondary structure characteristic of 16S rRNA(29).The results of sequence comparisons among P and S

symbionts and selected organisms are presented in Table 1.A phylogenetic tree based on the evolutionary distancematrix of Table 1 is presented in Fig. 4. The P and Ssymbionts are members of the y-subdivision of the classProteobacteria (27), one of the 10 major lineages of theeubacteria (29). (The class designation Proteobacteria hasbeen recently given for the lineage defined by the purplebacteria [27].) The S symbiont is slightly more related to E.coli than to Proteus vulgaris (Table 1; Fig. 4) and is clearlya member of the Enterobacteriaceae, a family within the -y-3subdivision. The high sequence similarity of the P symbiontto members of the Enterobacteriaceae (Table 1; Fig. 4), aswell as the possession of characteristic signature sequences(CT[Pu]ACCACTTTG, TACTTTCAG, ATCTCTAG, AACCCAG, CAAAAG, ATCATG) (30) indicates that the Psymbiont is also a member of the y-3 subdivision. The lengthof the branch between the P symbiont and the members ofthe Enterobacteriaceae is similar to that of the generaXenorhabdus (9) and Ruminobacter (20), which are alsomembers of the y-3 subdivision. Species of these two generahave signature sequences which indicate that they are dis-tinct from both the P and S symbionts.

J. BACTERIOL.

16S rRNA PEA APHID SYMBIONT RELATIONSHIPS 2973

P-s AAATTGAAGA GTTTGATCAT GGCTCAGATT GAACGCTGGCS-s AAATTGAAGA GTTTGATCAT GGCTCAGATT GAACGCTGGCEc AAATTGAAGA GTTTGATCAT GGCTCAGATT GAACGCTGGC

P-s CAAGCGGCAA ACGGGTGAGT AATATCTGGG GATCTACCCAS-s CGAGCGGCGG ACGGGTGAGT AATGTCTGGG AAACTGCCTGEc CGAGTGGCGG ACGGGTGAGT AATGTCTGGG AAACTGCCTG

P-s TGGGGGACCT TTTGGCCTCA TGCTTTTGGA TGAACCCAGAS-s TGGGGGACCT TCGGGCCTCA CGCCATCAGA TGTGCCCAGGEc AGGGGGACCT TCGGGCCTCT TGCCATCGGA TGTGCCCAGA

