Abstract
Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci. Two genes relevant to these intrageneric coaggregations were identified by transposon Tn916 mutagenesis of Streptococcus gordonii DL1 (Challis). A 626-bp sequence flanking the left end of the transposon was homologous to dltA and dltB of Lactobacillus rhamnosus ATCC 7469 (formerly called Lactobacillus casei). A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of S. gordonii DL1. This DNA encoded d-alanine-d-alanyl carrier protein ligase that was expressed in Escherichia coli from the fosmid clone. The cloned streptococcal dltA was disrupted by inserting an ermAM cassette, and then it was linearized and transformed into S. gordonii DL1 for allelic replacement. Erythromycin-resistant transformants containing a single insertion in dltA exhibited a loss of d-alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation. This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C. J. Whittaker, D. L. Clemans, and P. E. Kolenbrander, Infect. Immun. 64:4137–4142, 1996). The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations. The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs. The natural DNA transformation frequency was reduced 10-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the dltA probe revealed the presence of this gene in most viridans streptococci. Thus, it is hypothesized that d-alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin to mediate intrageneric coaggregation.
Coaggregation of human oral bacteria is thought to occur by specific interactions between complementary surface molecules on the partner cells (64). Cell-surface adhesins on one cell type may recognize and bind to complementary receptors on the partner cell type. Most of the interactions are among members of different genera, for example Streptococcus spp. and Actinomyces spp., and are termed intergeneric coaggregations. Streptococci also participate in intrageneric coaggregations, which occur among the human oral viridans streptococci, and these coaggregations are galactoside inhibitable (38). A 100-kDa putative adhesin on the surface of Streptococcus gordonii DL1 was proposed to mediate specifically these galactoside-inhibitable intrageneric coaggregations, because streptococcal insertional mutants as well as spontaneous coaggregation-defective mutants lost specifically the intrageneric coaggregation capability and lack this protein (9, 10, 63).
A potential role of lipoteichoic acid (LTA) in coaggregation is the proper presentation of adhesins and receptors on partner cells. LTAs are macroamphiphiles that contain alditolphosphates as integral parts of the hydrophilic chain (17). Most glycerol LTAs are substituted with d-alanyl ester residues (18). Because LTA is polyanionic, it binds Ca++ ions (53) and may contribute to the proper environment for coaggregation, which requires divalent cations, especially Ca++ (38, 46). Out of a total of 86 strains examined, LTA was found in all viridans streptococci except Streptococcus mitis and Streptococcus oralis (28). Since LTA inhibits the attachment of S. gordonii to substratum-located glucan polymer, the adhesion is thought to be mediated by LTA (60). LTA is reported to mediate binding of group A streptococci to fibronectin receptors on pharyngeal epithelial cells (4), and it inhibits binding of human oral viridans streptococci to fibronectin-coated spheroidal hydroxyapatite beads (27). LTA is thought to be important in the first of two steps of adhesion to human cells; the second step is postulated to occur by a specific adhesin(s) that determines tissue tropism (24). LTA exhibits properties of an enterococcal binding substance that is recognized by a mating cell-expressing aggregation substance (16), and the resulting union of the two cell types is part of the well-studied pheromone-inducible conjugation system in Enterococcus faecalis (12, 14).
The dlt operon involved in the synthesis of the d-alanyl esters of LTA was discovered in Lactobacillus rhamnosus (25, 26, 47) and subsequently identified in Bacillus subtilis (21, 50). The operons encode, respectively, four and five genes in these organisms. The first gene, dltA, encodes the d-alanine–d-alanyl carrier protein ligase (Dcl), which catalyzes the d-alanylation of the d-alanyl carrier protein (Dcp) encoded by dltC. Dcp in turn transfers the d-alanine to a membrane acceptor for the d-alanylation of LTA. There is a coding sequence overlap between dltA and -B and between dltC and -D with putative ribosome binding sites preceding dltA and dltC (47). On the basis of the hydropathy profile of the putative DltB, it is hypothesized that DltB is located in the cytoplasmic membrane and displays 12 membrane-spanning domains. DltB is hypothesized to be involved in the efflux of activated d-alanine to the site of LTA acylation (47).
The function of the d-alanine esters has been a point of recent investigation for several genera of gram-positive bacteria. In Lactobacillus rhamnosus ATCC 7469 they appear to play a role in determining cell shape and cell septation (48), whereas in B. subtilis, the absence of d-alanine esters has no effect on ultrastructure or cell septation but does enhance autolytic and beta-lactam-induced cell lysis (62). d-Alanine esters provide limited protection to B. subtilis JH642 against methicillin but do not protect against phagocytosis and degradation of the bacterium in macrophages (61). In Staphylococcus aureus mutant strains defective in formation of d-alanine esters, the cells exhibited reduced autolysis and enhanced expression of methicillin resistance (49). Increased resistance to vancomycin in Enterococcus faecium D366 was accompanied by a doubling of the d-alanyl ester content of LTA (22). It was proposed that this event would reduce the ability of autolysins to bind to the heavily d-alanylated LTA, which may affect a later step in the pathway that triggers autolytic and beta-lactam-induced cell lysis. Insertion of ISS1 into dltD resulted in Lactococcus lactis MG1363 becoming UV-sensitive suggesting that cell envelope integrity and the ability to repair DNA are related (15). The L. lactis dltD mutant also grew more slowly and formed longer chains than the parent strain. Thus, the d-alanyl esters of LTA would appear to play a variety of roles in gram-positive organisms.
