Abstract
Phenotypic diversity is an important trait of bacterial populations and can enhance fitness of the existing genotype in a given environment. To characterize different subpopulations, several studies have analyzed differential gene expression using fluorescent reporters. These studies visualized either single or multiple genes within single cells using different fluorescent proteins. However, variable maturation and folding kinetics of different fluorophores complicate the study of dynamics of gene expression. Here, we present a proof-of-principle study for an alternative gene expression system in a wbaP mutant of Salmonella Typhimurium (S. Tm) lacking the O-sidechain of the lipopolysaccharide. We employed the hemagglutinin (HA)-tagged inverse autotransporter invasin (invAHA) as a transcriptional reporter for the expression of the type three secretion system 1 (T1) in S. Tm. Using a two-reporter approach with GFP and the InvAHA in single cells, we verify that this reporter system can be used for T1 gene expression analysis, at least in strains lacking the O-antigen (wbaP), which are permissive for detection of the surface-exposed HA-epitope. When we placed the two reporters gfp and invAHA under the control of either one or two different promoters of the T1 regulon, we were able to show correlative expression of both reporters. We conclude that the invAHA reporter system is a suitable tool to analyze T1gene expression in S. Tm and propose its applicability as molecular tool for gene expression studies within single cells.
Highlights
Most previous reports studying the expression of individual promoters of interest within single cells have used fluorophores as reporter system [2, 22, 37, 43]
Using strains expressing gfp and invAHA reporters from the same promoter (PsicA, Fig 5) or from two Salmonella pathogenicity island 1 (SPI-1) promoters (PprgH and PsicA, Fig 3), which were thought to be co-regulated, we could verify the suitability of this approach
It is conceivable that establishing a set of InvAHA variants with equivalent maturation dynamics may allow such multi-gene expression analysis if one inserts multiple invA
Summary
Different phenotypes have increasingly been recognized within bacterial cultures that harbor the same genotype and share the same microenvironment This phenotypic diversity (heterogeneity) within isogenic bacteria has been recognized as common trait among bacterial species and has recently developed into an important research field [2,3,4,5]. Phenotypic diversity within isogenic bacterial populations is caused by stochastic (random) events during gene expression, molecular segregation during cell division and/or metabolic activity. We performed a proof-of-concept study to assess if autotransporters may be used as reporters for gene expression in Gram-negative bacteria. We show correlative expression of both reporters when driven under the control of one or two different promoters of the T1 regulon This expands the molecular toolbox for future studies of gene expression within a single bacterial cell
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