GAGGATAACC AGCCACACTGGAGGATGACC AGCCACACTGGAGGATGACC AGCCACACTG

GCCGCGTGTA TGAAGAAGGCGCCGCGTGTG TGAAGAAGGCGCCGCGTGTA TGAAGAAGGC

P-s AGCACCGGCT AACTCCGTGCS-s AGCACCGGCT AACTCCGTGCEc AGCACCGGCT AACTCCGTGC

P-s CAGGTGTGAA ATCCCTAGGCS-s CAGATGTGAA ATCCCCGCGCEC CAGATGTGAA ATCCCCGGGC

GAACTGAGAC ACGGTCCAGAGAACTGAGAC ACGGTCCAGAGAACTGAGAC ACGGTCCAGA

CTTAGGGTTG TAAAGTACTTCTTCGGGTTG TAAAGCACTTCTTCGGGTTG TAAAGTACTT

CAGCAGCCGC GGTAATACGGCAGCAGCCGC GGTAATACGGCAGCAGCCGC GGTAATACGG

TCAACCTAGG AACTGCATTT

GGCAAGCCTA ACACATGCAAGGCAGGCCTA ACACATGCAAGGCAGGCCTA ACACATGCAA

AAAGAGGGGG ATAACTACTAATGGCGGGGG ATAACTAGTGATGGAGGGGG ATAACTACTG

CGAGATTAGC TTGTTGGTAGTAGGATTAGC TGGTAGGTGGTGGGATTAGC TAGTAGGTGG

CTCCTACGGG AGGCAGCAGTCTCCTACGGG AGGCAGCAGTCTCCTACGGG AGGCAGCAGT

TCAGCGGGGA GGAAAAAAATTCAGCGAGGA GAAAGGGTAATCAGCGGGGA GGAAGGGAGT

AGGGTGCAAGAGGGTGCAAGAGGGTGCAAG

GAAACTGGAATCAACGTGGG AACGGCATTT GAGACTGGCATCAACCTGGG AACTGCATCT GATACTGGCA

P-s CGTAGATATC TGGAGGAATA CCCGTGGCGAS-s CGTAGAGATC TGGAGGAATA CCGGTGGCGAEC CGTAGAGATC TGGAGGAATA CCGGTGGCGA

P-s GGTAGTCCAT GCCGTAAACG ATGTCGACTTS-s GGTAGTCCAC GCTGTAAACG ATGTCGATTTEc GGTAGTCCAC GCCGTAAACG ATGTCGACTT

P-s CAAGGCTAAA ACTCAAATGA ATTGACGGGGS-s CAAGGTTAAA ACTCAAATGA ATTGACGGGGEC CAAGGTTAAA ACTCAAATGA ATTGACGGGG

P-s CACAGAATTC TTTAGAAATA AAGAAGTGCCS-s CAGAGAACTT TCCAGAGATG GAGAGGTGCCEC CACGGAAGTT TTCAGAGATG AGAATGTGCC

P-s GCAACGAGCG CAACCCTTAT CCCCTGTTGCS-s GCAACGAGCG CAACCCTTAT CCTTTGTTGCEC GCAACGAGCG CAACCCTTAT CCTTTGTTGC

TCATCATGGCTCATCATGGCTCATCATGGC

AGTCCGGACTAGTCCGGATTAGTCCGGATT

P-s CCGCCCGTCAS-s CCGCCCGTCAEC CCGCCCGTCA

AAGCGGCCTC CTAAACGAAAAGGCGGCCCC CTGGACAAAGAGGCGGCCCC CTGGACGAAG

GGAGGTTGTTGGAGGTTGCGGGAGGTTGTG

GCC-GCACAAGCCCGCACAAGCCCGCACAA

TTCGGGAGCTTTCGGGAGCTTTCGGGAACC

CAGCGGTTCGCAGCGATAAACAGCGGTCCG

CCTTACGACC AGGGCTACAC ACGTGCTACACCTTACGAGT AGGGCTACAC ACGTGCTACACCTTACGACC AGGGCTACAC ACGTGCTACA

GGAGTCTGCA ACTCGACTCC ACGAAGTCGGGGAGTCTGCA ACTCGACTCC ATGAAGTCGGGGAGTCTGCA ACTCGACTCC ATGAAGTCGG

CACCATGGGA GTGGGTTGCA AAAGAAGCAGCACCATGGGA GTGGGTTGCA AAAGAAGTAGCACCATGGGA GTGGGTTGCA AAAGAAGTAG

CGTTAATCAGCGTTAATCGGCGTTAATCGG

AACTAGAGTTAGCTAGAGTCAGCTTGAGTC

ACTGACACTGACTGACGCTCACTGACGCTC

GTCGAGCGGCGTCGAGCGGTGTCGAACGGT

GAAATGGTAGGAAACGGTAG

AGCGAGAAGA GAGCTTGCTC TCTTTGTCGGAGCAC-AAGA GAGCTTGCTC TCTG-GGTGA 100AACAGGAAGA -AGCTTGCT- TCTTTGCTGA

CTAATACCGC ATAATGTTGA AAAACCAAAGCTAATACCGC ATAACGTCGC AAGACCAAAG 200

GAAACGGTAG CTAATACCGC ATAACGTCGC AAGACCAAAG

AGTAATAGCC TACCAAGGCAGGTAACGGCT CACCTAGGCGGGTAACGGCT CACCTAGGCG

GGGGAATATT GCACAATGGGGGGGAATATT GCACAATGGGGGGGAATATT GCACAATGGG

AAAACTAATA ATTTTATTTCTGTGTTAATA AGACATTGCAAAAGTTAATA CCTTTGCTCA

AATTACTGGG CGTAAAGAGCAATTACTGGG CGTAAAGCGCAATTACTGGG CGTAAAGCGC

TCGTAGAGGG AGGTAGAATTTTGTAGAGGG GGGTAGAATTTCGTAGAGGG GGGTAGAATT

ACGATCTCTA GCTGGTCTGAACGATCCCTA GCTGGTCTGA 300ACGATCCCTA GCTGGTCTGA

CGAAAGCCTG ATGCAGCTATCGCAAGCCTG ATGCAGCCAT 400CGCAAGCCTG ATGCAGCCAT

GTGACGTTAC CCGCAGAAGATTGACGTTAC TCGCAGAAGA 500TTGACGTTAC CCGCAGAAGA

GCGTAGGTGG TTTTAAGTACGCAGGCGG MGTTAAGT 600ACGCAGGCGG TTTGTTAAGT

CTAGGTGTAG CGGTGAAATGCCAGGTGTAG CGGTGAAATG 700CCAGGTGTAG CGGTGAAATG

AGGCGCGAAA GCGTGGGGAG CAAACAGGAT TAGATACCCTAGGTGCGAAA GCGTGGGGAG CAAACAGGAT TAGATACCCT 800AGGTGCGAAA GCGTGGGGAG CAAACAGGAT TAGATACCCT

TCCAAGAGAA GTGACTTCCG AAGCTAACGC ATTAAGTCGACCCTTGAGGG GTGGCTTCCG TAGCTAACGC GTTAAATCGACCCTTGAGGC GTGGCTTCCG GAGCTAACGC GTTAAGTCGA

GCGGTGGAGC ATGTGGTTTA ATTCGATGCA ACGCGAAAAAGCGGTGGAGC ATGTGGTTTA ATTCGATGCA ACGCGAAGAAGCGGTGGAGC ATGTGGTTTA ATTCGATGCA ACGCGAAGAA

GTGAGACAGG TGCTGCATGG CTGTCGTCAG CTCGTGTTGTCTGAGACAGG TGCTGCATGG CTGTCGTCAG CTCGTGTTGTGTGAGACAGG TGCTGCATGG CTGTCGTCAG CTCGTGTTGT