Here we report that inhibition of d-alanyl-LTA biosynthesis by specific insertional mutagenesis in S. gordonii DL1 resulted in undetectable levels of a 100-kDa putative adhesin on the cell surface, prevented intrageneric-galactoside-inhibitable coaggregation, contributed to altered cellular division and morphology, and reduced DNA transformation frequency. In contrast, intergeneric coaggregation with actinomyces was unchanged. We propose that the absence of d-alanyl esters disrupts one or more of the normal scaffolding functions of LTA in adhesin presentation on the streptococcal cell surface and in a variety of cellular processes including transformation, adherence, and cell division.
MATERIALS AND METHODS
Cultivation of bacteria.
All bacterial strains used in this study are listed in Table 1. Streptococci and actinomyces were cultured in CAMG medium (9) at 37°C under anaerobic conditions with the GasPak system (BBL Microbiology Systems, Cockeysville, Md.) (2). S. gordonii strains containing transposon Tn916 were grown on CAMG medium supplemented with 10 μg of tetracycline (Sigma Chemical Co., St. Louis, Mo.) per ml of medium. All incubations for the generation of competent S. gordonii DL1 were carried out at 37°C. Transformants in the dlt operon were obtained by the method previously described (63) by using the transformation medium of LeBlanc and Hassel (39). The resultant dlt insertion mutants were tested for competence and transformation frequency by the method of Lunsford (42). All Escherichia coli strains were cultured aerobically at 37°C in Luria-Bertani (LB) broth or on LB agar (Gibco-BRL). LB medium supplemented with ampicillin (100 μg per ml), tetracycline (4 μg per ml), erythromycin (300 μg per ml), or chloramphenicol (20 μg per ml) was used to select for E. coli strains containing various plasmids (Table 2). E. coli CG120 was cultured in medium containing 100 μg of ampicillin and 4 μg of tetracycline per ml.
TABLE 1.
Organism and strain | Relevant characteristic(s) | Reference or source |
---|---|---|
S. oralis | ||
34 | Reference strain for streptococcal coaggregation group 3 | 38 |
C104a | Reference strain for streptococcal coaggregation group 3 | 38 |
Streptococcus sp. strain SM PK509 | Reference strain for streptococcal coaggregation group 5 | 38 |
A. naeslundii ATCC 51655 (formerly called A. naeslundii PK606) | Reference strain for actinomyces coaggregation group D | 38 |
S. gordonii | ||
DL1 (Challis) | Reference strain for streptococcal coaggregation group 1 | 38 |
PK1897b | Cog−, selected with S. oralis C104 | 10 |
G7a | DL1 (Cog− Tn916 insertion mutant selected with S. oralis 34) | This study |
PK3241c | DL1 (Cog− insertion mutant containing dltA::ermAM gene disruption in chromosome) | This study |
PK3242c | DL1 (Cog− insertion mutant containing dltA::ermAM gene disruption in chromosome) | This study |
E. coli | ||
DH1 | F−recA1 endA1 gyrA96 thi-1 hsdR17 supE44 | 23 |
DH5α | F− f80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk− mk+) supE44 λ− thi-1 gyrA96 relA1 | Gibco-BRL |
XL1-Blue | supE hsdR17 recA1 endA1 gyrA46 thi relA1 lac F′ [proAB+ lacIq lacZΔM15 Tn10(Tcr)] | Stratagene |
DH10B | F−mcrA Δ(mrr hsdRMS mcrBC) endA recA | Gibco-BRL |
CG120 | DH1 containing plasmid pAM120 | D. Clewell; 19 |
DH10B + pFOS1 | DH10B containing fosmid pFOS1 | 43 |
XL1-Blue + pDAE1 | XL1-Blue containing plasmid pDAE1 | 25 |
PK3322 | DH5α containing plasmid pDC3 | This study |
PK3324 | DH10B containing plasmid pDC5 | This study |
PK3327 | DH5α containing plasmid pDC8 | This study |
PK3330 | DH5α containing pDC11 | This study |
Resistant to tetracycline (10 μg/ml).
Resistant to rifamycin (25 μg/ml), streptomycin (100 μg/ml), and spectinomycin (500 μg/ml).
Resistant to erythromycin (10 μg/ml).
TABLE 2.
Plasmid | Relevant characteristic(s) | Reference or source |
---|---|---|
pAM120 | pGL101 carrying EcoRI F′ (F::Tn916) fragment of pAM211, Tcr | D. Clewell; 19 |
pFOS1 | Fosmid, Cmr | 36 |
pBSa | Apr | Stratagene |
pDAE1 | pBS KS+ with 1,568-bp L. casei dltA gene cloned in sense mRNA orientation | 25 |
pKSerm2 | 922-bp ermAM cassette, Apr Emr | 43 |
pDC3 | pUC19 with a 7.1-kb HindIII containing the left end of Tn916 and S. gordonii DL1 flanking DNA | This study |
pDC5 | pFOS1 with 40-kb S. gordonii (Challis) insert containing the dlt operon | This study |
pDC8 | pBS KS+ with 1.4-kb BamHI-KpnI PCR fragment of S. gordonii DL1 dltA gene | This study |
pDC11 | pDC8 HincII (ermAM, blunt) | This study |
pBS, pBluescript II KS.
