Insertional inactivation of binding determinants of Streptococcus crista CC5A using Tn916

Dental plaque is a complex microbial ecological system in which the temporal changes from the initial pioneer community to the final climax community (20) follow a recognized bacteriological pattern. Among the major pioneer populations associated with these changes are species in the sanguis group of streptococci. This group is of interest because it contains some species with higher affinity for salivary pellicle and some that bind to other bacteria. These distinctive activities provide a biological basis for the formation of the plaque community by allowing incoming bacteria to fix to the tooth surface and grow to form the mature climax community. In the absence of these binding capacities, the bacteria would be dislodged by the mechanical forces in the mouth. Without some form of physical removal, these communities eventually induce the inflammatory changes associated with some types of periodontal disease. One of the major efforts for control of this disease is directed at removal and/or control of dental plaque formation and maturation (11). Such control could be achieved with agents that block various phases of the attachment process. It is generally believed that the adhesins responsible for the attachment process are located in the fimbriae of the streptococci. One of the most unusual fimbrial arrangements is that found in the streptococci associated with “corncob” formation. This is a distinctive two-cell microbial community that resembles an ear of corn and is a prominent feature of dental plaque. The corncob consists of a central filamentous organism surrounded by streptococci bound to the filament by a polar tuft of fimbriae. In previous studies (15, 16), we have shown that the central filaments could be either the gram-positive, aerobic species Corynebacterium matruchotii (formerly Bacterionema) or the gram-negative, anaerobic bacterium, Fusobacterium nucleatum. The species designation of the streptococci associated with corncobs was recently changed from Streptococcus sanguis to Streptococcus crista (9). The relative simplicity of this community as well as its distinctive morphology are properties that make the complex an ideal model for the study of both the binding process as well as the possible communication that may take place between the partners in this bi-cellular complex. We have concentrated on the fusobacterial corncobs because this model might be archetypal of mechanisms associated with the conversion of the pioneer plaque community to the climax community dominated by anaerobic bacteria. Our initial studies have focused on the adhesins of S. crista. We hypothesized that if the synthesis or assembly of the polar fimbriae of S. crista were altered then corncob formation would be disrupted. Theoretically, such disruption could be accomplished by using a transposon to direct the insertional inactivation of genes associated with adhesin synthesis. Tn916 is a conjugative transposon that is able to insert at different sites in a range of bacterial species (2, 5). The insertion of Tn916 into a gene inactivates it. Gawron-Burke & Clewell (7) have suggested that these transposable elements would be useful for the targeting of genes and their subsequent cloning. This strategy has been used to identify a number of genes in gram-positive bacteria (1, 4, 27). The simplest method for introducing Tn916 into a bacterium is by conjugation. However, this method was unsuccessful with S. crista. Therefore, we have concentrated on developing a transformation system to insert Tn916 into S. crista. Among the sanguis group of streptococci, only Streptococcus gordonii Challis has been transformed at a high efficiency, using either plasmid or chromosomal DNA (25). The objectives of our study were to develop a similar transformation system for S. crista and to obtain mutants of the bacterium that are deficient in binding to F. nucleatum.