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

Most human oral viridans streptococci participate in intrageneric coaggrega tions, 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 Lactobacill us casei). A 60-kb probe based on this Banking sequence was used to Identif y the homologous DNA in a fosmid library of S. gordonii DL1. This DNA encod ed D-alanine-D-alanyl carrier protein ligase that was expressed in Escheric hia 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 trans formants 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 s pecificity of the mutation in altering intrageneric coaggregations. The mut ants were altered morphologically and exhibited aberrant cell septa in a va riety of pleomorphs. The natural DNA transformation frequency was reduced 1 0-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the dltA probe revealed the presence of this ge ne in most viridans streptococci, Thus, it is hypothesized that D-alanyl LT A may provide binding sites for the putative 100-kDa)a adhesin and scaffold ing for the proper presentation of this adhesin to mediate intrageneric coa ggregation.