Shuttle expression plasmids for genetic studies in Streptococcus mutans

Abstract

A set of shuttle plasmids containing four different constitutive promoters was generated to facilitate overexpression of foreign and native genes in streptococci, such as Streptococcus mutans. The four promoters that were chosen were: P(ami), P(spac), P(23) and P(veg). These promoters are active in many Gram-positive bacteria, and allow various levels of gene expression depending on the host bacterium. Shuttle plasmids were constructed based on two types of broad-host-range replication origins: a rolling-circle replicon (pSH71) and a theta replicon (pAMbeta1). Shuttle plasmids derived from the pAMbeta1 replicon were generated to avoid the structural and segregational stability problems associated with rolling-circle replication, since these problems may be encountered during large gene cloning. In a complementation assay, we used one such plasmid to express a gene in trans to show the utility of these plasmids. In addition, a series of plasmids was generated for the expression of recombinant proteins with an N-terminal 6xHis tag or a C-terminal Strep-tag fusion, and, using a gene derived from S. mutans, we showed a high level of recombinant protein expression in S. mutans and Streptococcus pyogenes. Since these plasmids contain broad-host-range replication origins, and because the selected promoters are functional in many bacteria, they can be used for gene expression studies, such as complementation and recombinant protein expression.