Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA Correspondence Indranil Biswas ibiswas{at}kumc.edu 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 (pAM β 1). Shuttle plasmids derived from the pAM β 1 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 6 x His 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. Abbreviations: CSP, competence-stimulating peptide; GUS, β -glucuronidase; MCS, multiple-cloning site; MU, Miller units; RCR, rolling-circle replicon