Classification of phenotypic subpopulations in isogenic bacterial cultures by triple promoter probing at single cell level

Phenotypic heterogeneity, defined as the unequal behavior of individuals in an isogenic population, is prevalent in microorganisms. It has a significant impact both on industrial bioprocesses and microbial ecology. We introduce a new versatile reporter system designed for simultaneous monitoring of the activities of three different promoters, where each promoter is fused to a dedicated fluorescent reporter gene (cerulean, mCherry, and mVenus). The compact 3.1 kb triple reporter cassette can either be carried on a replicating plasmid or integrated into the genome avoiding artifacts associated with variation in copy number of plasmid-borne reporter constructs. This construct was applied to monitor promoter activities related to quorum sensing (sinI promoter) and biosynthesis of the exopolysaccharide galactoglucan (wgeA promoter) at single cell level in colonies of the symbiotic nitrogen-fixing alpha-proteobacterium Sinorhizobium meliloti growing in a microfluidics system. The T5-promoter served as a constitutive and homogeneously active control promoter indicating cell viability. wgeA promoter activity was heterogeneous over the whole period of colony development, whereas sinI promoter activity passed through a phase of heterogeneity before becoming homogeneous at late stages. Although quorum sensing-dependent regulation is a major factor activating galactoglucan production, activities of both promoters did not correlate at single cell level. We developed a novel mathematical strategy for classification of the gene expression status in cell populations based on the increase in fluorescence over time in each individual. With respect to galactoglucan biosynthesis, cells in the population were classified into non-contributors, weak contributors, and strong contributors. (C) 2015 Elsevier B.V. All rights reserved.