Identification of a cis-acting determinant limiting expression of sphingomyelinase genesph2inLeptospira interroganswith agfpreporter plasmid

Abstract

1Many Pomona strains of the spirochete Leptospira interrogans express the osmotically-2 inducible sphingomyelinase gene, sph2, at much higher levels than non-Pomona strains. We 3 developed a new green fluorescent protein (GFP) reporter plasmid to examine sph2 gene 4 expression determinants. The vector allows fusion of the test promoter to the ribosome-binding 5 site and coding region of gfp. We fused the sph2 promoters from the serovar Lai strain 56601 6 and from the serovar Pomona strain LC82-25 to gfp to examine the molecular determinants of 7 differential sph2 expression between the two strains. Similar to what is observed with the native 8 sph2 genes, introduction of the plasmids into the Lai 56601 strain resulted in near background 9 levels of gfp expression from the Lai sph2 promoter, while expression from the Pomona sph2 10 promoter was high. Expression from both fusions increased at physiologic levels of osmolarity 11 achieved by addition of sodium chloride to the culture medium. We examined the role of a 17 12 bp upstream element found in all L. interrogans strains expressing low basal levels of sph2 and 13 missing from Pomona strains that express sph2 at high levels. When the 17 bp sequence 14 present upstream of the Lai sph2 promoter was deleted or scrambled, fusion expression 15 increased substantially. Conversely, insertion of the 17 bp sequence upstream of the Pomona 16 sph2 promoter diminished fusion expression. In contrast, removal of an insertion sequence-like 17 element that is found only in the Pomona sph2 upstream sequence had no effect on expression 18 from the Pomona sph2 fusion in the Lai strain. These findings demonstrate the utility of the gfp 19 reporter plasmid in analyzing gene expression in L. interrogans. 20 21 IMPORTANCE 22Genetic tools are needed to examine gene expression in the pathogen Leptospira 23 interrogans. We developed a reporter plasmid that replicates in L. interrogans with green 24 fluorescent protein (GFP) as the read out of promoter activity. We demonstrated an application 25 of the new reporter plasmid by identifying an upstream element responsible for the poor basal 26 3 expression of the sph2 sphingomyelinase gene in a serovar Lai strain of L. interrogans. This 27 new tool is useful for discovery of the molecular determinants of L. interrogans gene expression. 28