Bacterial cells used for coaggregation assays were pelleted by centrifugation at 10,000 × g for 10 min at 4°C, washed three times in coaggregation buffer (1 mM Tris [pH 8.0], 0.1 mM CaCl2, 0.1 mM MgCl2, 150 mM NaCl, and 0.02% NaN3), and stored in coaggregation buffer at 4°C. The visual assay for coaggregation has been described in detail elsewhere (63).
Isolation of Tn916-insertion mutants.
The procedure of Behnke (5) was followed for the transformation of S. gordonii DL1 cultures with plasmid DNA as described previously (63). Tetracycline-resistant transformants were picked onto fresh CAMG agar medium containing tetracycline and incubated at 37°C under anaerobic conditions. The colonies were screened (63) for the absence of coaggregation with Streptococcus oralis 34 by the microtiter plate assay described previously by Kolenbrander (37). Cells of coaggregation-defective cultures were frozen in a dense suspension in CAMG broth and stored at −40°C.
Recombinant DNA methods.
The procedures for preparing plasmid DNA from E. coli have been described previously (63), and genomic DNA from S. gordonii was prepared by the procedure described by Andersen et al. (2). To clone the region adjacent to the Tn916 transposon, we used the single HindIII site located 6.5 kb from the left end (19). The 7.1-kb fragment from HindIII-digested (New England BioLabs, Beverly, Mass.) genomic DNA from the Cog− Tn916 insertion mutant G7 was cloned into the HindIII site of pUC19 (Gibco-BRL, Gaithersburg, Md.) (63). The recombinant plasmid containing the 7.1-kb HindIII fragment was called pDC3 (Table 2), and the E. coli DH5α strain containing pDC3 was designated PK3322.
S. gordonii DL1 DNA flanking the left end of Tn916 on the cloned HindIII fragment in pDC3 was sequenced by dideoxy sequencing by using the Sequenase version 2.0 DNA sequencing kit (United States Biochemical, Cleveland, Ohio) and [35S]dATPαS (Dupont, NEN Research Products, Boston, Mass.). Oligonucleotide primers were prepared on a PCR-MATE model 391 DNA synthesizer (Applied Biosystems, Inc., Foster City, Calif.). A radioactively labeled probe was generated by nick translation of the 7.1-kb cloned fragment or by end labeling the 60-base oligonucleotide for G7 (5′-AACATCCTCCAGCTCGATGCGGTACGCATTAAACTTAATTTGGAAATCCATTCGACCACC-3′).
A complete genomic library of S. gordonii DL1 was constructed in the fosmid pFOS1 by using Sau3A1 partially digested genomic DNA as described previously (36, 43). The library represented in excess of 2.5 genome equivalents.
Southern analysis of dltA homologs in genomic DNAs from representative oral streptococci with S. gordonii dltA as a probe was performed according to Whittaker et al. (63) and Andersen et al. (2). Hybridizations were performed at 50°C with Quick-Hyb hybridization solution (Stratagene, La Jolla, Calif.) according to the manufacturer’s instructions. The strains tested were S. gordonii DL1, S. oralis H1, S. oralis 34, S. oralis J22, Streptococcus sp. strain SM PK509, S. gordonii PK488, Streptococcus sanguis 12, Streptococcus parasanguis FW213, S. sanguis ATCC 10556, S. oralis ATCC 10557, S. gordonii ATCC 10558, Streptococcus milleri K44Y, Streptococcus sobrinus 6715-10, Enterococcus faecalis GF590, Streptococcus crista PK1408, Streptococcus mutans LM7, Streptococcus pyogenes JRS4, Streptococcus rattus BHT, Streptococcus cricetus AHT, Streptococcus pneumoniae R6, Streptococcus constellatus PK2819, Streptococcus salivarius ATCC 25975, and Streptococcus intermedius PK2821.
Insertional inactivation of the S. gordonii DL1 dltA gene with ermAM.
Restriction sites for KpnI and BamHI were incorporated into the DNA primers with the respective 5′ and 3′ sequences of the dltA gene and used to amplify a 1.4-kb fragment of the dltA gene from pDC5 as template. The two primer sequences were as follows: PCR1, 5′-CCGGATCCTGACCTCGCTGATTAAGCCC-3′; PCR2, 5′-GGGGGTACCTCTCCTGTCGTGGTCTATGGTGGGC-3′. PCR of the S. gordonii DL1 dltA homolog was performed with the GeneAmp PCR core reagents (Perkin-Elmer Cetus, Norwalk, Conn.). pDC5 DNA (5 ng) was mixed with 1 μM each primer, 2 mM MgCl2, and 2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer Cetus) in a final reaction volume of 50 μl. The thermocycler program (Perkin-Elmer Cetus) was 4 min at 95°C; 30 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C; and a final elongation of 5 min at 72°C. The 1.4-kb dltA fragment contained a single HincII site near the center and was used to clone a blunt-ended 922-bp ermAM cassette from pKSerm2 (43). The recombinant plasmid was linearized with BamHI and used to transform S. gordonii DL1 (63) to disrupt the dltA gene with ermAM. Two transformants, PK3241 and PK3242, were chosen for study. Southern analyses of AccI and ScaI restriction endonuclease-digested genomic DNAs from strains DL1, PK3241, and PK3242 were performed according to the procedure of Whittaker et al. (63) and were conducted in order to confirm insertion of ermAM into the S. gordonii dltA homolog. Both the 0.9-kb ermAM and the 1.4-kb dltA probes were end labeled with 32P by using T4-polynucleotide kinase (Lofstrand Labs Limited, Gaithersburg, Md.).
