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Appl Environ Microbiol. 2018 Nov 15;84(23). pii: e02068-18. doi: 10.1128/AEM.02068-18. Print 2018 Dec 1.

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

Matsunaga J1,2, Haake DA3,2,4,5.

Author information

1
Research Service, VA Greater Los Angeles Healthcare System, Los Angeles, California, USA jamesm@ucla.edu.
2
Department of Medicine, UCLA, David Geffen School of Medicine, Los Angeles, California, USA.
3
Division of Infectious Diseases, VA Greater Los Angeles Healthcare System, Los Angeles, California, USA.
4
Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, California, USA.
5
Department of Urology, UCLA, Los Angeles, California, USA.

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.

KEYWORDS:

Leptospira; gene expression; leptospirosis; reporter gene; sphingomyelinase; transcription

PMID:
30266732
PMCID:
PMC6238058
DOI:
10.1128/AEM.02068-18
[Indexed for MEDLINE]
Free PMC Article

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