INFECTION AND IMMUNITY, Oct. 1994, p. 4469-4480 Vol. 62, No. 100019-9567/94/$04.00+0Copyright ©) 1994, American Society for Microbiology

Nucleotide Sequence of the Streptococcus gordonii PK488Coaggregation Adhesin Gene, scaA, and

ATP-Binding CassettePAUL E. KOLENBRANDER,* ROXANNA N. ANDERSEN, AND N. GANESHKUMARt

Laboratory of Microbial Ecology, National Institute of Dental Research,National Institutes of Health, Bethesda, Maryland 20892

Received 18 March 1994/Returned for modification 16 June 1994/Accepted 21 July 1994

Human oral viridans group streptococci that coaggregate with Actinomyces naeslundii PK606 express surfaceproteins related to ScaA, the coaggregation-mediating adhesin of Streptococcus gordonii PK488 (R. N. Andersen,N. Ganeshkumar, and P. E. Kolenbrander, Infect. Immun. 61:981-987, 1993). The nucleotide sequence of the6,125-bp EcoRI insert of pRA1, containing scaA, the gene encoding ScaA, was determined. Six open readingframes (ORFs) were identified. The orientation of four ORFs, two upstream (ORF 1 and ORF 2) and onedownstream (ORF 4) ofscaA (ORF 3), indicated transcription in one direction, whereas ORF 5 and ORF 6 weretranscribed divergently. Computer analysis of the deduced amino acid sequences identified a consensusbinding site for ATP (GxxGxGKS) in the putative 28,054-Da protein encoded by ORF 1. ORF 2 potentiallyencoded a hydrophobic protein of 29,705 Da with six potential membrane-spanning regions. ScaA was 310amino acids, 34,787 Da, and contained the lipoprotein consensus sequence LxxC, also reported for theScaA-related proteins SsaB, FimA, and PsaA from Streptococcus sanguis 12, Streptococcus parasanguis FW213,and Streptococcus pneumoniae R36A, respectively. ORF 4 potentially encoded a 163-amino-acid protein of 17,912Da, which was nearly identical to the downstream adjacent gene products of ssaB, fimA, and psaA. Nosignificant homology with other proteins was found with the putative ORF 5 gene product, a 229-amino-acidprotein of 25,107 Da. ORF 6 was incomplete and encoded a protein larger than 564 amino acids. This putativeprotein had a consensus Zn2+ binding motif, HExxH, found among bacterial thermolysins and mammalianneutral endopeptidases and was 40% identical to a homologous 210-amino-acid region of human enkephali-nase. The genetic organization of ORFs 1, 2, and 3 was similar to those of the bacterial periplasmic-bindingprotein-dependent transport systems of gram-negative bacteria and binding-lipoprotein-dependent transportsystems of gram-positive bacteria, and these genes appeared to encode ABC (ATP-binding cassette) proteins.This report describes a cell-to-cell adherence function associated with an ATP-binding cassette.

One of the most striking adherence-relevant features of themajority population of viridans group streptococci in humandental plaque is that nearly all isolates (Streptococcus sanguis,Streptococcus SM, Streptococcus gordonii, and Streptococcusoralis) so far tested coaggregate with actinomyces (Actinomycesnaeslundii) and many other genetically distinct oral bacteria(33). The properties of the coaggregations with actinomyceshave delineated six streptococcal (coaggregation groups 1 to 6)and six actinomyces coaggregation groups (coaggregationgroups A to F), which characterize more than 95% of thestrains so far tested (33). While five of the streptococcal groupscoaggregate with four or more of the actinomyces groups,group 6 streptococci coaggregate only with coaggregationgroup D actinomyces (33). Because it exhibits the least com-plex coaggregation pattern of the streptococci, S. gordoniiPK488, the representative strain of group 6, was chosen forfurther study of adherence-relevant characteristics.

Identification and characterization of adherence-relevantstreptococcal surface proteins (5, 15, 17, 28, 30, 36, 40, 42, 43,46, 48-50, 54, 56, 60, 64) are rapidly expanding. The proteinadhesins may be subunits of fimbrial structures or minor sub-units attached to the tips of fimbriae, as has been shown for

* Corresponding author. Mailing address: Building 30, Room 310,National Institutes of Health, Bethesda, MD 20892. Phone: (301)496-1497. Fax: (301) 402-0396. Electronic mail address: kolenbrander@yoda.nidr.nih.gov.

t Present address: Forsyth Dental Center, Boston, MA 02115.

several gram-negative bacteria (26, 57, 65). In gram-positivebacteria, surface orientation of nonfimbrial adhesins may bethrough anchorage of a hydrophobic region of the adhesinpolypeptide in the cytoplasmic membrane and presentation ofa portion of the remainder of the protein to the surface. Mem-brane anchoring may involve the combination of an LPxTGxmotif, a C-terminal hydrophobic domain, and a charged tail inthe protein (56); this combination has been found on a widevariety of gram-positive bacteria including many oral strepto-cocci and actinomyces (56). A second method, and the focus ofthe current report, involves glyceride-containing lipoprotein-adhesins, which were first reported by Jenkinson (28). Theseproteins are likely to be anchored in the cytoplasmic mem-brane by their N-terminal lipid-modified cysteine (24) and arepresumably exposed to the cell surface near the C terminus ofthe lipoprotein.Among the oral streptococci, there appear to be four groups

or families of surface-exposed proteins with adherence-rele-vant functions. The first group has a single member, CshA,which is about 290 kDa and contains the sequence LPxTGxnear its C terminus (40) for proper anchoring (56). Mutants ofS. gordonii that are disrupted in expression of cshA are alteredin their ability to coaggregate with actinomyces (40). Thesecond group has been broadly called the antigen IJI group ofpolypeptides. They have a molecular mass of approximately180 to 210 kDa and are found on S. gordonii, Streptococcusmutans, and Streptococcus sobrinus. Several of the proteinsfrom these strains are known to possess the LPxTGx motif for

4469

4470 KOLENBRANDER ET AL.

anchoring (56). These proteins are adhesins that bind tosalivary glycoproteins and may mediate binding of the strep-tococcus to a saliva-coated tooth surface (42, 43) or to otheroral bacteria (30). This potential dual function of streptococcalsurface proteins, recognizing salivary receptors as well as

receptors on actinomyces and other streptococci, has beensuggested elsewhere (30, 36).The third and fourth groups of surface-exposed proteins are

lipoproteins (2, 5, 15, 17, 28, 49, 54) and, by homology toproteins encoded by the ami locus (2), the first reportedstreptococcal ABC import proteins, they are likely to be ABCproteins (4, 45). The third group is composed of proteins in therange of 45 to 93 kDa and includes AmiA, an oligopeptide-binding protein (2), AliA and AliB, two homologs of AmiA (1),and PlpA (39, 49), all from Streptococcus pneumoniae, andSarA (29) from S. gordonii. A 45-kDa sugar-binding lipopro-tein, MsmE (52, 58), from S. mutans may also be an adhesinthat recognizes sugar-containing receptor molecules.The fourth group consists of lipoproteins of about 35 kDa

from four species of streptococci, FimA from Streptococcusparasanguis FW213 (14), SsaB from S. sanguis 12 (18), PsaAfrom S. pneumoniae R36A (54), and the coaggregation-medi-ating ScaA from S. gordonii PK488 (5) (we report here that thisis a lipoprotein and part of an ABC protein complex). Onlyfragments of the set of ABC genes have been cloned to datefrom the first three species, with the fragment from S. para-

sanguis FW213 being the most complete (13-15).The evidence that ScaA of S. gordonii PK488 functions as a

mediator of coaggregation with human oral actinomyces isthat (i) coaggregation-defective (COG-) mutants of strainPK488 that fail to coaggregate with actinomyces are missingthe 38-kDa ScaA protein in sodium dodecyl sulfate-polyacryl-amide gel electrophoresis gels of cell surface sonicates (34), (ii)COG- mutants are still capable of coaggregation with otherpartner cell types (34), and (iii) antiserum against PK488 cellsthat is absorbed with COG- cells blocks coaggregation be-tween PK488 and actinomyces but not coaggregations betweenPK488 and other partners (34). This absorbed antiserum alsoidentifies a protein of about 38 kDa in immunoblots of cellsurface sonicates of all six reference strains for the six strep-tococcal coaggregation groups (34), as well as Streptococcuscrista PK1408, Streptococcus milleri K44Y, and several otherstreptococci (5), all of which coaggregate with actinomyces.Only the combination of absorbed antiserum and 100 mMlactose could block bimodal coaggregations between other-wise-untreated oral streptococci (these cells bear ScaA ho-mologs as well as the receptors for lactose-sensitive adhesins)and actinomyces (these cells bear lactose-sensitive adhesinsand receptors for ScaA); previously, blocking these coaggre-gating pairs required both inactivation of the streptococci withheat or protease treatment and addition of lactose (34). Thisindirect evidence strongly implicates ScaA as the coaggrega-tion mediator. Direct evidence by use of purified ScaA has notbeen possible to obtain, since ScaA appears to be a minorprotein on the streptococcal surface (34).

Here, we report the complete sequence of a putative ABCoperon consisting of single copies of the genes encoding an

ATP-binding protein, a hydrophobic membrane protein withsix transmembrane regions, and the coaggregation-mediatingadhesin ScaA, which recognizes receptors on actinomyces cellsurfaces. This putative operon is distinct from those consideredabove in the third group of streptococcal surface proteins, inthat the msm and ami operons consist of two genes for thehydrophobic membrane proteins and one or two genes, respec-tively, for the ATP-binding proteins (2,52). In the fourth groupof streptococcal surface proteins, the genetic organization of

the putative ABC operon in the cloned fragments from theother three species appears to be the same as that describedhere for S. gordonii PK488. The reported nucleotide sequencesfor cloned fragments containing ssaB, fimA, and psaA will becompared with the putative ABC operon containing scaA fromS. gordonii PK488. We also examine the distribution of scaA-like sequences in a large collection of streptococcal strains.

