Construction of a tightly regulated plasmid vector for Streptococcus pneumoniae:: Controlled expression of the green fluorescent protein

We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible PU promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the P-M promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the P-M-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer. (C) 2000 Academic Press.