New mechanisms for regulating the response to photooxidative and oxidative stress in the bacterium Myxococcus xanthus and their evolutionary conservation

Abstract

The transmembrane protein CarF is a key player that acts early in the complex response to photooxidative stress of the Gram-negative soil bacterium Myxococcus xanthus. Recent findings revealed that CarF is required in the M. xanthus light response because it corresponds to plasmanylethanolamine desaturase (PEDS1), the enzyme that generates the vinyl-ether bond in plasmalogens (special glycerophospholipids that signal photooxidative stress in M. xanthus). The first objective has been to demonstrate that the animal CarF sequence homologs, known as TMEM189, correspond to the long-sought mammalian PEDS1, and to explore the role of these lipids in human cell lines and in two animal models. Human TMEM189-knockout cells showed a complete depletion of plasmalogens with a concomitant accumulation of their precursors and of the minor signaling species C14-Cer and C14-CerP, thus confirming TMEM189 as the exclusive PEDS in humans. Additionally, the EGFP-TMEM189 fusion protein localized to the endoplasmic reticulum (ER) and complemented the lack of TMEM189, while plasmalogens potentially contributed to GPI-anchored protein transport from the ER to the Golgi. In the invertebrate model C. elegans, TMEM189 localized at the intestinal wall and at two potential sensory neurons, suggestive of a sensory role for plasmalogens, which do not appear to have an antioxidant role in C. elegans. In the vertebrate model zebrafish, plasmalogen deficiency delayed development, increased basal inflammation, and triggered myeloid cell apoptosis. The second objective was inspired by the observation that FAD4, the plant CarF homolog, requires a peroxiredoxin (Prx) for activity. Thus, we tested whether AhpC, the only Prx conserved in both M. xanthus and humans, affects CarF activity, and whether it participates in the peroxide stress response in M. xanthus. The highly conserved and widespread AhpC generally scavenges low H2O2 levels, while catalases detoxify high levels of extracellular H2O2. Interestingly, ahpC deletion did not impair plasmalogen biosynthesis but produced pleiotropic effects, including growth and plating defects and enhanced tolerance to H2O2. Transcriptomic analyses revealed upregulation of the KatB catalase, as well as alterations in iron and sulfur homeostasis, and in DNA and protein repair systems. Only complementation with ahpC under the control of its own promoter restored the wild-type phenotype, while plating defects were counteracted by sodium pyruvate, a H2O2 scavenger. Since M. xanthus lacks the peroxide sensor OxyR, which generally controls kat and ahpC expression in Gram-negative bacteria, we aimed to identify the factor behind katB upregulation in ahpC-deficient cells. This led us to discover PexR (peroxide regulation), a bacterial enhancer-binding protein encoded by the gene immediately upstream of ahpC, that controls the peroxide stress response in M. xanthus. PexR is a member of a class of AAA+ ATPase DNA-binding proteins involved in activating σ54-dependent promoters. While, under peroxide stress, PexR activated katB and ahpC expression by binding to (pseudo)palindromic repeats upstream of their σ54-dependent promoters, PexR partially repressed the σ70-dependent expression of ahpC under normal growth conditions. Interestingly, deletion of ahpC was synthetic lethal with that of katB or pexR, highlighting the crucial role of PexR-mediated katB upregulation for viability of ahpC-deficient cells. Removal of the sensory GAF domain in PexR produced a constitutively active form (PexR∆GAF) that could be countered by expressing the GAF domain in trans, suggesting an inhibitory role for this domain. Additionally, mutations in conserved residues that potentially form a non-heme iron center yielded a partially constitutively active PexR. Transcriptomic and DNA-protein interaction analyses with purified PexR and PexR∆GAF showed that the regulatory action of PexR is restricted to katB and ahpC. Overall, this work reveals a novel mechanism for regulating the peroxide stress response in bacteria, which plays a crucial role for M. xanthus cell survival.