MATERIALS AND METHODS

Strains and culture conditions. All strains used in this studyare listed in Table 1. Streptococci were cultured in a mediumconsisting of tryptone, yeast extract, Tween 80, and glucosebuffered to pH 7.5 with K2HPO4 (38), with the exception of S.pneumoniae R6, which was grown in brain heart infusion broth(Difco Laboratories, Detroit, Mich.). Streptococcal cultureswere grown at 37°C under anaerobic conditions with theGasPak system (BBL Microbiology Systems, Cockeysville, Md.),with the exception of Streptococcus pyogenes JRS4, which wasgrown statically in air. Escherichia coli XL1-Blue (StratageneCloning Systems, La Jolla, Calif.) containing pRAl was cul-tured aerobically at 37°C in Luria-Bertani broth or on Luria-Bertani agar (Gibco BRL, Gaithersburg, Md.) with 100 ,ug ofampicillin (Sigma Chemical Co., St. Louis, Mo.) per ml.Recombinant DNA methods. The methodology used to

obtain pRAl containing scaA and adjacent genes has beenreported in detail (5). Sequencing data obtained during thispresent study indicated that the insert in pRAl was 6.125 kb ofstreptococcal DNA instead of the previously reported 6.6 kb(5).DNA preparation. Plasmid DNA for sequencing was pre-

pared by using the Magic Minipreps DNA Purification System(Promega Corp., Madison, Wis.). The preparation of strepto-coccal chromosomal DNA has been described previously (5).

Southern blotting. Between 2 and 4 ,ug of chromosomalDNA was digested with PstI (Boehringer Mannheim, India-napolis, Ind.), electrophoresed in a 0.7% agarose gel, andtransferred by capillary action onto nitrocellulose membranes.Radioactively labeled probes were generated by end labelingthree 30-base oligonucleotides. The first had the sequenceCAAGATCCGCACAAATACGAACCTCTGCCC and corre-sponded to nucleotides 193 to 222 of scaA; the second se-quence (TACATCTGGGAAATCAACACTGAAGAAGAA)was also from scaA and corresponded to nucleotides 664 to693; and the third sequence (TACATCTGGGAAATCAACACCGAAGAAGAA) was from ssaB, the homolog of scaA,from S. sanguis 12. It is identical to nucleotides 664 to 693 ofscaA except for a T-to-C nucleotide change in the 21st positionof the 30-mer. Both the hybridization of these probes to thetransferred DNA and all subsequent washing were done at50°C. For probing the same blot with a second probe, the firstprobe was stripped from the nitrocellulose membrane by themethod described by Sambrook et al. (53). Before beingreprobed, the blot was shown by autoradiography not to haveany residual label. Other procedures were reported previously(5).

Sequencing and computer-assisted sequence analyses. Dide-oxy sequencing (55) was performed with Sequenase version2.0 (United States Biochemical, Cleveland, Ohio) and 35S-dATPaLS (Dupont, NEN Research Products, Boston, Mass.).Both strands of the 6.125-kb insert of pRAl were sequenced.Sequence analyses, translations, manipulations, and compari-sons were conducted with the Genetics Computer Groupsequence analysis software package (20). The computer pro-gram BLAST was used to search for amino acid sequences that

INFECTr. IMMUN.

S. GORDONII ADHESIN AND ATP-BINDING CASSETlE 4471

TABLE 1. Strains used

Strain Relevant property or properties Reference(s) or source

S. gordonii DL1 Reference strain for coaggregation group 1 5, 34S. oralis Hi Reference strain for coaggregation group 2 5, 34S. oralis 34 Reference strain for coaggregation group 3 5, 34S. oralis C104 Reference strain for coaggregation group 3 5, 34S. oralis J22 Reference strain for coaggregation group 4 5, 34Streptococcus SM PK509 Reference strain for coaggregation group 5 5, 34S. gordonii PK488 Reference strain for coaggregation group 6 5, 34S. gordonii PK1804 Coaggregation-defective mutant of PK488 34S. sanguis ATCC 10556 Type strain ATCCaS. oralis ATCC 10557 Coaggregates with actinomyces (5) ATCCS. gordonii ATCC 10558 Type strain ATCCS. sanguis 12 Adherence to S-HA B. C. McBrideS. parasanguis FW213 Formerly S. sanguis FW213; adherence to S-HA P. Fives-TaylorS. crista PK1408 Formerly S. sanguis CCSA, "corncob" formation C. MoutonS. mileri K44Y Coaggregates with actinomyces (5) J. MizunoS. sobrinus 6715-10 Coaggregates with actinomycesS. pneumoniae R6 Noncoaggregating control strain R. D. LunsfordS. pyogenes JRS4 Noncoaggregating control strain J. R. ScottS. pyogenes JRS75 Noncoaggregating control strain J. R. ScottS. mutans LM7 Coaggregates with actinomyces J. DonkerslootS. rattus BHT Noncoaggregating control strain J. DonkerslootS. cricetus AHT Coaggregates with actinomyces J. DonkerslootS. constellatus PK2819 Noncoaggregating control strain L. V. H. MooreS. salivarius ATCC 25975 Noncoaggregating control strain ATCCS. intermedius PK2821 Coaggregates with actinomyces L. V. H. MooreEnterococcus faecalis GF590 Noncoaggregating control strain D. ClewellEscherichia coli XL1-Blue recA mutant strain used for cloning StratageneActinomyces naeslundii PK606 Coaggregation partner of streptococci 34

a American Type Culture Collection, Rockville, Md.

were homologous to any of the gene products of open readingframes (ORFs) 1 to 6 (3).

Nucleotide sequence accession number. The DNA sequenceof the 6.125-kb fragment has been assigned the GenBankaccession number L11577.

RESULTS

DNA sequence of the adhesin gene, scaA, and the surround-ing region. We determined the nucleotide sequence (Fig. 1) ofa 6.125-kb DNA fragment of S. gordonii PK488 that expresseda 38-kDa protein previously identified as the streptococcalcoaggregation adhesin ScaA (5). The sequence contained sixORFs. ORF 3, the scaA gene, started at nucleotide 3418 andended at 4347. The nucleotide sequence of scaA was 86%identical to that of the ssaB adhesin gene from S. sanguis 12(18), 75% identical to that of the psaA adhesin gene from S.pneumoniae R36A (54), and 73% identical to that of the fimAadhesin gene of S. parasanguis FW213 (14). The proteinencoded by scaA contained amino acids LAAC (shown inboldface type in Fig. 1) in positions 17 to 20, which agrees withthe consensus cleavage site (shown by the small vertical arrowin Fig. 1) sequence, LxxC, of signal peptidase II for prolipopro-teins (24). Immediately downstream of the termination of scaAis a palindromic sequence, nucleotides 4355 to 4383, shown byconverging horizontal arrows, which is followed by a run of sixadenines. The free energy for this potential rho-independentterminator was -13.9 kcallmol (ca. -58.2 kJ/mol). ORF 4(nucleotides 4500 to 4988) appeared to be transcribed in thesame direction as scaA. The start codon of the downstreamputative gene ORF 5 (nucleotides 5358 to 6044) was on thecomplementary strand, oriented in the opposite direction andlocated near the 3'-terminus of the 6.125-kb clone at nucle-otide 6044.

Immediately upstream of scaA, between nucleotides 2560and 3393, was ORF 2, a gene encoding a hydrophobic proteinthat contained a 17-amino-acid sequence (shown in boldfacetype in Fig. 1) similar to sequences found in several membraneproteins known to be involved in peptide transport or othersmall-molecule transport functions (10). The next gene up-stream, called ORF 1 (nucleotides 1803 to 2555), possessed theGxxGxGKS/T sequence (shown in boldface type in Fig. 1) usedfor binding ATP (63). A partial gene, ORF 6, was oriented inthe opposite direction on the complementary strand, started atnucleotide 1692 just upstream of the start of ORF 1 andextended beyond the EcoRI site at the 5' terminus of the6.125-kb clone. A sequence of amino acids, HEISH, followedby another glutamic acid 21 amino acids downstream waspresent in the deduced amino acid sequence from nucleotides261 to 247 and 186 to 184, respectively, and was consistent withthe consensus sequence of HExxH for Zn2+-dependent met-allopeptidases (31).

Organization and partial restriction map of pRAl. The totalnumber of amino acids and corresponding calculated molecu-lar weights of the proteins encoded by the six ORFs of pRAlare listed at the bottom of Fig. 2. The three-gene motif (ORFs1, 2, and 3) encoding an ATP-binding protein, a hydrophobicmembrane protein, and a substrate-binding protein is similarto that of the binding-protein-dependent transport systems ofgram-negative bacteria (4, 45) and the binding-lipoprotein-dependent transport systems of gram-positive bacteria (2, 52).This type of genetic organization has been termed the ABC(ATP-binding cassette) system (27, 45) or traffic ATPases (4).The directions of transcription of the six ORFs, on the basis ofthe orientation of the start codons in the nucleotide sequenceof pRAl (Fig. 1), are shown by the single-headed arrows.Except for the gene product of ORF 5, which had no significanthomology with other proteins in the GenBank database, the

