Zoocin A facilitates the entry of antisense constructs into Streptococcus mutans

Abstract

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.