Computer analyses.
Sequence assembly and analyses were performed with Assembly-LIGN and MacVector (International Biochemicals, Inc., New Haven, Conn.). Database searches were conducted with the BLAST algorithm (1) by using the current version of the nonredundant protein database at the National Center for Biotechnology Information (NCBI-NLM, Bethesda, Md.). Protein alignments were performed with PILEUP, GAP, and PRETTY programs (Genetics Computer Group, version 9.1, for VMS-VAX).
Immunoblot analyses.
The proteins from streptococcal surface-sonicate extracts were separated on sodium dodecyl sulfate–6% polyacrylamide gel electrophoresis gels (Novex, San Diego, Calif.) as described previously (63). Detection of the 100-kDa putative adhesin was done by previously published methods with anti-DL1 polyclonal serum that had been adsorbed with spontaneously occurring coaggregation-defective mutant strain PK1897 (9, 63).
For the determination of d-alanine esters, cells of S. gordonii were transferred to Whatman no. 42 filter paper. Cells were lysed by incubating the filter paper on Bio-Rad ultra-thick blotting paper saturated with lysis solution (10 mM EDTA [pH 8.0] and lysozyme [10 mg/ml]). The transfer of the lysate to Bio-Rad Trans-Blot polyvinylidene difluoride (PVDF) membrane was accomplished with the Bio-Rad Trans-Blot Semi-dry transfer cell. The PVDF membrane was treated with serum containing antibodies against d-alanine esters (40) and visualized with a secondary antibody (immunoglobulin G) conjugated with peroxidase. The blot was developed with the luminol reagent by using the ECL kit (Amersham, Arlington Heights, Ill.).
Streptococcal surface protein preparations.
Streptococcal cells were washed three times with distilled water by centrifugation (10,000 × g for 10 min at 4°C) and resuspended to a concentration of 0.5 g (wet weight) cells per ml of distilled water up to a maximum of 1 ml and sonicated on ice for 1 min at maximum power (50 W) with a Micro Ultrasonic cell disrupter (Kontes, Vineland, N.J.). The sonicated suspension was centrifuged at 20,000 × g for 15 min at 4°C, and the supernatant fluid was stored frozen until used. Total protein was determined with the Bio-Rad protein assay (Bio-Rad, Richmond, Calif.).
Electron microscopy.
Exponential-phase cells were fixed overnight in 0.1 M cacodylate buffer (pH 7.4) containing 2.5% (wt/vol) glutaraldehyde at 4°C. For scanning microscopy fixed samples were washed four times with 0.1 M cacodylate buffer. A drop of each sample on a glass coverslip was dehydrated in a graded series of ethanol concentrations, critical point dried, and sputter coated with gold-palladium. Specimens were examined with a JEOL JSM-1 or an ultra-high-resolution Hitachi S-4500 microscope. For transmission microscopy the fixed cells were washed four times with 0.1 M cacodylate buffer and postfixed for 1 h at 25°C with 0.5% (wt/vol) osmium tetroxide–0.8% (wt/vol) potassium ferricyanide in 0.1 M cacodylate (pH 7.4). After washing the samples three times with water, they were stained with 0.5% (wt/vol) uranyl acetate for 1 h at 25°C and washed three more times. The samples were pelleted in 2% (wt/vol) agar, cut into 1-mm cubes, and dehydrated in increasing concentrations of ethanol (34). The samples were infiltrated with Spurr’s resin (56), embedded in BEEM capsules, and sectioned (80 nm, silver) on a Reichert Ultracut-E. The sections were poststained with 1% (wt/vol) uranyl acetate for 20 min and examined with a JEOL JEM-100CX 11 microscope at 80 kV.
d-Alanine incorporation assay.
Incorporation of d-[14C]alanine into toluene-treated cells was performed in a reaction mixture (20 μl) which contained 30 mM bis-Tris (pH 6.5), 10 mM ATP, 10 mM MgCl2, 1 mM dithiothreitol, 0.11 mM d-[14C]alanine (43 mCi/mmol), and 4 μl of permeabilized cell suspension. For the preparation of permeabilized S. gordonii, a modification of the procedure of St. Martin and Wittenberger (57) was used. The cells (0.65 gm/ml), suspended in 100 mM phosphate buffer (pH 7.0), were treated with 0.15 volumes of toluene-acetone (1:9 [vol/vol]) for 1 min of vigorous agitation on a Vortex mixer. The agitation was repeated three more times with intermittent cooling in an ice bath. The reaction mixture was incubated at 37°C, and the reaction was terminated by the addition of 0.9 ml of 30 mM bis-Tris (pH 6.5). The labeled cells were collected on a GN-6 Metricel filter and washed with three 1 ml and one 10 ml portion of 30 mM bis-Tris (pH 6.5). The filters were dissolved in ethyl acetate and assayed for radiolabel.
Nucleotide sequence accession number.
The assigned GenBank accession number is AF059609.
RESULTS
Isolation of S. gordonii DL1 Cog− mutant.