VOL. 62, 1994

GAATTCCTCTGCAGAGAAATCAGCTCTCTTFTGC1CTTCTAAGGCTGC¶GCAATACCGCCCAAATC CCACGTTICTGAAACGGTCAGCTTIGCC<F E E A S F D A E K K A A E L A A A I G G L D A V N E S V T L K GATTGACCTTAGCTCCATAGGAATCCTG.ACCTTCAAACTGGTCAATGACCTTT7GAGTCCGCTCGGTG.AAGGCTTGGTAGTCGTGCTCCGTCCACCAGTT<N V K A G Y S D Q G E F Q D I V K Q T R E T F A Q Y D H 3 T W W NGTTGAGGCTACCATTTTCATCAAAGGAAGCGCCATTGGTATCAAAAGCATGAGAAATCTCATGGGCGATGACCGCTCCGATACCACCGTAATTGGCTGA<N L S G N E D F S A G N T D F A 3 3 X 3 3 A I V A G I G G Y N A S1GAGGATSGIGCAAGTCATAGAAAGGCGCCTSCAGGATAGCTGCCGSAAAGACGATCAGGTITl'rTGCGGATTATAGTAGGCATIGACCATATGGGC<S S Q H L D Y F P A Q L I A A P F V I L N K Q P N Y Y A N V M H ATGGCATTICCCCATTCCTTATAATCTACTGGTTGATTCCACTTGCTCCAGCTATAAGCAATATCAATCAGGCTCAGTTTCTGAGCATTrTCAAAGAGCGT<P H G W E K Y D V P Q N W K S W S Y A I D I L S L K Q A N E F L TCAGATTTCGTCCACGATCTTGCGAGAATAACGCTCAGGCAACTCGTCTGGATAACCAATATAAGGCTTGATGACATTAAGTTTGACAATGGCTTTGTC<L N E D V I K R S Y R E P L E D P Y G I Y P K I V N L K V I A K DACGAGTTTCCGGTGTTAACCAGTCATTCTAGCTAGGCGGTTTTTTAAACTTCAATCATCGTCACTACTTTCT'CTICTACATCAGCCTTGGCTTCGG<R T E P T L W D N Q A L R N K Y V E I M T V V K Q E V D A K A E PAGAGAACTTCTGACCGGCATACCAGAGACCGATCGCTTrATTAAAAGGTvCCCTGAGCTAGGTAATAAGCAGCTT'rTTCT'TTCCTGGGCTTGAGGAGT<S F K Q G A Y W L G I A Q N F P G Q A L Y Y A A K K K D Q A Q P TTCCCGATAAAGCACGTTGIATAAGCACCTSCCAAGACACGGATCATCCGTTAGATA3QGGGGTTrTATIQCGAACCGCAGAGAGAATrAGCAAAGCATG<G S L A R Q Y A G A L V R I D D T L Y P T T N R V A S L I L L A HGAGTTrATCCCAGTTTGCAGCTGTATAAATATCCTTGGCTGC'13CAGAAGCGTTCCTCAGGCACAATSACT3GGTCGGGTTCTrGGCCGATIAACTC<L K D W N A A T Y I D K A A K W F R E E P V I V Q D P E Q G I L KGGTGAAGATATCTGTCAAAGGCAATTCCGGCACCAAGGCCTTGAAATCGTCCCACTTATAAGGATGGTAGAGCTTAGCATACT1CAGAGCTCTCCTCATT<T F I D T L P L E P V L A K F D D W K Y P H Y L K A Y E S S E E NAGAGAGAACATAC3TGGCAAAAACAGCATCCAAGTCCAAAACCTTGTCTAAAAGATCCTTGATTCICTCTTCAGTAAAGTCAAATT'TGCTAGTAAATC<S L V Y Q A F V A D L D L V K D L L D K I E E E T F D F K A L L DTTCCTGGGCCTTACGCCATTTTCAAGAAGATCTGCTTTCTGAGGATGGTCTTCCGCGTAGTAGGTCGTATCAGGCAAAATAGTGCCTGGTCCATCCGC<E Q A K R W K A L L D A K Q P H D E A Y Y T T D P L I T G P G D ACCAGAGAACATTrGATCCGAGCGTCCATAAAGTCAGGTGICCACACCAAAAGGAAAATAAGTCGGCTrSCCAGCAAGCTCGAAGTCTGCAATCTTAGAAGC<W L V N I R A D M F D P A V G F P F Y T P K G A L E F D A I K S AATAThCTTCAAAAGACGCTAGGTISCGAATCTCATCAATATAAGCACGCGCTGGCTCAGTCCCTGCTSC¶ICCCGC¶TGTCATAATCCGCTGCTAGGCG<Y E E F S A L N R I E D I Y A R A P E T G A A E R K D Y D A A L RATGTAAGCAACGAAGTTCTCAGAATAGCATCTTCTGGTACTCCATCGCCAGCCAACCATTTGCAGTCGTAGAGAGCATGAGATCTTCAATTTICCTC<H Y A V F N Q L I A D E P V G D G A L W K D T T S L M L D E I E EAGCCAAGTCCATAAAGCCACCTGTAACTGGTTTATCGTCGGGAATAACAGCTGTCTGGCCCATGTCCCATTTATTGCATCATAAAAATCATCTTGCAG<A L D H F G G T V P K D D P I V A T K A W E G N I A D Y F D D Q L<start orf 6<TCGTGTCATCIrTTCIIACTTTCATAC3TTIATATAACCTTATCAAAAATAGCTrTTA3'rM&TCTTTGT'TAAATATTTCCCAA=GCAAA<R T M -35 -10

>start orf 1>ATTAAC7TrACMTrTATTAAGGTATATATTAATACAG C;G2GTSATATACAIQATT'AGATACAAAACCTAASTGTCAGCTATCAGGGCCAGC

rbs M L R Y I N T V G V I Y H I E I Q N L S V S Y Q G Q>7TGCATTAGATAAGGCAAACGTAACTATAAAAGGCCCTACTATTACAGGAATTATCGGCCCAAACGGGGCCGGAAAATCCACCCTTATTAAAGGTTTATL A L D K A N V T I K G P T I T G I I 0 P 1 0 A 0 8 T L I K G L>TGGGAATISTCGACCACCAAGGCCAAGCIACTSSGATGGTCAGCCCTTAGACAAAGAACTAAAACGAATTICCTACGTAGAACAAAAAATCAATATTGL G I V D H Q G Q A L L D G Q P L D K E L K R I A Y V E Q K I N I>ACTATAACTNCCTATCAAGGTAAAAGAGTGTGTTCCTCT3GGGCTCrATCCTAASATAAASCTCFTCCAAAGATTAAAGAC7TCTGACTGGGACAAGGD Y N F P I K V K E C V S L G L Y P K I K L F Q R L K T S D W D K>TCAACCAGGCTCTAAAAATCGT7IGGTTTAGAGGATTTTGCCGAACGTCAAATCAGTCAACTGTCTGGCGGTCAATTCCAACGGGTCTFSATTGCGCGCTV N Q A L K I V G L E D F A E R Q I S Q L S G G Q F Q R V L I A R>GCCTAGTCCAAGAAGCGGATTATATr1rCCTCSATSAGCCTPGISGGAT3GACTCGGTCAGCGAAGAGATrATTAIAAAAACACTCCGTCAGCTAAC L V Q E A D Y I F L D E P F V G I D S V S E E I I M K T L R Q L>GAAAAGACGGGAAGACGATTGATTGTCCACCATGACTTGAGCAAGG7GGTCGCTTAMTTTGATCAGGTCTTGCTACTTAACAAAAAAGTCGTCGCTTR K D G K T I L I V H H D L S K V V A Y F D Q V L L L N K K V V A>

>start orf 2>TCGGATCAACCGAGAGTACC3TCACCAAAGAAAATA3r.CAGCAGACCTACGGCTCTCAGCCTTTATGAA7QGA^GGCCTAGGATGATFCAGGAATTTF G S T E S T F T K E N M Q Q T Y G S Q L F M N G G A *>

rbs M I Q E F>ATT3CAAGGCTTACASACTTTCAT7TCCTGCAAAATGCTTTrATTACAGCCATTCATGA AGTCTTGGCCGG7rCCGTNGC7CCTNTATCATTCTI Q G L H D F H F L Q N A L I T A I V I G V V A G A V G C F I I L>

CGGGGCATGTCASCTCA7GG A7GCCA CACGCGGCTGCTCCSGSGrGGCCC3GTCTTfTAT'rGGGGATTAATNT'CTrCA7TCGGr.CCATTR G M S L M G D A I S H A V L P G V A L S F I L G I N F F I G A I>

ACCTTT1GGCTTACTAGCTTCCATTATTATCACTrATATCAAGGGCAATTCTATrATCAAAAGTGACACGGCTATCGGAATTACTTTTCTTCCTTCCTGT F G L L A S I I I T Y I K G N S I I K S D T A I G I T F S S F L>

GCTCTGGGGATCATTTAATTAGCGTTGCCAAGAGTTCGACCGACCTCTTCCATATCCTCTTTGGGAATATCTTGGCGGTACAAGACCTTGATAT7GGGA L G I I L I S V A K S S T D L F H I L F G N I L A V Q D L D M W>ATTAGTATGGCG7TGGGTATTSSGTGCTGCTGGTCATTASGTATICTTCAAAGCCAACT'GCTCACGTCCTTIGACCCGCTITTGSCCCAAGCTATGI S I G V G I L V L L V I S I F F K Q L L L T S F D P L L A Q A M>

GGAATIAAGGTTAATTTTACCACTATrTICTrAISGA'ITCTSACTGGTFTC7TGCACAGCTATGCAAAGSTGCGGAACGATTC7GATTGTGGCCG H K V N F Y H Y L L M I L L T L V S V T A Y Q 3 V 0 T X L I V ArATGCIGATTACACCAGCTGCGACAGCCTATCTCTA3SCCAAGAGTCTCAAGACCATGATTTTATTIGTCATCTGCCTSGGGGGCTGGS¶GCATCTGI'TFAY L X T P A A T A Y L Y A K S L K T H I L L S S A L G A G A S V L>GGCCTCTTCATCGGTTATAGTTTTAATGTTSGCAGCAGGTTCTAGTATCGTTCTAACCTCTGCCC7GATCTrTTTGGTTTCATTTTTrATCGCACCAAAAG L F I G Y S F N V A A G S S I V L T S A L I F L V S F F I A P K>

DraI >start scaA>CAGCGGTATTTAAAAAGAAAAGTAAAATAATATrrATATATTTTCCAATGAAAAAATGTCGTTTCTTAGTICTGCTFII5CTGGC'rr3TTTCGGCQ R Y L K R K V r* > rbs M K K C R F L V L L L L A F V G>CTAGCAGCCTGCTCTAGTCAAAAAAGCAGTACTGACTCTAGCTCTTCCAAGCTAAATGTCGTTGCAACCAATTCAATTATCGCAGATATCACAAAAAATL A A C S S Q K S S T D S S S S K L N V V A T N S I I A D I T K N>

