cis -Acting Determinant Limiting Expression of Sphingomyelinase Gene sph2 in Leptospira interrogans, Identified with a gfp Reporter Plasmid

Abstract

Many strains of the spirochete Leptospira interrogans serovar Pomona express the osmotically inducible sphingomyelinase gene sph2 at much higher levels than strains from other serovars. We developed a new green fluorescent protein (GFP) reporter plasmid to examine sph2 gene expression determinants. The vector enables the fusion of the test promoter to the ribosome-binding site and coding region of gfp. We fused the sph2 promoters from the L. interrogans serovar Lai strain 56601 and from the L. interrogans serovar Pomona strain LC82-25 to gfp to examine the molecular determinants of differential sph2 expression between the two strains. Similar to what was observed with the native sph2 genes, the introduction of the plasmids into the Lai 56601 strain resulted in near background levels of gfp expression from the Lai sph2 promoter, while the expression from the Pomona sph2 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 L. interrogans strains expressing low basal levels of sph2 and missing from Pomona strains that express sph2 at high levels. When the 17-bp sequence present upstream of the Lai sph2 promoter was deleted or scrambled, the fusion expression increased substantially. Conversely, the insertion of the 17-bp sequence upstream of the Pomona sph2 promoter diminished fusion expression. In contrast, the removal of an insertion sequence-like element that is found only in the Pomona sph2 upstream sequence had no effect on the expression from the Pomona sph2 fusion in the Lai strain. These findings demonstrate the utility of the gfp reporter plasmid in analyzing gene expression in L. interrogans. IMPORTANCE Genetic tools are needed to examine gene expression in the pathogen Leptospira interrogans. We developed a reporter plasmid that replicates in L. interrogans 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 sph2 sphingomyelinase gene in an L. interrogans serovar Lai strain. This new tool is useful for the discovery of the molecular determinants of L. interrogans gene expression.

4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogenLeptospira interrogans

“…The mutation in the coding sequence is silent. The resulting allelic constructs containing of the first 150 bp of the sigX gene were translationally fused to GFP ( 23 ), cloned into the pathogenic Leptospira vector pRAT724, and conjugated into wild-type and lomA L. interrogans . The resulting fluorescence was measured to compare background expression of the sigX gene with or without methyl groups (Figure 5B ).…”

“…All plasmid constructs used in this study are based on the leptospiral replicative vector pMaOri ( 22 ) and pRAT724 ( 23 ). A list of all strains created and used in the course of these studies can be found in Supplementary Table S4 , and the primers used in Supplementary Table S5 .…”

“…Bacteria were prepared as previously described ( 23 ) with some minor modifications. Briefly, bacteria were grown to mid-log phase as described above, and an aliquot of 2 ml was removed and centrifuged at 6500g for 10 min at room temperature.…”

Identification of σE-Dependent Promoter Upstream of clpB from the Pathogenic Spirochaete Leptospira interrogans by Applying an E. coli Two-Plasmid System

“…Therefore, it was of great interest to compare the activity of the potential clpB Li promoter (clpB Li P1) under basal and increased levels of σ E . To our knowledge, there has been only one report to date, describing the construction and utility of the gfp reporter plasmid for assessing promoter activity in L. interrogans [22]. Therefore, due to the still existing limitations of genetic tools that could be easily utilized for examining gene expression in L. interrogans, experiments were performed in E. coli cells.…”

Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes

“…Such limitations have hampered efforts to identify proteins involved in cell shape, spatial localization of proteins, and transport across the periplasmic space while accommodating flagellar rotation. To address some of the molecular mechanisms underpinning these and other cellular processes, fluorescent proteins and dyes have proven to be valuable tools, permitting detection of spirochete-host interactions (Moriarty et al, 2008; Norman et al, 2008; Dunham-Ems et al, 2009; Bockenstedt et al, 2012; Carrasco et al, 2015; Teixeira et al, 2016; Krishnavajhala et al, 2017), localization or transport of proteins within the bacterial cell (Schulze and Zuckert, 2006; Schulze et al, 2010; Xu et al, 2011; Zhang et al, 2015), or as reporter systems for gene expression (Carroll et al, 2003; Bykowski et al, 2006; Clifton et al, 2006; Eggers et al, 2006; Miller et al, 2006; Gautam et al, 2008, 2009; Whetstine et al, 2009; Aviat et al, 2010; Cerqueira et al, 2011; Grove et al, 2017; Matsunaga and Haake, 2018; Takacs et al, 2018). However, the use of fluorescent protein fusions, most commonly with green or red fluorescent protein (GFP or RFP, respectively), has limitations: the bulkiness of these proteins, typically 25–30 kDa, can lead to atypical protein localization and irregular cellular trafficking (Senf et al, 2008), and some FP alleles have specific pH or oxygen requirements that are not always compatible with the targeted location or underlying biological question.…”