Transposon mutagenesis was conducted (63) to isolate Cog− mutants that failed to coaggregate with streptococcal partners of the wild-type strain DL1. Transposon Tn916 was inserted into the chromosome of S. gordonii DL1 by transformation of strain DL1 with the donor plasmid pAM120 (19, 63). This process yielded mutant strain G7, which lost specifically the ability to coaggregate with streptococcal partners while exhibiting wild-type levels of coaggregation with an actinomyces partner (Table 3). Intergeneric coaggregations of the mutant or wild type with Actinomyces naeslundii ATCC 51655 are not inhibited by lactose, whereas the intrageneric coaggregations between the wild-type DL1 with S. oralis 34, S. oralis C104, and Streptococcus sp. strain SM PK509 are inhibited by lactose (38). In these galactoside-inhibitable coaggregations, S. gordonii DL1 was sensitive to heat and protease treatment and was presumed to contain a putative adhesin which recognizes a carbohydrate-containing receptor on the surface of its partner strains (38). On the basis of the current work with Cog− mutant G7, it was discovered that Tn916 inserted into a new coaggregation-relevant locus, which is different from the locus in the Cog− insertion mutant described previously by Whittaker et al. (63).
TABLE 3.
Strain | Coaggregation scorea with:
|
|||
---|---|---|---|---|
S. oralis 34 | S. oralis C104 | Streptococcus sp. strain SM PK509 | A. naeslundii ATCC 51655 | |
DL1 | 30 | 30 | 30 | 44 |
G7 | 0 | 0 | 0 | 44 |
PK3241 | 0 | 0 | 0 | 44 |
PK3242 | 0 | 0 | 0 | 44 |
The method for assigning coaggregation scores has been described by Kolenbrander, Andersen, and Moore (38). The maximum score is 4; no coaggregation is given a zero score. Coaggregation scores are given in two parts: the first score is that given after mixing the two strains together, and the superscript is the score after adding lactose (final concentration, 60 mM) to the coaggregates.
Cloning and sequencing of the streptococcal DNA flanking the Tn916 insert.
To obtain the nucleotide sequence of the coaggregation-relevant locus, the HindIII junction fragment (7.1 kb), which included the left end of Tn916 and flanking S. gordonii DNA, was isolated and cloned into the HindIII site of plasmid pUC19 to yield pDC3. The 626-bp sequence of the streptococcal DNA flanking the left end of the HindIII fragment in strain G7 was obtained and is presented as a partial restriction map (Fig. 1A). Translation of the two partial open reading frames (ORFs) showed significant sequence identity to DltA (d-alanine–d-alanyl carrier protein ligase [Dcl]) and DltB (13, 25, 26). The two partial ORFs were 519 bp (ORF 1, dltA homolog) and 111 bp (ORF 2, dltB homolog) in length with the 3′ end of ORF 1 sharing four nucleotides with the 5′ end of ORF 2. A similar overlap of dltA and dltB in the dlt operon from L. rhamnosus was reported by Heaton and Neuhaus (25). Both ORFs appear to be transcribed towards the Tn916 insertion with dltB being directly interrupted by the transposon insert. To examine the possibility that there was more than one streptococcal locus homologous to dltA from L. rhamnosus, a 60-bp probe (Fig. 1A) was used for Southern blot analysis of BamHI-, ClaI- and HindIII-digested genomic DNA from S. gordonii. A single hybridizing band was observed in genomic digests with each of the three restriction enzymes (data not shown), suggesting a single locus for the dlt operon. The 60-bp probe (G7) hybridized with L. rhamnosus dltA cloned into pDAE1. These data confirmed the presence of a single homologous sequence in S. gordonii.
The translated sequences of truncated dltA and dltB were aligned (Fig. 1B) with corresponding proteins from other bacteria by using PILEUP (20). DltA consensus regions II and III (Fig. 1B) described by Heaton and Neuhaus (25) are conserved. These regions are characteristic of nonribosomal-peptide condensation via a thiolester intermediate (25). Alignment of the truncated fragments of DltA and DltB of S. gordonii DL1 by using GAP (20) showed that DltA was 41, 44, 50, 75, and 79% identical to DltA of S. aureus, B. subtilis, L. rhamnosus, S. pyogenes, and S. mutans, respectively. Truncated DltB also shows significant similarities with the DltB of other organisms.
Cloning and expression of the S. gordonii DL1 dltA.
To determine if a functional dltA gene product could be expressed from a cloned streptococcal dltA, we used the 60-bp G7 probe to locate the putative dltA locus in the fosmid-constructed genomic library of wild-type S. gordonii DL1 (Challis). Three fosmids hybridized with the 60-bp G7 probe; one was selected for further study and designated pDC5. Recombinant E. coli PK3324 containing the fosmid pDC5 exhibited Dcl activity (6.2 U/mg of protein) which was comparable to that of the cloned L. rhamnosus dltA in E. coli (11.2 U/mg of protein). These data indicated that the cloned streptococcal dltA in pDC5 encodes Dcl.
Insertional inactivation of the S. gordonii DL1 dltA with ermAM.