ATCGCTGGCGATAAAATCAATCTGCACAGTATITGTCCCTGTTCGGTCAAGATCCGCACAAATACGAACCTCTGCCCGAAGATGTCAAAAAGACTTCTAAGH S I V E P T S K>

kAACAAGGACN K D>

,GAAAATGGTY Y A V S E G V D V I Y L E G Q N E K G K E D P H A W L N L E N G>

ATCATCTATGCGCAAAATATTGCCAAACGCTT,GA:'GAAAAuGACCC':ACAATAAGGCTACTrACGAGAAAAATCTCAAAGCTTATATAGAGAAGCTAI I Y A Q N I A K R L I E K D P D N K A T Y E K N L K A Y I E K L>

AC7SCTlGACAAGGAAGCCAAAGAGAAATTCAACAACATCCCAGAAGAAAAGAAAATGA?IS3 ACCAGTGAAGGTT1TCCTAAATACTTCTCTAAGT A L D K E A K E K F N N I P E E K K H I V T S E G C P K Y F S K>

GCATACAATGTCCATCGGCCTACATCTGATATCAACACTG .AAGAAGAAGGGACTCCAGACCAAATTAAAAGCTTAGTTGAAAAACTGCGCAAGACCA Y N V P S A Y I W E I N T E E E G T P D Q I K S L V E K L R K T>

AAAGTCCCATCTCTCTCGAATCAAGTGTAGACGACCGTCCGATGAAGACCGTTTCTAAAGACACTAACATCCCAATCTACGCTAAAATCTTCACCK V P S L F V E S S V D D R P M K T V S K D T N I P I Y A K I F T>

GACTCAATCGCT3AAAAAGGCGAAGAT GAGACAGCTACTACAGCATGATGAAATACAATCTGGATAAAATTTCTGAAGGATTGGCCAAATAATAACAAD S I A E K G E D G D S Y Y S M H K Y N L D K I S E G L A K *> --

AAGGTTrAGGAAGCAATCCTAACCTTTA1CAAAGAAAAAATTGGGCTGAAAT4TAGGCGTTCTCAAAT<TCAGTCAAATTATTCGAAAAAAATACT

>start orf 4>TATrCGGAGTACAATGAAGCTATCAAACATGATG^A3&CTI'TI

rbsGCGACACAGCCCAT'GATTTAGCCIGACTGCAACGGATCTATCAUG D T A H D F S L T A T D L S

ClaICTATCGATACTrGGTGTCTG,CTCAACTCAGACTCGCCGClT7CAACI

T G V TCTlTTICACAAGGCAAGCSGTGCGC7KP F A Q G K W C ATCATCAATGAATGGCACC7TTG3SGCC(

99

198

297

396

495

594

693

792

891

990

1089

1188

1287

1386

1485

1584

1683

1782

1881

1980

2079

2 178

2 277

2376

2475

2574

2673

2772

2871

2970

3069

3168

3267

3366

3465

3 564

3 663

3 762

3861

3960

4059

4158

4257

4356

4455

'ATGACAAC1STFCT'IGAAATCCTG TAACCTTTACTGGGAAACAACTGCAAGTAG 4554M T T F L G N P V T F T G K Q L Q V>LAAGAAAACTC¶SGCIACTTMCTGGCAAAAAGAAAGTCCTGAGCATCATCCCAT 4653K K T L A D F A G K K K V L S I I P>

'CAAGAACTCTCTGACT'IGGACAATACCGTTGTTATCACAGTT'ICAGTTGATTTGC 4752Q E L S D L D N T V V I T V S V D L>

'GCTGTTATGCTATC3TGACTACTTCGACCATTCTTTCSGGTCGCGACTACGCITGTCC 4851A V M L S D Y F D H S F R D Y A VrGATGAGAACAACACTGTGACCTATGCCGAATATIGCGACAATATCAACACTGAAC 4950D E N N T V T Y A E Y V D N I N T E>LTCACGACCACTCAAACGAGTGTTTAATGTCTAATSAAAAAGAGAGT 5049

LCTTACGAAACAAAGCTTCTTAAGATCTGACACACTACAAAAGAAAACTCTCCCTG 5148'TAAACAATFCTTCCTISATCGACAACTAGTTTTCCTGCCTTGTAGACCIGATGGG 5247SGCATCTAGGAISTAA3TGCAGCCTGCTTGCCAGTATCAAAGCTACCAATCTTAT 5346AGGACTTCCACTGGCGTCAGAGCGCATCATAAAGCAGCCCAGCTSCATAACGAAC 5445P S G S A D S R M F C G L Q V F

'CAGAGTGATGGCCATACCAGCTTCCAGCATCT'TCGGGCTGGTGCATAGGTATCT 5544L T I A M G A E L K R A P A Y T D

'CTSGCTTCSCSCATCTTGCGAATACCTICGTCTGTCGCCATCATCAAGTSTTICC 5643K A E A M K R I G E D T A M L H E

'AGACTCCATTTCATCAGCATGGATACGAAGCTAAGCCCATCTCCTTAGCTTTA 5742S E M E D A H I R L K F G M E K A K

'ACAGAAAATATCACAGAACTCAGCTAGATITTCTGCTTTTACCCGTGGCAGCATT 5841C F I D C F E A L N E A K V R P L MITTCTSGGTGAACGGCGSIGGCAGCCATAAAGGTSCAAACTAGGTCAATATCAIGG 5940E P P V A H A A F T S V L D I D H

'TfCCCAATCTAAACCATAGCCACTCT1CGCCTCAACCGTSTTCACCCCATGAAGC 6039E W D L G Y G S K A E V T T V G H L

ATAGSCTGAATTTATCATACAGATTTTCAAAAGAAGCTTCCCTTGTTGCTCGAACTGTGCTAAGAATACC<L M rbs

6125

4472 KOLENBRANDER ET AL. INFECT. IMMUN.

N E W H

<Q L N A TTCCATCAGACTGAA

<E H L S FGCACTGGTTGCTCC

<A S T A GGAAAGTAAGTAGCG

<S L L Y RTCCTCGACGATSAG

<E E V I LTCACGGTCCAAAGC

<D R D L A<start orf 5<AACATATAGTCCAA

EcoRI

S. GORDONII ADHESIN AND ATP-BINDING CASSE1TTE 4473

FIG. 1. Nucleotide sequence of S. gordonii PK488 scaA and adjoining regions. The locations and the likely directions of transcription of ORFs1 to 6 are indicated by arrowheads (>). These starts are proposed on the basis of the DNA sequence and not on the basis of any direct evidence.Potential ribosome binding sites (rbs) and putative promoter regions (-35 and -10) are underlined. The deduced amino acid sequence is shownwith the one-letter code. Stop codons are indicated by asterisks. Four amino acid consensus sequences are shown in bold type: the Zn2+-bindingregion HEISH of the product of ORF 6, the nucleotide-binding region GPNGAGKST of the product of ORF 1, the conserved regionAMQSVGTILIVAMLITP of hydrophobic membrane proteins (the product of ORF 2) of binding-protein-dependent transport systems, and theLAAC recognition sequence for prolipoprotein signal peptidase II cleavage (indicated by a small vertical arrow) of ScaA. The proposedrho-independent terminator immediately following scaA is shown by converging arrows.

other potential gene products were compared with homolo-gous proteins or sequences in the GenBank release 80.0 (Dec.1993) and PROSITE release 11.0 (Oct. 1993) databases (7, 20)with the Genetics Computer Group programs (version 7.3,June 1993) MOTIFS, PILEUP, and GAP (20).The putative ATP-binding protein (ATPase) encoded by

ORF 1. Comparison of the primary sequence of the putativeATP-binding protein encoded by ORF 1 with the partialsequence of pVT618 ORF 5 peptide of S. parasanguis FW213(15) indicated that the two proteins were 80% similar and 67%identical (Fig. 3). At amino acid 46 from the N terminus ofORF 1 was the start of the first nucleotide-binding site, GPNGAGKST (Fig. 3, the sequence below the first horizontal line),which agrees with the consensus sequence GxxGxGKS/T in theglycine-rich loop of ATP-binding enzymes (63). A glutamine-,glycine-rich motif, LSGGQFQR, started at amino acid 146(Fig. 3, the sequence below the middle horizontal line). Thisregion may be a peptide linker that joins separate domainswithin proteins and acts as a signal transducer (4), or it mayfunction within the second nucleotide-binding pocket, RCLVQEADYIFLDE (27), which immediately follows, starting atamino acid 158 (Fig. 3, the sequence below the third horizon-tal line). The second aspartic acid residue is 100% conservedand is thought to bind MgATP (4, 25). The aspartic acid ispreceded by four hydrophobic amino acids which help toexclude water from the nucleotide-binding pocket (25), afeature that is also conserved in pRAl ORF 1. For pRAl ORF1 and pVT618 ORF 5 (Fig. 3), only one amino acid wasdifferent in the three conserved regions described above (firstnucleotide-binding site, glutamine- and glycine-rich motif, and

second nucleotide-binding site; the regions below the horizon-tal lines in Fig. 3), indicating that the function for these twostreptococcal proteins includes binding ATP.The putative hydrophobic membrane protein encoded by

ORF 2. The putative 278-amino-acid protein (ORF 2, Fig. 2)contained a conserved 17-amino-acid sequence, AMQSVGTILIVAMLITP, starting at amino acid 191 that was similar to asequence found in numerous other hydrophobic membraneproteins involved in transport of small molecules (sugars,amino acids, or oligopeptides) in a variety of gram-negativeand gram-positive bacteria (2, 10, 22, 52, 61). The averagelocation of this 17-amino-acid sequence relative to the N and Ctermini of ten of these proteins was 183 amino acids from theN terminus and 85 amino acids from the C terminus. Thesedata suggest that these proteins act at similar steps in theirrespective transport processes.

Analysis of potential transmembrane helices was done bycriteria described by Engelman et al. (12). This method waschosen because it was designed especially to accommodateamino acid side chains in nonpolar environments. As suggestedby the authors, a window of 20 amino acids was used (Fig. 4,top panel), and six transmembrane regions were easily dis-cerned as the inverted peaks below the value of zero. The sixhydrophobic regions exhibit a regular periodicity. The pair ofarrows above the fifth transmembrane region defines thelocation of the 17-amino-acid sequence given above. Whilemost of the protein was clearly hydrophobic, it had a veryhydrophilic C-terminal tail.

