Ulijasz, A. T. | Search bioRxiv

bioRxiv2026

A glyoxal sensing Pseudomonas aeruginosa transcription factor enables lung infection

Aldehydes are a class of normally unwanted toxic electrophilic compounds that mainly arise from oxidation of glucose, lipids or DNA. However, it has recently come to light that they can also be weaponized by professional phagocytes to kill engulfed bacteria. How microbes subvert these assaults remains largely enigmatic. Here we describe the function, atomic structure and mechanism of the first bacterial transcription factor able to directly sense the dicarbonyl glyoxal (GO), which we aptly named the Glyoxal Regulator (GloR, from Pseudomonas aeruginosa PAO1), We show that GloR directly senses GO through a reversible cysteine modification that results in its binding to a conserved DNA regulatory motif (a glo box), which then triggers a transcriptional activation of a defined set of genes to help counter GO toxicity and enable acute lung infection. Despite substantial evolutionary divergence, when unmodified gloR and a glo box-regulated reporter were transferred into E. coli, a strikingly tight GO-specific regulation was maintained, suggesting this system could be readily transferred between unrelated microbial species. As homologs of GloR were identified in diverse bacterial species we anticipate its use to be widespread in both pathogens and environmental bacteria. Taken together, we present the first bona fide bacterial aldehyde regulator which senses host GO to enable survival during infection.

microbiology↗

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↗