Characterization and control of bacterial phenotypes at the single cell level

Abstract

<p indent=0mm>Microbial biofilms comprise surface-associated, multicellular, highly structured matrix-enclosed, morphologically complex microbial communities. The subject of biofilms has received much attention, in part owing to scientific communities’ acknowledgements that biofilm formation of pathogen can cause many acute or chronic diseases that usually cannot be treated by antibiotic therapies, such as <italic>Pseudomonas aeruginosa</italic>, one of the highly prevalent opportunistic human pathogens, that is the leading cause of morbidity and mortality in immunocompromised patients and in patients suffering from cystic fibrosis (CF). We have learned that biofilm-forming bacteria are phenotypically distinct from those of planktonically grown cells, because they express genes in a pattern differs profoundly from that of their free-swimming, planktonic counterparts. Therefore, understanding the roles played by bacterial behaviors and phenotypes in the development of biofilms is an emerging link to disease pathogenesis. In the first half of this review, we present how to develop characteristic approaches by using high-throughput microscopical techniques together with automatic image-processing methods and customized microfluidic system to in situ visualize bacterial cells and investigate their behaviors at the single cell scale. We mainly focused on the pathogen of <italic>P. aeruginosa</italic> and provided an overview of recent related progress using those approaches in characterization of bacterial phenotypes at the single cell level in the process of biofilm formation, especially in areas of bacterial twitching motility, attachment phenotypes, social behaviors and architecture of biofilms. For instance, by monitoring single-cell motility behaviors, researchers observed single-cell twitching chemotaxis in developing biofilms and revealed that cells can modulate twitching motility behaviors to survive or thrive in slowly changing and locally heterogeneous natural environments. We then referred to optogenetics, which is a technology that allows targeted, fast control of precisely defined events in biological systems by expressing exogenous genes coding for light-sensitive proteins. In optogenetics, light as inducer can be applied more precisely in the concentration, time and space dimensions than traditional effectors such as chemical molecules. In the field of microbiology, different light responsive sensors, such as UV, blue, green, red and far-red transcriptional regulation systems, have been engineered, and application of these optogenetic systems enables little perturbations and unprecedented spatiotemporal resolution in controlling bacterial behaviors that can reveal new insights into biological function. For example, researchers have used blue light-switchable gene regulation system to optogenetic control of gene expression to spatiotemporally manipulate biofilm formation and pattern cells with high-resolution. These studies provide the ability to grow structured biofilms, with applications toward an improved understanding of natural biofilm communities, as well as the engineering of living biomaterials and bottom-up approaches to microbial consortia design. In addition, optogenetics enables characterization of bacterial gene circuit dynamics with optically programmed gene expression signals, which could advance understanding of gene circuit dynamics and cell signaling pathways. We also proposed that the development of optogenetics in bacteriology is limited by the requirement of engineered light-sensitive proteins for specific regulation networks and new methods for manipulation of living bacterial cells at the single-cell level. This review concludes with some expectations on the foreseeable future application of those single-cell analysis approaches in biofilms-related fields, and a discussion of innovative anti-biofilm strategies that are inspired from the studies of characterization and control of single-cell phenotypes.