To prepare plasmid for insertional inactivation, it was necessary to sequence dltA. Fosmid pDC5 was chosen as the template for sequencing the 5′-region flanking the 626-bp fragment described in Fig. 1A. From the sequence two PCR primers were constructed to amplify a fragment containing partial dltA (Fig. 1C). This product, which hybridized with the 60-bp G7 probe, was cloned into pBluescript to form pDC8. To insertionally inactivate dltA, the 0.9-kb streptococcal erythromycin resistance determinant (ermAM) was ligated into the unique HincII site (Fig. 1C, Hc) within the 1.4-kb fragment of cloned DNA in pDC8 (43, 45), and the resulting plasmid was designated pDC11. By digesting this plasmid with ScaI, which cuts the ermAM cassette asymmetrically (Fig. 1C), it was shown that the ermAM determinant was cloned in the opposite transcriptional orientation to the dltA. Linearized plasmid pDC11 DNA was transformed into S. gordonii DL1. Two Emr isolates, PK3241 and PK3242 (Table 3), unable to coaggregate with streptococcal partners but still able to coaggregate with A. naeslundii ATCC 51655, were identified and used for further study.
To confirm that ermAM had integrated into the chromosome, DNA was prepared from PK3241 and PK3242 and the wild-type strain DL1 and digested with either AccI or ScaI. AccI does not cut within dltA or ermAM, while ScaI yields a single asymmetrical cut within ermAM (Fig. 1C). Southern analysis using either the ermAM determinant or the 1.4-kb S. gordonii DL1 dltA homolog as probe revealed that both the ermAM and dltA probes recognized identical bands after digestion of the genomic DNA of PK3241 and PK3242 (data not shown). When the dltA probe was used, the ScaI digest of DL1 showed a higher-molecular-size band than found in the mutants, and the AccI-digested DNA showed a lower-molecular-size band in the parent compared to those of the mutants. No hybridization was seen in S. gordonii DL1 wild-type genomic DNA blots probed with ermAM. These data indicated that ermAM integrated into dltA in transformants PK3241 and PK3242.
Characterization of Emr S. gordonii DL1 dltA insertion mutants PK3241 and PK3242.
The insertion mutants exhibited an inability to participate in galactoside-inhibitable coaggregation with other streptococci while maintaining strong noninhibitable coaggregations with actinomyces, for example A. naeslundii ATCC 51655 (Table 3). The wild-type strain DL1 coaggregated strongly with the streptococcal partners, and these coaggregations were reversed by adding lactose. Also, the growth of the mutants was slower than that of the wild type, with doubling times at 37°C for S. gordonii DL1, PK3241, and PK3242 of 51, 96, and 117 min, respectively. Thus, insertional inactivation of dltA results both in a loss of galactoside-inhibitable coaggregation and in a slowing of the growth rate compared with that of the parent strain DL1.
The insertion mutants PK3241 and PK3242 were deficient for d-alanine incorporation into LTA (Fig. 2). To define further the defect(s) in the incorporation system of the mutants, we (i) assayed the activity of Dcl in each of the mutant supernatant fractions and (ii) assayed either the supernatant fraction or membrane fraction in the presence of its cognate fraction from DL1 for d-alanine incorporation. No significant d-alanine activation as measured by the hydroxamate assay was observed (Table 4) and thus, it was concluded that Dcl is absent in the mutants. However, further analysis of the membrane fractions revealed that membranes from the mutants were also not able to reconstitute d-alanine incorporation with the supernatant fraction from the parent. This result is most likely the consequence of translational coupling of dltA and dltB (47); the polar effect of the dltA insertion causes a lack of dltB expression. Since DltB is a putative membrane protein involved in d-alanine incorporation, insertional inactivation of dltA could also lead to inactive membranes for d-alanine incorporation.
TABLE 4.
Strain | D-[14C]alanine incorporation (cpm)
|
Dcl (U/mg)c | ||
---|---|---|---|---|
M + S | Mm + Sp | Mp + Sm | ||
DL1 | 726 | NAb | NA | 0.530 |
PK3241 | 6 | 47 | 52 | 0.065 |
PK3242 | 21 | 29 | 33 | 0.015 |
Data are averages of two experiments; membranes and supernatant fractions were prepared as described by Ntamere et al. (48). M (membrane fragment), 2.6 μg of protein/μl; Mp, membrane fragment preparation from parent strain DL1; Mm, membrane fragment preparation from dltA mutant strain; S (supernatant fraction), 2.6 μg of protein/μl; Sp, supernatant fraction from parent strain DL1; Sm, supernatant fraction from dltA mutant strain.
NA, not applicable.
Dcl was assayed according to the procedure described by Heaton and Neuhaus (25). One unit of enzyme activity is defined as that amount of enzyme that catalyzes the formation of 1 μmol of d-alanine hydroxamate per h. Specific activity is expressed as the number of units per milligram of protein.
In accord with the absence of Dcl activity, the mutants have no detectable d-alanyl LTA on their cell surface (Fig. 3). To monitor the presence of d-alanyl esters in the LTA of S. gordonii DL1 and the mutant strains, a serum preparation containing antibody to d-alanyl LTA (40) was used. As shown in the immunoblot in Fig. 3, mutants PK3241 and PK3242 are both deficient. The low background in the mutants may reflect the presence of antibody(s) for poly (glycerol phosphate) or other surface antigens.