GCCGGGAACT CAGAGGAGAC TGCCGGTTAT AAACCGGAGGGTCGGGAACT CAAAGGAGAC TGCCGGTGAT AAACCGGAGGGCCGGGAACT CAAAGGAGAC TGCCAGTGAT AAACTGGAGG

ATGGTTTATA CAAAGAGAAGATGGCGTATA CAAAGAGAAGATGGCGCATA CAAAGAGAAG

AATCGCTAGT AATCGTGGATAATCGCTAGT AATCGTAGATAATCGCTAGT AATCGTGGAT

GTATCCTAAC CCTTTAAAAGGTAGCTTAAC C----TTCGGGTAGCTTAAC C----TTCGG

CAAATCTGCA AAGACAAGCACGACCTCGCG AGAGCAAGCGCGACCTCGCG AGAGCAAGCG

CAGAATGCCA CGGTGAATACCAGAATGCTG CGGTGAATACCAGAATGCCA CGGTGAATAC

CCGCCTGGGG AGTACGGCCGCCGCCTGGGG AGTACGGCCG 900CCGCCTGGGG AGTACGGCCG

CCTTACCTGG TCTTGACATCCCTTACCTAC ACTTGACATC 1000CCTTACCTGG TCTTGACATC

GAAATGTTGG GTTAAGTCCCGAAATGTTGG GTTAAGTCCC 1100GAAATGTTGG GTTAAGTCCC

AAGGTGGGGA CGACGTCAAGAAGGTGGGGA TGACGTCAAG 1200AAGGTGGGGA TGACGTCAAG

AACCTCATAA AGTAAATCGTGACCTCACAA AGTACGTAGT 1300GACCTCATAA AGTGCGTCGT

GTTCCCGGGC CTTGTACACAGTTCCCGGGC CTTGTACACA 1400GTTCCCGGGC CTTGTACACA

GAAGGCGCTT ACCACTTTGT GATTCATGAC TGGGGTGAAGGAGGGCGCTT ACCACTTTGT GATTCATGAC TGGGGTGAAG 1500GAGGGCGCTT ACCACTTTGT GATTCATGAC TGGGGTGAAG

P-s TCGTAACAAG GTAACCGTAG GGGAACCTGC GGTTGGATCA CCTCCTTAS-s TCGTAACAAG GTAACCGTAG GGGAACCTGC GGTTGGATCA CCTCCTTAEC TCGTAACAAG GTAACCGTAG GGGAACCTGC GGTTGGATCA CCTCCTTA

FIG. 3. Sequences of 16S rDNAs from P symbiont (P-s), S symbiont (S-s), and E. coli(Ec) (6). Spaces (hyphens) have been introduced tomaintain optimal alignment.

The class Proteobacteria contains a number of lineageswith members that are intimately associated with plant or

animal cells. The a subdivision includes the rhizobacteria,the agrobacteria, and the intracellular parasite Rhochalimaequintana (Fig. 4) and is also the origin of plant mitochondria(31). The -y division also contains the sulfur-oxidizing endo-symbionts of marine invertebrates (7), which are, however,distinct from the -y-3 subdivision. The P and S symbionts arealso distinct from the obligate intracellular parasites, thechlamydiae and the mycoplasmas (29). The latter are mem-bers of the gram-positive branch of the eubacteria (Fig. 4).

Speculations. Ochman and Wilson (23) have suggested atentative relationship between the percent sequence differ-ence of the 16S rRNAs and the time at which divergenceoccurred. Based on this calibration, the P symbiont diverged

from E. coli about 420 million years ago and the S symbiontdiverged about 250 million years ago. If the initial impetusfor the divergence was an adaptation to a symbiotic associ-ation (resulting in sequestration of the endosymbiont in themycetocyte), then it would appear that the infection of thehost by the ancestor of the P symbiont occurred in theSilurian-Devonian period and preceded the appearance ofthe Homoptera in the Upper Carboniferous period (2). Theinfection with the ancestor of the S symbiont was a laterevent which occurred during the Triassic period at a time forwhich fossils of aphids have been detected (13). Thesespeculations suggest that the association between the aphidsand the P and S symbionts is of great antiguity and involvesa high degree of adaptation and interdependence on the partof the host and the endosymbiont.

P-sS-sEC

P-sS-sEc

P-sS-sEc

P-sS-sEc

VOL. 171, 1989

2974 UNTERMAN ET AL.

Y

C. psittaci

0 0.05

\ Myc. capricolum

FIG. 4. Phylogenetic tree illustrating the relationships of the Pand S symbionts to selected species of eubacteria, based on the datapresented in Table 1. Scale represents estimated number of nucle-otide substitutions per sequence position (7). a, 1, y, 8, Subdivisionswithin the class Proteobacteria (27, 29).

ACKNOWLEDGMENTS

We thank E. F. DeLong and N. R. Pace for the phylogenetic tree,H. F. Noller for pkk 3535, L. Baumann and M. G. Kinsey forassistance in growing aphids, and B. C. Campbell for his advice.

B. M. Unterman was a recipient of an Eastman Kodak graduatefellowship. This research was in part supported by the University ofCalifornia, Agricultural Experiment Station.

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