Menaquinone synthesis genes are critical for the decolorization of acid yellow 36 in Shewanella putrefaciens CN32.

The anaerobic decolorization of azo dyes by Shewanella species relies on an intricate electron transfer network, yet the full complement of genes involved remains incompletely defined. In this study, we used mini-Tn5 transposon mutagenesis to identify genes required for the decolorization of the azo dye acid yellow 36 in Shewanella putrefaciens CN32. Two genes were uncovered: menD, encoding a key enzyme in menaquinone biosynthesis, and lutB, involved in lactate metabolism. Deletion of menD caused a nearly complete loss of decolorization capacity without affecting growth. Supplementation with vitamin K2 or spent supernatant from the wild-type strain restored decolorization, demonstrating that menaquinone is essential. We systematically identified the menaquinone biosynthesis gene cluster of S. putrefaciens CN32, including menA, menB, menC, menE, menF, menG, menH, and paaI. Deleting most of these genes severely impaired decolorization, whereas deletions of menH, paaI, or an adjacent LysR-family regulator gene had no significant effect. Furthermore, acid yellow 36 induced biofilm formation in a dose-dependent manner, and this induction was abolished in most menaquinone synthesis mutants. Acid yellow 36 did not substantially upregulate transcription of the men gene cluster, suggesting that the dye promotes biofilm formation through a direct mechanism rather than by enhancing menaquinone synthesis. Collectively, our results establish menaquinone as a critical component for both azo dye decolorization and biofilm formation in S. putrefaciens CN32, and they point to the existence of a CymA-independent electron transfer branch in this strain.IMPORTANCEAzo dyes are environmental pollutants that can be effectively removed by Shewanella bacteria through anaerobic respiration. Understanding the genetic basis of azo dye decolorization is essential for improving bioremediation strategies. This study identifies menaquinone, a respiratory chain cofactor, as indispensable for the decolorization of acid yellow 36 in Shewanella putrefaciens CN32. By systematically mapping the menaquinone biosynthesis gene cluster, we show that an intact synthesis pathway is required for both dye reduction and biofilm formation, a lifestyle that enhances bacterial survival and metabolic activity in wastewater treatment systems. Importantly, the severe decolorization defect of menaquinone-deficient mutants contrasts with the relatively mild effect of deleting cymA, the canonical electron hub, suggesting the existence of a CymA-independent electron transfer route from menaquinone to azo dyes. These findings expand the current model of extracellular electron transfer in Shewanella and provide new genetic targets for engineering more efficient azo dye-degrading strains.