Further examination of the primary sequence of the putative

Ec Sc Si

I

Dr Cl SI Sp Sc

I 11I 1

ORF6 ORFI ORFOR>F3 O> F> OORF 6 ORF I ORF 2 ORF 3 ORF 4 ORF 5

Zinc-Binding ATP- Hydrophobic ScaAEndopeptkds Bincing Membrane Upoprotein

Protein Protein1 kb

aa 564+ 251 278 310 163 229mw 63706+ 28054 29705 34787 17912 25107

FIG. 2. Restriction map of pRA1, showing the location of thelipoprotein adhesin gene, scaA (ORF 3), relative to the genes for theputative ATP-binding protein (ORF 1) and the hydrophobic mem-brane protein (ORF 2) of the proposed ABC operon. ORFs 1 to 4 areputatively transcribed in one direction and ORFs 5 and 6 are orientedin the opposite direction and putatively transcribed from the comple-mentary strand. ORF 6 is a partial sequence. The 1-kb bar indicatesthe proportional sizes of the ORFs. The deduced number of aminoacids (aa) for each ORF is shown below along with the calculatedmolecular weights (MW) of the putative proteins. At the top of thefigure are some of the pertinent restriction sites: Ec, EcoRI; Sc, SacI;SI, SalI; Dr, DraI; Cl, ClaI; Sp, SpeI.

pRAlorfl 1 MLRYINTVGVIYMIEIQNLSVSYQGQLALDKANVTIKGPTITGIIGPNGA 50

pVT618orf 5 1. ILGPNGA 7

pRAlorfl 51 GKSTLIKGLLGIVDHQGQALLDGQPLDKELKRIAYVEQKINIDYNFPIKV 100

pVT618orf 5 8 GKSTLIKAMLGLLPHSGRVQLDQKDLSQVLQRVAYVEQKSAIDFHFPITV 57

pRAlorfl 101 KECVSLGLYPKIKLFQRLKTSDWDKVNQALKIVGLEDFAERQISQLSGGQ 150

pVT618orf5 58 RECVSLGLYPHLSIFKRKSKEDLKKVENALKLVNLLDLADRQIGQLSGGQ 107

pRAlorf 1 151 FQRVLIARCLVQEADYIFLDEPFVGIDSVSEEIIMKTLRQLRKDGKTILI 200

pVT618orf5 108 FQRVLIARCLVQEADVIFLDEPFAGIDSVSEDIIMQTLQTLKQEGKTILI 157

pRAlorf 1 201 VHHDLSKVVAYFDQVLLLNKKVVAFGSTESTFTKENMQQTYGSQLFMNGG 250

pVT618orf 5 158 VHHDLSKVPAYFDKVLLLHRKLIAFGKTEETFTKENLHAAYGHELFIGGG 207

pRAlorf 1

pVT618orf 5

251 A. 251

208 VG 209

FIG. 3. Amino acid alignment of the complete sequence of theputative ORF 1 protein of pRAl and the partial sequence of thehomologous protein from ORF 5 of pVT618. The respective aminoacid numbers starting from the N termini are listed at the left and rightmargins. Identical ( ), conserved (:) and similar (.) amino acidresidues are indicated. The sequences below the three horizontal linesare the putative first nucleotide-binding site (amino acids 46 to 54), theglutamine- and glycine-rich motif (amino acids 146 to 153), and thesecond nucleotide-binding site (amino acids 158 to 172).

mlimim iiil i

VOL. 62, 1994

4474 KOLENBRANDER ET AL.

10.00 - _> 7.50-Z 5.001-_= 2.50 - -£ 0.00- v

-2. 50 -0S - 5.00z -7.50

-10.00-12.50 I l

50 100 150 200 250

1 MIQZFIQGLNDFNFLQNALITAIVIGWAGAVGCFIIL

39 RGMSLMGDAISHAVLPGVALSFILGINFFIGAITFGLLASIIITYI

85 KGNSIIKSDTAIGITFSSFLALGIILISVA

115 KSSTDLFNILFGNILAVQDLDMWISIGVGILVLLVISIFF

155 1QLLLTSFDPLLAQAMGMXVNFYNYLLMILLTLVSVTAMQSVGTILIVAMLITPAATAYLYA

217 KSLETMILLSSALGAGASVLGLFIGYSFNVAAGSSIVLTSALIFLVSFFIAP

269 IQRYLKKVK

FIG. 4. Hydropathy index for the putative hydrophobic membraneprotein encoded by ORF 2 (top panel). The hydrophilicity values (yaxis) were calculated by the method of Engelman et al. (12) with awindow length of 20 and are plotted against the amino acid numberstarting from the N-terminus (x axis). Hydrophobic domains havenegative values and appear below the zero value, and hydrophilicdomains have positive values and appear above zero. The pair ofarrowheads indicates the location of the 17-amino-acid conservedsequence, AMQSVGTILIVAMLITP, found in several hydrophobicmembrane proteins of ABC transporters (see the text). The bottompanel shows the distribution within the putative hydrophobic mem-brane protein of the proposed alternating aqueous-exposed polarregions and transmembrane domains with a C-terminal hydrophilic tailas calculated according to the method of Engelman et al. (12). Theamino acid number is listed at the beginning of each of the six sets ofdomains, 1 to 38, 39 to 84, 85 to 114, 115 to 154, 155 to 216, and 217to 268. The hydrophilic tail is composed of amino acids 269 to 278.Boldface type indicates charged polar amino acids. The horizontal lineabove the fifth putative transmembrane domain is the 17-amino-acidconserved sequence.

ORF 2 peptide by the criteria described by Engelman et al.(12) characterized seven amino acid segments (Fig. 4, bottompanel) that corresponded to the six transmembrane regionsand the hydrophilic C-terminal tail. Counting from the Nterminus, the starting amino acid of each respective segment islisted on the left-hand side (Fig. 4, bottom panel). Eachsegment consisted of a putative aqueous-exposed polar regionand a putative transmembrane region. The segments arearranged to show these two regions from the left to the right,respectively (Fig. 4, bottom panel). The charged polar aminoacids are shown in boldface type and indicate the beginningand end of the probable aqueous-exposed turn between trans-membrane domains. Transmembrane segments 1 to 6 con-tained 25, 34, 21, 19, 38, and 48 amino acids, respectively. Theconserved 17-amino-acid sequence seen below the heavy linein the fifth transmembrane region is buried within the trans-membrane domain (Fig. 4, bottom panel). This internal posi-tioning of a conserved sequence is consistent with the obser-vations by Yeates et al. (67), who compared numerous othermembrane proteins and determined that interior residues inthe transmembrane proteins are better conserved than themembrane-exposed residues among homologous proteins.Two regions within the putative sixth transmembrane seg-

ment could be distinguished. Adjacent to the short, charged,polar end was a stretch of 31 amino acids, numbers 221 to 252,that comprised a Gly-Ser-rich region with 11 polar but un-

1 50pRAlorf2 NIQEFIQGLH DFHFLQNALI TAIVIGWAG AVGCFIILRG MSLMGDAISHpSA2orf2 --L--F---R ---------- ------I--- ---------- ----------

pVT618orfl --T---D--Q Q--------- --- A--I--- ---------- ----------

pSTR3lorfl --T---D--Q Q--------- --- A--I--- ---------- ----------

51 100pRAlorf2 AVLPGVALSF ILGINFFIGA ITFGLLASII ITYIKGNSII KSDTAIGITFpSA2orf2 --------- ---------- ---------- ---------- ----------

pVT618orfl ---------- -----L------V------- L -----S---- ----------

pSTR3lorfl ---------- ---------- - A-------L -----S--------- M-..Y

101 150pRAlorf2 SSFLALGIIL ISVAKSSTDL FHILFGNILA VQDLDMWISI GVGILVLLVIpSA2orf2 ------- V-- ---- N----- ---------- ---I------ ----A--- I-pVT618orfl ------- V-- -G -------- ---------- --Q---MT- ---VT----pSTR31orfl L-LPRPRSHS DR-------- ---------- ---Q---VT- ---VA-----

151 200pRAlorf2 SIFFKQLLLT SFDPLLAQAM GMKVNFYHYL LMILLTLVSV TAMQSVGTILpSA2orf2 TL------I- ---------- --P-S----- ---------- ----------

pVT618orfl CLL-RP---- ----V---S- -VR-KI---- --V------- ----------

pSTR31orfl VLL-RP---- ----V---S- -VR-KL---- --V------- ----------

201 250pRAlorf2 IVAMLITPAA TAYLYAKSLK TMILLSSALG AGASVLGLFI GYSFNVAAGSpSA2orf2 ---L------ ------N--- -------G-- PL-------- ----------

pVT618orfl ---------- ------N--W S-M----S-- -L--I----- ---L-I-V--pSTR3lorfl -A----------.--- N--W S-M ----G-- -L--I----- ----- I-V--

251pRAlorf2 SIVLTSALIF LVSFFIAPKQ RYLKRKVK ......278pSA2orf2 ---------- - I-------- ----L-NRPK LK. .282pVT618orfl C----- VF- -I-------- -KN-HALSSH .... 280pSTR31orfl C------IF- -I-------- -KN-HALSPH ....278

FIG. 5. Alignment of the deduced amino acid sequence of ORF 2from S. gordonii PK488(pRAlorf2) with those of homologous proteinsfrom three other streptococci, S. sanguis 12(pSA2orf2), S. parasanguisFW213(pVT618orfl), and S. pneumoniae R36A(pSTR31orfl). Theonly gap to improve alignment is in the pSTR31orfl sequence at aminoacid 98 (indicated as two dots). Dashes indicate amino acids identicalto those directly above them in the pRAlorf2 sequence. The solid lineindicates the 17-amino-acid conserved sequence (see the text).

charged Gly-plus-Ser residues, or 35% Gly-plus-Ser residues.Some or all of this region, along with the short, polar, chargedsegment (amino acids 217 to 220) may constitute the turn inthe protein between the proposed fifth and sixth transmem-brane segments. In accord with this idea, the region aroundamino acids 217 to 220 possessed a very strong surfaceprobability by computer analysis. The remaining 17 aminoacids (numbers 253 to 268) consisted primarily of uncharged,nonpolar residues, suggesting that they are buried within themembrane bilayer.

