Visick, K. L. | Search bioRxiv

bioRxiv2025

A versatile dual-color bacterial reporter system highlights two distinct Pseudomonas aeruginosa Type 3 secretion system intracellular populations.

Since their discovery, fluorescent reporters have revolutionized our ability to track gene and protein expression in real time. Ideally, two reporters are used, one constitutive signal for tracking viable bacteria and the other for measuring the expression of the gene/protein of interest. Unfortunately, these valuable tools are not available for most bacterial species, and if available are often not optimized for fluorophore protein folding rates and fluorescence intensity. Here we present a versatile dual reporter system, pCG-VmS, optimized for both transcriptional and translation fusions in Gram-negative bacteria. Using the important pathogen Pseudomonas aeruginosa for proof of concept, we demonstrate pCG-VmS utility in tracking transcriptional expression with flow cytometry and within a complex biofilm, and using a translational reporter fusion, monitor protein expression and visualize subcellular protein localization. We then analyzed T3SS-associated exoS toxin expression in infected host cells, which highlighted two distinct T3SS-dependent intracellular populations, one where the exoS promoter is turned on and the other where it is turned off in a smaller sub-population of bacterial cells (hereon referred to as T3SS-on and T3SS-off, respectively). Finally, we demonstrate the feasibility of spatiotemporal imaging in whole animals by using our system to monitor expression of an alternative sigma factor during Vibrio fischeri colonization of its squid host. Our findings demonstrate the versatile uses for the pCG-VmS vectors in microbiology, and that this vector can be used to visualize and separate distinct populations with precision for both in vitro and in vivo applications.

microbiology↗

bioRxiv2022

High levels of cyclic diguanylate interfere with beneficial bacterial colonization

During colonization of the Hawaiian bobtail squid (Euprymna scolopes), Vibrio fischeri bacteria undergo a lifestyle transition from a planktonic motile state in the environment to a biofilm state in host mucus. Cyclic diguanylate (c-di-GMP) is a cytoplasmic signaling molecule that is important for regulating motility-biofilm transitions in many bacterial species. V. fischeri encodes 50 proteins predicted to synthesize and/or degrade c-di-GMP, but a role for c-di-GMP regulation during host colonization has not been investigated. We examined strains exhibiting either low or high levels of c-di-GMP during squid colonization and found that while a low c-di-GMP strain had no colonization defect, a high c-di-GMP strain was severely impaired. Expression of a heterologous c-di-GMP phosphodiesterase restored colonization, demonstrating that the effect is due to high c-di-GMP levels. In the constitutive high c-di-GMP state, colonizing V. fischeri exhibited reduced motility, altered biofilm aggregate morphology, and a regulatory interaction where transcription of one polysaccharide locus is inhibited by the presence of the other polysaccharide. Our results highlight the importance of proper c-di-GMP regulation during beneficial animal colonization, illustrate multiple pathways regulated by c-di-GMP in the host, and uncover an interplay of multiple exopolysaccharide systems in host-associated aggregates. IMPORTANCEThere is substantial interest in studying cyclic diguanylate (c-di-GMP) in pathogenic and environmental bacteria, which has led to an accepted paradigm in which high c-di-GMP levels promote biofilm formation and reduce motility. However, considerably less focus has been placed on understanding how this compound contributes to beneficial colonization. Using the Vibrio fischeri-Hawaiian bobtail squid study system, we took advantage of recent genetic advances in the bacterium to modulate c-di-GMP levels and measure colonization and track c-di-GMP phenotypes in a symbiotic interaction. Studies in the animal host revealed a c-di-GMP-dependent genetic interaction between two distinct biofilm polysaccharides, Syp and cellulose, that was not evident in culture-based studies: elevated c-di-GMP altered the composition and abundance of the in vivo biofilm by decreasing syp transcription due to increased cellulose synthesis. This study reveals important parallels between pathogenic and beneficial colonization and additionally identifies c-di-GMP-dependent regulation that occurs specifically in the squid host.

microbiology↗