A dual plasmid system, consisting of a soxS-promoter, single-copy “transduction" plasmid and a pET-derived “amplification” plasmid, was constructed to sense oxidative stress such as bacterial toxin pyocyanin. This system enhanced the output signal by ~15 times compared to a single-copy reporter plasmid, although it did not lower the detection limit for many redox-active compounds. Due to the modularity of the system, it was simple to generate sensing systems with different output modes by pairing with various “amplification” plasmids. A luminescent E. coli reporter was shown to be capable of detecting 1-8 mM of herbicide dicamba in 1.5 hours. We also discovered that this system had distinct sensing ranges and output magnitudes when transformed into E. coli strains of different genetic modifications, enabling a reverse engineering approach towards reporter development. For example, the ΔsoxRS reporter displayed higher sensitivity toward all oxidative stressors; and the ΔptsH reporters which lacked the protein HPr in the phosphotransferase system (PTS) showed lower output magnitude and sensitivity. With the deletion of global oxidative stress regulon soxRS, we expected lower levels of stress-relieving genes fumC/sodA, potentially enabling a more oxidized cell state, consistent with our results. Similarly, an active PTS was previously reported to influence the cell’s oxidative state; soxS was shown to be upregulated, consistent with diminished output from our system. Interestingly, we also observed that a ΔluxS strain (lacking the terminal synthase for quorum sensing (QS) autoinducer, AI-2) displayed higher output than WT. Quantitative PCR results confirmed our findings that soxS expression in ΔluxS reporters was 10-fold lower than WT when exposed to 5 µM of pyocyanin. Hypotheses were postulated on the mechanisms of how QS might affect E. coli’s intra- and even inter-cellular response to stress: (i) QS-kinase, LsrK, binds to HPr and when bound inhibits kinase activity, QS could influence PTS thus affecting soxS expression. (ii) The mutant cannot generate homocysteine through the LuxS-pathway, which could alter its ability to recycle methionine/thiol groups. This could lower the internal glutathione levels, changing cellular responses toward stress. The dual-plasmid sensing system enhanced the output for reporting external oxidative stress, it also helped shed light on how E. coli manages its internal oxidative state including in response to environmental cues.

Dual plasmid system for sensing external oxidative stress and illuminating internal oxidative states resulting from genetic deletions – a curious interaction with quorum sensing

ACS Spring 2022

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