Considering a bilayer thickness of about 30 A (3.0 nm), ana-helix of about 21 residues is required to span the bilayer (12).Longer hydrophobic sequences can be accommodated if thea-helix is tilted with respect to the bilayer plane, and it isknown that lipid bilayers in the fluid state can be readilydistorted up to twice the thickness. Transmembrane helices ofup to 36 amino acids have been reported (12), as may be thecase for transmembrane regions 1, 2, 5, and 6 of this hydro-phobic protein (Fig. 4, bottom panel).A comparison of the amino acid sequences of the ORF 2

protein with its homologs in S. sanguis 12 (18), S. parasanguisFW213 (15), and S. pneumoniae R36A (54) showed closerelatedness throughout most of the sequence (Fig. 5). TheORF 2 protein was 91, 83, and 79% identical, respectively, toits homologs in S. sanguis 12(pSA2orf2), S. parasanguis FW213(pVT618orfl), and S. pneumoniae R36A(pSTR31orfl). Whenconservative amino acid substitutions were included in theanalysis, even greater relatedness among these ScaA homologswas observed; there was 96, 93, and 89% similarity to ScaA,respectively. The underline at amino acids 192 to 208 (Fig. 5)indicates the conserved 17-amino-acid sequence describedabove and shows the near identity of the four peptides in thisregion.The streptococcal coaggregation adhesin encoded by scaA.

The deduced amino acid sequence indicated a 34,787-Da

6. NEFF---!= I -an&"

INFECT. IMMUN.

S. GORDONII ADHESIN AND ATP-BINDING CASSETTE 4475

ScaA MKKCRFLVLL LLA. FVGLAA CSSQKSSTDS SSSKLNVVAT NSIIADITKNSsaB ---LG--S-- ---.VCT-F- --N--NAS.- D-----K---- ----------

FimA ---IASVLA- FV-LLFG-L- --.. -G-SSG A-G--K--T- ---L-----PsaA ---IASVLA- FV-LLF--L- --KGT-- K- --D--K--T- ---L------

50ScaA IAGDKINLHS IVPVGQDPHK YEPLPEDVKK TSKADLIFYN GINLETrGNASsaB ------D--- -----K---E ---------- - Q------- ----------

FimA ------E--- -----K---E ---------- - Q------- ----------

PsaA --2----E- ---------E- ---_ ---- Q---

100ScaA WFTKLVENAQ KKENKDYYAV SEGVDVIYLE GQNEKGKEDP HAWLNLENGISsaB ------ K--N -E------- - D -------- - S------- ----------

FimA ------K--N -V-----F-- ---------- --- QA----- ---------

PsaA ------K--N -V---- F-A -D--E----- ---QA----- ----------

150ScaA IYAQNIAKRL IEKDPDNKAT YEKNLKAYIE KLTALDKEAK EKFNNIPEEKSsaB ---------- ---------- --------V- ---------- ----------

FimA L--K----Q- -A---K--DF -----A--T- --SK--QKA- QA-K----D-PsaA ---K----Q- -A---K--DF -----A--T- --SK--Q--- QA-----A--

200ScaA KMIVTSEGCP KYFSKAYNVP SAYIWEINTE EEGTPDQIKS LVEKLRKTKV

SsaB ---------F ---------- ---------- ---------- ----------

FimA ---------F -------G-- ---------- -----E---T ------Q --

PsaA ---------F -------G-- ----------V.---E---T -L----Q---

250ScaA PSLFVESSVD DRPMKTVSKD TNIPIYAKIF TDSIAEKGED GDSYYSM14KYSsaB ---------- ---------- ----- H---- ----- DQ--E --T------FimA -A-------- E------A ------------ ---- KE--K ---------WPsaA ---------- E--------- S----F---- -----KE--E ---------W

300ScaA NLDKISEGLA K ... .310SsaB ---------- - 309

FimA -----A---S Q.. .309

PsaA --E--A---N -... 310

FIG. 6. Alignment of ScaA and its homologs SsaB (S. sanguis 12),FimA (S. parasanguis FW213), and PsaA (S. pneumoniae R36A).Dashes indicate amino acids identical to those directly above them inthe ScaA sequence. One or two amino acid gaps (dots) are inserted inthe N-terminal regions of the sequences to improve alignment. Thetwo horizontal lines below QDPHKYEPLP and YIWEINTEEE indi-cate the respective 10-mer sequences corresponding to the 30-mernucleotide probes used to survey the presence of scaA homologs inother oral bacteria (cf. Fig. 7).

protein of 310 amino acids containing the sequence LAAC atamino acids 17 to 20 (Fig. 6), which is in agreement with theLxxC consensus sequence for signal peptidase II cleavage ofprolipoproteins. ScaA adhesin was 91, 80, and 80% identical toSsaB, FimA and PsaA, respectively, which also exhibit theconsensus sequence (Fig. 6). Most of the dissimilarity wasfound in the first 20 amino acids, which constitute the respec-tive signal peptides and are not part of the mature proteins.Two underlined regions of 10 amino acids (numbers 66 to 75and 223 to 232) (Fig. 6) indicate the sequences correspondingto the nucleotide probes used for screening genomic digests ofseveral streptococci for homologs of this gene (Fig. 7).

Southern blot analysis of streptococcal genomic DNA di-gested with PstI and probed with the 30-mer probe correspond-ing to amino acids 66 to 75 of ScaA (probe 1) showed thepresence of reactive fragments in 14 of the 23 streptococciexamined, including a 3.9-kb fragment from S. gordonii PK488(Fig. 7A, lane 6). The streptococci showing reactive fragmentsconsisted of five of the six coaggregation-group-representativestreptococci (S. gordonii DL1, S. oralis Hi, S. oralis 34 [weaklyhybridizing fragment], S. oralis J22, and S. gordonii PK488), S.sanguis 12 (weakly hybridizing fragment), S. parasanguisFW213, S. sanguis ATCC 10556 (type strain) (weakly hybrid-izing fragment), S. gordonii ATCC 10558 (type strain), S.milleri K44Y, S. crista PK1408, S. pneumoniae R6, and S.cricetus AHT. Probe 1 reacted with two fragments in both S.gordonii ATCC 10558 (type strain) and S. pneumoniae R6,which suggests the presence of two distinct scaA homologs.The probe did not react with S. oralis ATCC 10557, S. sobrinus6715-10, Enterococcus faecalis GF590, S. mutans LM7, S.pyogenes JRS4, Streptococcus rattus BHT, Streptococcus con-

stellatus PK2819, Streptococcus salivarius ATCC 25975, orStreptococcus intermedius PK2821. The agarose gel from whichthis blot was made contained adequate loads of all genomicdigests except in lane 5 (Streptococcus SM PK509). When theamount loaded was similar to that in the other lanes, afragment reacting at the same position as the upper fragmentof S. pneumoniae R6 was observed (35). Probe 1 also reactedwith a 3.9-kb fragment of S. gordonii PK1804, a COG- mutantof S. gordonii PK488 (35).The same blot was stripped and reprobed with the 30-mer,

called probe 2, corresponding to amino acids 223 to 232 ofScaA but with the nucleotide sequence of ssaB of S. sanguis 12(Fig. 7B). It reacted with fragments in 19 of 23 of thestreptococci (Fig. 7B) including all of the streptococci recog-nized by probe 1 (Fig. 7A) as well as with fragments in S. oralisATCC 10557, S. pyogenes JRS4, S. constellatus PK2819, S.salivarius ATCC 25975, and S. intermedius PK2821. Probe 2 didnot react with S. sobrinus 6715-10, E. faecalis GF590, S. mutansLM7, or S. rattus BHT. Probe 2 hybridized only to the lowerreactive fragment hybridized by probe 1 with S. gordonii ATCC10558 (Fig. 7A) and with both fragments of S. pneumoniae R6(Fig. 7A). All of the fragments recognized by both probes werecoincident (Fig. 7A and B), with the exception of the weaklyreacting S. cnicetus AHT (Fig. 7B).

Lastly, the blot was stripped and probed with the scaA30-mer, called probe 3, of the sequence encoding amino acids223 to 232 of ScaA (Fig. 7C). It differed from probe 2 by asingle C-to-T nucleotide change at position 21. This probehybridized with only 15 of the 19 recognized by probe 2. Thefour strains recognized by probe 2 but not by probe 3 were S.parasanguis FW213, S. pyogenes JRS4, S. cnicetus AHT, and S.intermedius PK2821. As with probe 2, probe 3 hybridized onlywith the lower fragment of S. gordonii ATCC 10558 (Fig. 7C).The results of these three probes indicated significant diversityin their hybridization patterns with the same streptococcalgenomic digests. Probe 3 also reacted with a 3.9-kb fragment ofS. gordonii PK1804 and a 5.0-kb fragment of S. oralis C104(formerly S. sanguis C104) (35).Computer analysis revealed that a 25-amino-acid segment at

number 161 to 186 was 42% identical and 65% similar toamino acids 357 to 382 of the S. mutans P1 (I/II) surfaceantigen (32). P1 is a much larger protein of 1,561 amino acidsand contains a repeat of this region at 440 to 465, which is 34%identical and 61% similar to the 25-amino-acid segment ofScaA. Nearly the same identity and similarity were found forhomologous proteins from other mutans group streptococci;these proteins comprise a cluster of salivary agglutinin-bindingproteins in the mutans group streptococci, described above inthe introduction as the second group of streptococcal surface-exposed proteins with adherence-relevant functions (9, 32, 42,43).The binding function of ScaA appeared to be limited to

recognition of a receptor molecule on the surface of actino-myces, since cell walls of coaggregation partner strain A.naeslundii PK606 coaggregated the streptococcal parent strainbut not the COG- mutant (35). Further fractionation of thecell walls has not yet been done. More than 20 sugars havebeen tested, and none inhibit coaggregation with actinomyces(34, 35). We have tested all amino acids separately and inseveral combinations for their ability to block coaggregation ofS. gordonii PK488 with A. naeslundii PK606, and none wereeffective. Several dipeptides (Arg-Leu, Gly-Val, Gly-His, Gly-Leu, Leu-Arg), tripeptides (Gly-Gly-Leu, Gly-Gly-Ala), andprotein hydrolyzates such as Casamino Acids had no effect onthis coaggregation. Although we have not rigorously investi-gated the possibility that the ScaA lipoprotein has a transport

