Insertional inactivation of genes responsible for the D-alanylation of lipoteichoic acid in Streptococcus gordonii DL1 (Challis) affects intrageneric coaggregations

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.

FIG. 1

(A) Partial restriction map of the streptococcal flanking region and the left end of Tn916 from mutant G7. The location of the 60-bp G7 probe is indicated. Putative transcription orientation is from left to right. Abbreviations of restriction enzyme sites: C, ClaI; H, HindIII. (B) Comparison of deduced amino acid sequence of the 626-bp fragment from S. gordonii (panel A) with truncated sequences of DltA and DltB homologs from L. rhamnosus, S. aureus, B. subtilis, S. mutans, and S. pyogenes. Upper case reverse font indicates amino acid sequence identity in five or more proteins; lower case reverse font indicates identity among all three streptococcal strains. Regions II and III are indicated and are discussed in the text. The 60-bp G7 probe includes the sequence encoding the first 19 amino acids of region II. S. pyogenes, contig 320 (51a); S. mutans, accession no. AF049357 (55a) accession no. AF051356 (5a); B. subtilis, accession no. X73124 (21); Staphylococcus aureus, accession no. D86240 (46a) (nucleotide 3939 was changed from A to C to remove false stop codon); L. rhamnosus, accession no. U43894 (25). (C) Strategy for obtaining the ermAM insertion in the dltA gene of S. gordonii DL1. The 1,385-bp region was obtained by PCR with primers PCR1 and PCR2 (Materials and Methods). The location of the KpnI (K) and BamHI (B) restriction sites are shown. A portion of panel A is shown for orientation. The HincII site (Hc) of insertion of the 922-bp ermAM cassette is indicated. The transcriptional orientation of the cassette and the ScaI site (S) are indicated. The dltA fragment was cut with KpnI and BamHI and cloned into KpnI- and BamHI-digested pBluescript IIKS(+) (Stratagene).

FIG. 2

Incorporation of d-[¹⁴C]alanine into the parent and mutant strains. The d-alanine incorporation assay (described in Materials and Methods) was used with permeabilized cells of the indicated strains.

FIG. 3

d-Alanine ester content of S. gordonii wild-type DL1 and mutant strains PK3241 and PK3242. As described in the text, a Western blot analysis using serum with antibodies to d-alanyl esters (40) was used to detect the presence of d-alanyl esters. (A) Photograph of plate; (B) colony immunoblot.

FIG. 4

Scanning electron micrographs of DL1 (A and D) and PK3241 (B and C). The specimens shown in panels A to C were examined with a JEOL JSM-1 microscope. Bar = 1.0 μm. The specimen shown in panel D was examined with a Hitachi S-4500 microscope after fixing and coating with gold-palladium (Materials and Methods). Bar = 0.5 μm. The arrow in panel B indicates nonlinear fission and the arrows in panel C indicate multiseptate pleomorphs.

FIG. 5

Aberrant morphology of PK3241. Exponential-phase cells of DL1 (A) and PK3241 (B to D) were fixed, embedded, sectioned, and examined with a JEOL-100CX 11 microscope as described in Materials and Methods. Bars = 0.5 μm.

FIG. 6

Immunoblot analysis of sonic surface extracts of PK3241 and PK3242, ermAM insertion mutants in dltA, compared with wild-type S. gordonii DL1. The position of the putative adhesin (arrow) was determined by using prestained molecular weight standards (Bio-Rad). The standards of 112 and 84 kDa are indicated by dashes.