Zoocin A facilitates the entry of antisense constructs into Streptococcus mutans.

The use of antisense oligodeoxyribonucleotides (asODNs) to inhibit gene function has proven to be an extremely powerful tool for establishing gene-function relationships. Diffusion limitations imposed by the thick peptidoglycan layer of Gram-positive bacteria have proven difficult to overcome for permeability of asODNs. Typically, introduction of the asODN is achieved by cloning the antisense sequence into a vector downstream of an inducible promoter and transforming this construct into the cell of interest. In this study, we report that the use of the streptococcolytic enzyme zoocin A facilitated entry of phosphorothioate oligodeoxyribonucleotides (PS-ODNs) into Streptococcus mutans, such that the degree of phenotypic response (cell growth inhibition) observed was sequence specific and correlated with the amount of zoocin A (R(2) =0.9919) or PS-ODN (R(2) =0.9928) used. Quantitative reverse transcriptase PCR was used to demonstrate that only the expression of the target gene against which the PS-ODN was designed was affected. We believe that the use of an appropriate bacteriolytic enzyme to facilitate entry of asODNs into bacterial cells provides a method that will be generally useful in the study of gene regulation in Gram-positive bacteria.