VOL. 62, 1994

4476 KOLENBRANDER ET AL.

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C

.Im

FIG. 7. Southern blot analysis of PstI-digested genomic DNA from various streptococcal strains using 30-mer probes corresponding either toamino acids 66 to 75 (A) or to amino acids 223 to 232 (B and C) of ScaA. The probe used in panel B is the homologous sequence in ssaB of S.sanguis 12 and differs from the probe used in panel C by one nucleotide (see the text). The strains were S. gordonii DL1 (lane 1), S. oralis Hi (lane2), S. oralis 34 (lane 3), S. oralis J22 (lane 4), Streptococcus SM PK509 (lane 5), S. gordonii PK488 (lane 6), S. sanguis 12 (lane 7), S. parasanguisFW213 (lane 8), S. sanguis ATCC 10556 (lane 9), S. oralis ATCC 10557 (lane 10), S. gordonii ATCC 10558 (lane 11), S. millei K44Y (lane 12),S. sobninus 6715-10 (lane 13), E. faecalis GF590 (lane 14), S. cnista PK1408 (lane 15), S. mutans LM7 (lane 16), S. pyogenes JRS4 (lane 17), S. rattusBHT (lane 18), S. pneumoniae R6 (lane 19), S. cicetus AHT (lane 20), S. constellatus PK2819 (lane 21), S. salivanius ATCC 25975 (lane 22), andS. intennedius PK2821 (lane 23). The two size markers (1.9 and 3.9 kb) on the left margins correspond, respectively, to the 1.9-kb reactive fragmentsin S. sanguis 12 (lane 7) and S. sanguis ATCC 10556 (lane 9) and to the 3.9-kb reactive fragments in S. gordonii DL1 (lane 1), S. gordonii PK488(lane 6), and S. gordonii ATCC 10558 (lane 11, lower fragment).

function, there was no noticeable difference in growth of theparent strain and COG- mutants, in tests under several growthconditions. These conditions included growth in brain heartinfusion and a completely defined medium (Dulbecco's mod-ified Eagle's medium; Gibco Laboratories, Grand Island,N.Y.) used for tissue culturing. Also, there was no noticeabledifference in the ability of S. gordonii PK488 to coaggregatewhen grown under different conditions (35).

Putative proteins encoded by ORF 4 and ORF 5. ORF 4encoded a 163-amino-acid protein of 17,912 Da. This proteinwas virtually identical (a different penultimate C-terminalamino acid) to the 20-kDa protein expressed from the gene

next to the ssaB gene in clone pSA2 (18) and was 78% identicalto the product of the ORF 3 gene adjacent tofimA on pVT618(14-16) (Fig. 8). Although only 50 amino acids from thehomolog from S. pneumoniae R36A were known (54), theywere 86% identical to the ORF 4 protein from S. gordoniiPK488. The closest homology of these proteins to any inGenBank was 26% identity over a 69-amino-acid overlap to abacterioferritin comigratory protein from E. coli (6). Nosignificant homology with other proteins was found with thegene product of ORF 5. This ORF could potentially encode aprotein of 25,107 Da that consisted of 229 amino acids.The putative zinc-binding endopeptidase encoded by ORF 6.

INFECT. IMMUN.

kb ;- .,

1.9_

S. GORDONII ADHESIN AND ATP-BINDING CASSElTE 4477

1 50pRAlorf4 MITFLGNPVT FTwGQLQVGD TAHDFSLTAT DLSKKTLADF AGKKKVLSIIpSA2orf4 ---------- ---------- ---------- ---------- ----------

pVT618orf3 -A-------- ---S-----E I------ITP A-E--S---- ----------

51 100pRAlorf4 PSIDTGVCST QTRRFNQELS DLDNTVVITV SVDLPFAQGK WCAAEGIENApSA2orf4 ---------- ---------- ---------- ---------- ----------

pVT618orf3 ------ I--M ---H--KT-- --ED---L-- ---------- ------ LD--

101 150pRAlorf 4 VMLSDYFDHS FGRDYAVLIN EWHLLARAVL VLDENNTVTY AEYVDNINTEpSA2orf4 ---------- ---------- ---------- ---------- ----------

pVT618orf3 I-----Y----- KA-GL-- ---------- --- AD-KI-- V--L----S-

151pRAlorf4 PDYDAAIAAV KNL. 163pSA2orf4 ---------- -S-. 163pVT6l8orf3 -N-----E-- -V-G 164

FIG. 8. Alignment of the deduced amino acid sequence of theproduct of ORF 4 from S. gordonii PK488(pRAlorf4) with homolo-gous proteins from two other streptococci, S. sanguis 12(pSA2orf4)and S. parasanguis FW213(pVT618orf3). Dashes indicate amino acidsidentical to those directly above them.

ORF 6 was an incomplete ORF, which could potentiallyencode a protein larger than 564 amino acids. At amino acids478 to 482, HEISH (Fig. 9, underline), this putative proteinhad a consensus Zn2+-binding motif (HExxH) found amongmetallo-endopeptidases (31) including the neutral endopepti-dase, human enkephalinase, EC 3.4.24.11 (37). The 217-amino-acid span containing the Zn2+-binding motif (Fig. 9)showed 58% identity to an endopeptidase, PepO, from Lacto-coccus lactis (formerly Streptococcus lactis) (41). The pepOgene is found immediately downstream and perhaps is part ofan operon involved in the transport of oligopeptides (opp)(61). The ORF 6 peptide was 40% identical to the homologousZn +-binding region, amino acids 460 to 670, of humanenkephalinase (Fig. 9). The peptide also contained a glutamicacid residue 21 amino acids downstream from the HExxHconsensus box, a property found among thermolysin andneutral peptidases (62), as well as the immunoglobulin Aprotease of S. sanguis ATCC 10556 (21). While the S. sanguisATCC 10556 immunoglobulin A protease contains 10 tandemrepeats of 20 amino acids in the amino-terminal region and is

Distancefrom

Protein N-term Sequence

1 50Orf 6 364 .ETRDKAIVK LNVIKPYIGY PDELPERYSR .KIVDENLTL .FENAQKLSLPepO 361 Q--AE---E- -DA-T-F--F --K---I--- L-TrSG .Y-D-L-FDE

EC 3.4.24.11 460 .KR-EE- ALA--ER-----DIVS ..ND N-LNN-Y-E- NYKEDEYFEN

51 100Orf6 IDIAYSWS .... KWNQPV DYKEWNPAH NVNAYYNPQK NLIVFPAAIL

PepO -LT-RTFE -....-FSED- -KTS-H---- ------S-DS -T--------EC 3.4.24.11 -IQNLKF-QS KQLK-LREK- -KD--ISG-A V---F-SSGR -Q-----G--

Orf6PepOEC 3.4.24.11

Orf6PepOEC 3.4.24.11

Orf 6PepOEC 3.4.24.11

101 Zn'+ 150

QAPFYDLHQS SSANYGGIGA VIAHEIAF D¶IGPSFDEN GSWNNWWIWTj-----S-E-- --Q ------T ---------- -N---Q--KE -N--K--LDE-P--FSAQ-- N-L------M --G---T-G- -D--RN-NRD -D-VD---QQ

151 200DYQAFTERTQ EVIDQFBGQ. DSYGAR.VN GRLTVSENVA DLGGIAAALE--E--E-EQE EM-AL-D-V. .ETEAGP.A- ---I----I- -Q---T---TSASN-R-QS- CNVY-YGNFS W-LA-GQHL- -IN-LG--I- -N--LGQ-YR

201 217AAK. .EADF SAEEF..---D. -K-V DLKA-..-YQNYIRKNG --RLLPG

FIG. 9. Alignment of a truncated portion of the deduced aminoacid sequence of the ORF 6 product with homologous truncatedsequences of PepO from L. lactis and human enkephalinase (EC3.4.24.11). The amino acid numbers from the respective N termini ofthe proteins are listed at the beginning of each truncated segment.Dashed lines indicate amino acids identical to those directly abovethem in the Orf6 sequence. Gaps (dots) are inserted into the sequences

to improve alignment. The 5-amino-acid consensus motif for bindingZn2+ is underlined and includes a glutamic acid residue at amino acid149 in the ORF 6 product.

a large protein of 186,000 Da, the ORF 6 peptide does notcontain repeating sequences. The ORF 6 peptide is likely to bethe same relative size as the PepO protein from L. lactis, sincethe locations of their homologous regions in their respectivepeptides are nearly identical (Fig. 9). Furthermore, ORF 6 andpepO are coincidentally adjacent to their respective ABCtransporter proteins.