The morphologies of DL1 and PK3241 were compared in scanning electron micrographs of exponential-phase cells (Fig. 4). The cell surface of the mutant PK3241 (Fig. 4B and C) appears smooth and nonstructured compared with that of the parent DL1 (Fig. 4A and D). In addition, a significant number of the cells appear to be multiseptated (Fig. 4C). In many cases, the septa are not parallel, leading to nonlinear binary fission (Fig. 4B). Multiseptation leads to “dumbbell-like” pleomorphs similar to those observed by Pucci et al. (51) in Enterococcus hirae ATCC 9790 (formerly called Streptococcus faecalis ATCC 9790) when treated with sublytic concentrations of beta-lactam antibiotics. For a more detailed morphological analysis of PK3241, transmission microscopy of sections was also used (Fig. 5). In the parent DL1, the new septal planes were formed parallel to the cell division plane and occurred at the midpoint of the elongating cells (Fig. 5A). A heavily stained layer was prominent in DL1 but was less obvious in the mutant PK3241 (Fig. 5B to D). The mutant exhibited a thick but uneven surface layer that was lightly stained. The reason for the difference in staining of this layer in the parent and mutant is unknown. In Fig. 5B the nascent divisional septa are not parallel to the completed septum, while the pleomorph in Fig. 5C has three septation sites at approximately the same stage of development. In Fig. 5D, the cells are poorly separated. This range of cellular morphologies as well as the related division-plane locations and geometry leads one to speculate that autolysins may not function properly. Thus, these results illustrate a number of pleiotropic defects resulting from insertional inactivation of dltA.
The two insertion mutants were tested for the presence of the putative 100-kDa adhesin that has been linked to mediating intrageneric coaggregations of the wild-type DL1 (9). This was done by immunoblot analysis as described previously (63) by examining the cell surface proteins obtained by mild sonication of cells (Fig. 6). This adhesin (Fig. 6) is present in the wild-type DL1 but is absent in the lanes containing cell surface proteins of the two insertion mutants PK3241 and PK3242. Some differences in the banding patterns of proteins with molecular sizes below 84 kDa are visible. However, only the 100-kDa protein is consistently absent (9, 63) in all mutants unable to exhibit intrageneric coaggregation, including PK3241 and PK3242 studied here. The data show that insertional inactivation of dltA results in the loss of this adhesin.
To test the possibility that the mutation in dltA may modulate the activity of other surface functions, we compared the ability of the parent and mutants to be transformed with homologous DNA from a spontaneous Smr mutant of DL1 (44). The transformation frequency of the parent was 10−3 per viable cell as expected (41); the transformation frequency of the mutants was 10-fold lower. Given the noticeable difference in cell septation and morphology in the mutants, this reduction may represent changes in the segregation of transformants or possibly a global effect on surface function.
Southern blot analysis of streptococcal genomic DNA digested with PstI and probed with the 1.4-kb dltA probe showed the presence of reactive fragments in all streptococci except S. sanguis ATCC 10556 and S. oralis ATCC 10557 (data not shown). Hybridizing bands of the same size were found for S. gordonii DL1, S. oralis H1, S. oralis 34, S. milleri K44Y, S. rattus BHT, and S. salivarius ATCC 25975. Another cluster of strains showing the same-size reactive fragment included S. mutans LM7, S. pneumoniae R6, S. cricetus AHT, and S. constellatus PK2819. Further studies will be necessary to characterize the dlt operon structure in these organisms.
DISCUSSION
This is the first report of a mutation in the dlt operon resulting in altered adherence properties. The dltA mutants, PK3241 and PK3242, of S. gordonii DL1 specifically lost the ability to participate in intrageneric coaggregations while maintaining intergeneric coaggregations. The concomitant loss of the 100-kDa putative adhesin suggests that this protein binds to d-alanyl LTA. LTA has been implicated in the adhesion of a number of gram-positive bacteria to a variety of target cells and surfaces (6, 24, 58). In these examples, the participation of LTA as a direct mediator of adhesion has been postulated. In contrast, the present report describes the function of a putative adhesin which binds to d-alanyl-LTA and is presented to its partner cell. Thus, the d-alanyl esters would appear to play a role in determining the binding and presentation of this adhesin.
Some cell surface proteins of S. gordonii may bind to d-alanyl LTA in a way analogous to the binding of choline-binding proteins of S. pneumoniae (54, 65). Among the family of surface-located choline-binding proteins on S. pneumoniae is CbpA, a 75-kDa adhesin and virulence determinant. The family of proteins is noncovalently bound to the phosphoryl-choline of the wall teichoic acid. By analogy, the 100-kDa putative adhesin that is lacking in the S. gordonii DL1 mutants studied here may normally bind to the d-alanine-substituted LTA. In support of a weakly bound protein is the observation that mild sonication of parent DL1 cells is sufficient to remove the 100-kDa protein and render them unable to participate in galactoside-inhibitable coaggregation with streptococci but still capable of other galactoside-noninhibitable coaggregations with actinomyces (data not shown). In this regard, the d-alanyl LTA may act as a scaffolding for presenting the bound 100-kDa protein on S. gordonii surface.
Another important class of protein ligands which bind to LTA are autolysins. These proteins are cell wall hydrolases, e.g. MurNAc l-ala amidase, which the bacterium must regulate for growth (11, 29). Gel-permeation chromatography demonstrated the binding of the amidase to LTA (30). In B. subtilis, insertional inactivation of the genes in the dlt operon results in an increased rate of autolysis (61, 62). The increased negative charge of LTA in the mutants resulting from a decrease in d-alanylation appeared to increase the amount of autolysin (s) bound. While the mechanism of adhesin binding would appear to be different from that of autolysin binding, the conclusion is made that d-alanylation of LTA provides a feature for regulating the ability of LTA to bind selected protein ligands.
