-Acting Determinant Limiting Expression of Sphingomyelinase Gene in Leptospira interrogans, Identified with a Reporter Plasmid.

Many strains of the spirochete serovar Pomona express the osmotically inducible sphingomyelinase gene at much higher levels than strains from other serovars. We developed a new green fluorescent protein (GFP) reporter plasmid to examine gene expression determinants. The vector enables the fusion of the test promoter to the ribosome-binding site and coding region of We fused the promoters from the serovar Lai strain 56601 and from the serovar Pomona strain LC82-25 to to examine the molecular determinants of differential expression between the two strains. Similar to what was observed with the native genes, the introduction of the plasmids into the Lai 56601 strain resulted in near background levels of expression from the Lai promoter, while the expression from the Pomona promoter was high. The expression of both fusions increased at physiologic levels of osmolarity achieved by adding sodium chloride to the culture medium. We examined the role of a 17-bp upstream element found in all strains expressing low basal levels of and missing from Pomona strains that express at high levels. When the 17-bp sequence present upstream of the Lai promoter was deleted or scrambled, the fusion expression increased substantially. Conversely, the insertion of the 17-bp sequence upstream of the Pomona promoter diminished fusion expression. In contrast, the removal of an insertion sequence-like element that is found only in the Pomona upstream sequence had no effect on the expression from the Pomona fusion in the Lai strain. These findings demonstrate the utility of the reporter plasmid in analyzing gene expression in Genetic tools are needed to examine gene expression in the pathogen We developed a reporter plasmid that replicates in with green fluorescent protein (GFP) as the readout of promoter activity. We demonstrated an application of the new reporter plasmid by identifying an upstream element responsible for the poor basal expression of the sphingomyelinase gene in an serovar Lai strain. This new tool is useful for the discovery of the molecular determinants of gene expression.