DISCUSSION

We propose that the 6.125-kb cloned fragment from S.gordonii PK488 contains six ORFs and that ORF 1, ORF 2, andscaA encode a set of ABC transporter homologs. The putativezinc-binding endopeptidase of ORF 6 may be associated withthe physiological function of the ABC transporter proteins.The functions of the proteins potentially encoded by ORF 4and ORF 5 are unknown; however, a protein homologous tothe ORF 4 gene product is found in three other streptococcalspecies, and its gene is found in exactly the same positionrelative to the scaA homologs, suggesting that it may berelevant to the ABC proteins.The importance of ScaA and its homologs to human oral

streptococcal adherence is evident from the presence of scaAhomologs in most of the 23 strains studied here (Fig. 7). Thehybridizations were run at high stringency. We used three30-mer probes. Probe 1 corresponded to a region near the Nterminus of ScaA, while probes 2 and 3 differed by a singlenucleotide and corresponded to the homologous regions in theC-terminal half of SsaB (probe 2) or ScaA (probe 3). Probe 1appeared to be the most restrictive in the fragments that itrecognized and hybridized especially strongly with the S.gordonii strains (Fig. 7A, lanes 1, 6, and 11) as well as S. milleiK44Y, S. crista PK1408, and S. pneumoniae R6 (Fig. 7A, lanes12, 15, and 19, respectively). Probe 2 reacted with the widestvariety of strains, but, surprisingly, probe 3 did not show areaction identical to that seen with probe 2. Our initial interestin using the three probes was to examine the range of strainsreactive with probes from the two halves of scaA. It appearsthat sequences in the C-terminal half are better conservedamong streptococci, whereas probe 1, from the N-terminalhalf, reacts especially strongly with the three S. gordonii strains.The observation that two fragments of S. gordonii ATTC

10558 reacted with probe 1 but only the smaller fragmentreacted with probes 2 and 3 suggests that the smaller fragmentis better conserved among the streptococci and a closerhomolog of scaA, while the larger fragment may be a secondgene related to scaA. Clearly, S. pneumoniae R6 (Fig. 7, lane19) has close homologs ofscaA which are likely to be related tothe scaA homolog reported for S. pneumoniae R36A (54). AliAand AliB, two lipoproteins highly homologous to AmiA, haverecently been reported in S. pneumoniae, supporting the notionthat the pneumococcus has multiple genes for oligopeptidetransporter proteins (1). Probe 2, and probe 3 to a lesserextent, reacted with fragments from several strains not recog-nized or poorly recognized by probe 1. This difference may inpart be explained by the number of mismatches in theirrespective 30 nucleotides. Examination of the nucleotide se-quences of ssaB, fimA, and psaA indicated that probe 1 had 7,6, and 6 mismatches, respectively, whereas probe 3 had only 1,3, and 2 mismatches, respectively, and probe 2 had 1, 2, and 3mismatches, respectively, for scaA, fimA, and psaA (35). Ex-tending this observation to the survey of other scaA homologsamong the streptococci (Fig. 7), it appears that scaA homologsare conserved among a wide variety of streptococci includingnoncoaggregating strains, but none of the three probes hybrid-ized to S. sobrinus 6715-10, E. faecalis GF590, S. mutans LM7,

VOL. 62, 1994

4478 KOLENBRANDER ET AL.

and S. rattus BHT (Fig. 7, lanes 13, 14, 16, and 18, respectively).Among these four strains, two mutans group streptococci,namely S. sobrinus 6715-10 and S. mutans LM7, did showcoaggregation withA. naeslundii PK606, the reference strain ofactinomyces coaggregation group D. It may be that thesemutans group streptococci mediate these coaggregationsthrough the antigen I/II group of polypeptides identified onseveral of these strains (32, 42, 43). We showed here that thereis 42% identity in a short segment of the S. mutans P1 surfaceantigen (32); the significance of this relatedness will requireadditional study.We reported previously that a homologous adhesin was

expressed in all coaggregation-positive streptococci (5). In ourcurrent study, we showed that scaA homologs were found insome of the noncoaggregating streptococci (S. parasanguisFW213, S. pyogenes JRS4, S. constellatus PK2819, and S.salivarius ATCC 25975), but it depended upon which of thethree probes were used to screen the genomic digests of thestreptococci. Their gene products may be adhesins with slightlydifferent specificities and, thus, may constitute a family ofScaA-related adhesins with functional heterogeneity. A familyof proteins related to PsaA of S. pneumoniae R36A was foundamong the 24 serotypes of S. pneumoniae examined but not inany of the 55 heterologous strains representing 19 genera and36 species of pathogens that also can cause acute lowerrespiratory tract disease and may colonize the nasopharynx(51, 54). The homolog FimA from S. parasanguis FW213, anoncoaggregating strain, is known to bind to salivary compo-nents (46). The homolog SsaB from S. sanguis 12 binds both tosalivary components (19) and to the actinomyces cells (5).ScaA has not been tested for binding to salivary components.Although some of the ScaA homologs may have considerableamino acid sequence identity and show some overlappingfunctional identity, they are functionally dissimilar.

This notion of functional heterogeneity among closely re-lated adhesins differing by as little as a single amino acidsubstitution has been carefully documented with the FimHfamily of type 1 fimbrial adhesins of E. coli (57). Some adhesinsmay have only one function, while other adhesins may have twoor more functions. This observation of multiple functions hasalso been reported by Jenkinson et al. (30), who proposed thatan S. gordonii DL1 surface protein, SspA, has the dual func-tions of recognizing salivary agglutinin and actinomyces cellsurface receptors. Additional examples of multiple functionalsurface proteins of streptococci include an enzymatically activeglyceraldehyde-3-phosphate-dehydrogenase which also bindsto fibronectin, lysozyme, actin, and myosin (48).The receptor for ScaA is likely to be a component on the

surface of A. naeslundii PK606 (34). Since S. gordonii PK488coaggregates with coaggregation group D actinomyces but notwith actinomyces of the other five coaggregation groups, itfollows that the receptor for ScaA is probably not found, atleast not exposed, on the other actinomyces. Besides its role inadherence, ScaA may function as a binding lipoprotein andrecognize soluble small molecules that can be transported bythe ABC transporter system. ScaA is not essential for growth incomplex media containing glucose, since ScaA-deficient, coag-gregation-defective mutants of S. gordonii PK488 have nonoticeable differences in growth rate or final growth yield.Other media have not been tested, although media limited inamino acids or peptides may be useful to try, since theZn2+-binding endopeptidase gene ORF 6 is adjacent to theABC genes and may function in the transport of short pep-tides.The pentapeptide HEISH in the potential ORF 6 gene

product indicates a Zn2+-binding protein, and the high degree

of identity to the pepO gene product of L. lactis and humanenkephalinase (41) suggests that the protein is a Zn2+-bindingendopeptidase. Furthermore, ORF 6 is located adjacent to thegenes encoding the ABC proteins, as is pepO of L. lactis. It isbelieved that PepO is located intracellularly (61), and it wasshown that the ABC proteins of L. lactis comprise an oligopep-tide transport system (61). Although the juxtapositioning ofthe putative endopeptidase and ABC transporter genes isidentical between S. gordonii PK488 and L. lactis, transcriptionof ORF 6 of S. gordonii is divergent from that of the putativeABC transporter genes, ORF 1, ORF 2, and scaA, whereasthese genes are transcribed in the same direction and appear tobe part of an operon in L. lactis. These results suggest that anoligopeptide transport system may lie in the direction oftranscription of ORF 6, and, therefore, ORF 6 may not be apart of the putative ORF 1, ORF 2, and scaA operon.The ScaA sequence was analyzed by comparison of the

signature sequences identified by Tam and Saier (59) for theeight binding protein families, and no homology was found. Bytheir criteria, ScaA and its homologs would be part of a familyyet to be characterized, as are the lipoproteins of Mycoplasmaspp. (11), evolutionarily close relatives of streptococci (44, 47).The prolipoproteins of Mycoplasma hyorhinis possess a recog-nition sequence AxxC instead of LxxC for cleavage by signalpeptidase 11 (66). The mature lipoproteins are exposed on thesurface and appear to be expressed as random genetic variants,perhaps to function as immune masks for other essentialfunctions of transport, adherence, or invasion (66).The anchoring of mature lipoproteins appears to occur

sequentially (23, 24) through (i) the hydrophobic N-terminalprolipoprotein signal sequence which terminates in a cysteineat about amino acid 20, (ii) the covalent attachment of diglyc-eride to the cysteine, (iii) the subsequent cleavage of the hydro-phobic tail by signal peptidase II, an integral membraneprotein that recognizes an LxxC motif, and (iv) the emergenceof the mature lipoprotein with an N-terminal anchoring sitecomposed of a lipid-modified cysteine. The lipid-modifiedcysteine is the likely reason for the N-terminal block observedin routine sequencing of these adhesins, as was reported forthe SsaB adhesin from S. sanguis 12 and the PsaA adhesin fromS. pneumoniae R36A (18, 54).Computer analysis of the sequences of ScaA, SsaB, FimA,

and PsaA showed that these four lipoproteins have significanthomology with each other but not with any other reportedprotein. Among these ScaA homologs, there appeared to betwo distinct groups. ScaA and SsaB were 91% identical, FimAand PsaA were 93% identical, and there was only 80% identitybetween the two groups. This difference between the twogroups may explain the inability of FimA-bearing (S. parasan-guis) and PsaA-bearing (S. pneumoniae) cells to coaggregatewith A. naeslundii cells (35). Each lipoprotein is about 35 kDaand surface exposed, but it remains unclear how these proteinsanchor at the surface and present their respective binding sitesto complementary receptors on partner cell surfaces. They maybe anchored in the cytoplasmic membrane and extend throughpossible thinner segments of the peptidoglycan. Alternatively,they may complex to form linear aggregates and penetrate thesurface layers. Recent evidence from our laboratory suggeststhat lipoteichoic acid, another surface-exposed polymer onstreptococci, may be involved in the presentation of a differentstreptococcal adhesin involved in intrageneric streptococcalcoaggregation (8). These results suggest that the ScaA ho-mologs also may be complexed with lipoteichoic acid through-out the peptidoglycan and presented at the surface of the cells.Additional studies with purified adhesins and monospecific

INFEcr. IMMUN.

S. GORDONII ADHESIN AND ATP-BINDING CASSETTE 4479

antibodies will provide more satisfying answers regarding thesurface-exposed nature of ScaA and its homologs.

ACKNOWLEDGMENTS

We thank H. Jenkinson, P. Fives-Taylor, and H. R. Masure for manyfruitful discussions of surface proteins, J.-P. Claverys for kindlyproviding a manuscript preprint, H. Metzger and S. Forsen for help inevaluating the structural properties of integral membrane proteins,R. D. Lunsford, C. Bouma and J. London for critical reading of themanuscript, and K. Klimpel for help with computer analyses.

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