Interestingly, many S. gordonii and S. sanguis strains in biovars that are positive for LTA (the Lancefield group H antigen [52]) also are positive for galactoside-sensitive adhesins detected by intrageneric coaggregation (32, 38). The group H antigen occurs in most strains of S. gordonii and S. sanguis and not at all in strains of S. oralis and S. mitis (35). These latter species include strains with GalNAc-containing cell wall polysaccharides, which are the receptors for the galactoside-sensitive adhesins, including the putative 100-kDa adhesin on S. gordonii DL1 (32, 38). Significantly, all streptococcal strains that are positive for the group H antigen are negative for GalNAc-containing cell wall polysaccharides and visa versa (7, 32). These findings implicate an association between the galactoside-sensitive 100-kDa putative adhesin and the group H antigen on the streptococcal cell surface. Moreover, the anti-DL1 polyclonal serum absorbed with spontaneous Cog− mutant PK1897 and used to detect the 100-kDa putative adhesin in DL1 (see Fig. 6) also identifies a 100-kDa protein in other streptococcal strains that also possess the group H antigen (9, 63). This absorbed antiserum does not react with S. oralis strains (9). The correlation of the reactivity of both anti-adhesin and anti-LTA sera with the same strains strongly supports the association of 100-kDa putative adhesin with LTA.
Previous results with d-alanine ester-deficient mutants of L. rhamnosus showed aberrant morphology and defective cell separation (48). However, these mutants may have resulted from multiple mutations or single mutations with pleiotropic effects. The results with the S. gordonii mutants show a correlation between d-alanine ester deficiency resulting from insertional inactivation of dltA and aberrant cell morphology, slower growth rate, and defective cell separation. Inactivation of the dltD of L. lactis also results in a mutant that grows more slowly and forms longer chains than the wild-type strain (15). In contrast to these results, mutations in dltA-dltD of B. subtilis did not result in changes in cellular morphology, cell growth, basic metabolism, and formation of flagella (61, 62). The only changes correlated with d-alanine ester deficiency in this organism were an enhanced rate of autolysis and a higher susceptibility to methicillin (61). Thus, at this time it cannot be concluded that d-alanine ester deficiency will have the same effect in each gram-positive organism.
A wide range of species exhibited dltA-reactive fragments in Southern blots (data not shown). Out of 23 strains tested, all but one strain of S. sanguis and one strain of S. oralis were positive for dltA. The surprising finding was that S. pneumoniae, S. oralis, and S. mitis possess dltA but do not have d-alanine esters of LTA. In fact, S. pneumoniae has the entire dlt operon (3). The expression of the dlt operon may be silent or may occur under environmental conditions that have not been tested in the laboratory. S. oralis and S. mitis do not express detectable amounts of glycerol LTA (28, 35), but S. oralis can incorporate radiolabelled choline into membrane components, suggesting that it may produce a choline-containing macroamphiphile similar to that of S. pneumoniae (31). Alternatively, these three species may use the dlt operon for d-alanylation of a different molecule or macroamphiphile than LTA (8). One possibility has been suggested for the DltA-DltD homologs of S. mutans (accession no. AF049357): mutants in genes encoding these proteins accumulate elevated levels of intracellular polysaccharide in the presence of fructose or sucrose (55). When grown in continuous culture, S. mutans Ingbritt produced two to six times as much LTA during growth on fructose compared to glucose, depending on the generation times of the cells (33). And, the LTA content of strain Ingbritt increased four- to fivefold when the pH of the culture medium was raised from 5.0 to 7.5, irrespective of the carbon source (33). Thus, environmental factors can greatly influence the amounts of LTA in streptococcal cells. A recent entry into GenBank, accession no. AF051356, reports a potential relationship of defects in the dlt operon to acid sensitivity in S. mutans (59). Taken collectively, the properties of adherence, intracellular polysaccharide accumulation, and acid sensitivity all being affected by mutations in the dlt operon suggest that the LTA macroamphiphile may act as a supporting matrix or scaffolding for binding of a family of proteins with specific functions for sensing the streptococcal environment.
ACKNOWLEDGMENTS
D.L.C. was supported by a postdoctoral fellowship awarded by the Warner-Lambert Company. This study was supported by Public Health Service grant R01 GM51623 (F.C.N.) from the National Institute for General Medical Sciences.
We are indebted to E. W. Minner for his generous help in the Electron Microscopy Facility of the Department of Neurobiology and Physiology, Northwestern University. We thank V. Dravid, Material Sciences, for the specimen examined in the Hitachi S-4500 microscope. We thank M. Levine, University of Oklahoma at Oklahoma City Health Sciences Center, for serum samples containing antibody specific for the d-alanyl esters of LTA and for discussions of the use of these sera; H. F. Jenkinson for useful discussions on the preparation and properties of LTA from oral streptococci; E. J. St. Martin for suggesting the novel procedure for permeabilization of S. gordonii; J. London, N. Ganeshkumar, and J. Cisar for helpful comments in preparing the manuscript; and R. Andersen for technical assistance.
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