Quorum Sensing Regulates Heterogeneous Gene Expression in V. cholerae Biofilms

Abstract

Coordination of gene expression is required for complex bacterial processes such as virulence, host infection, and biofilm formation. Such coordination is orchestrated via the cell-to-cell communication process called quorum sensing (QS). In the model bacterium and global pathogen Vibrio cholerae, the accumulation of autoinducers in the local environment facilitates the transition between the sessile biofilm lifestyle and the motile planktonic lifestyle. While previous studies on other bacterial species suggest biofilms are heterogeneous and contain cells that are physiologically distinct, gene expression heterogeneity in V. cholerae biofilms has yet to be explored. I examined spatial patterns of the QS master regulator genes AphA (protein) and hapR, as well as the QS-controlled genes luxC and vpsL using advanced microscopy techniques that enable spatiotemporal analyses of living, growing biofilms at single cell resolution. Furthermore, I developed a biofilm segmentation method that integrates mathematical image processing and segmentation algorithms to (i) identify cell positions and gene expression levels at single cell resolution, to (ii) track the evolution of spatial gene expression gradients over time and to (iii) identify heterogeneous populations of cells. I demonstrated that QS-regulated genes exhibit distinct and dynamic spatial distribution patterns across the biofilm: AphA (protein) and hapR form spatial gradients that reflect their reciprocal transcriptional regulation; spatial patterns of luxC mimic those of hapR, but at a 10-fold lower magnitude; and vpsL expression produces a dynamic spatial gradient with decreasing expression from the biofilm center to the periphery. I further discovered that contrary to the supposition that cell density regulates the expression of QS-implicated genes, cells contained within the same region, and that are thus of identical cell density, exhibit a broad range of gene expression levels. Additionally, I report that both inter-regional and intra-regional gene expression heterogeneity are maintained in QS mutant biofilms, shedding light on the complex and redundant regulatory logic underlying gene expression in V. cholerae biofilms. The present study yields an understanding of how QS mechanisms regulate gene expression patterns, which, going forward, will allow manipulation of such patterns possibly for antimicrobial interventions.