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 inducibl e PU promoter of the S. pneumoniae malMP operon which, in turn, is regulate d 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 re gulator in cis. Pneumococcal cells harboring this vector gave a linear resp onse of GFP synthesis in a maltose-dependent mode without detectable backgr ound levels in the absence of the inducer. (C) 2000 Academic Press.