Peptidoglycan remodelling improves salt resilience of Zymomonas mobilis

Abstract The alpha-proteobacterium Zymomonas mobilis is one of the most efficient microbial producers of ethanol and has the potential to be stablished as biofuel producer at industrial scale. However, a bottleneck hindering the full use of Z. mobilis in biorefinery is its sensitivity to environmental stresses, including sodium chloride (NaCl), a common component present in biomass from various sources. To address this limitation, we need to deepen our understanding of the cell envelope, the crucial barrier between bacteria and external stressors. To date, the cell envelope of Z. mobilis has remained largely uncharacterized. Here we show that the deletion of ctpA , which encodes a periplasmic protease, increases the salt resilience. Salt resilience is mediated by an increased level of the peptidoglycan endopeptidase MepM, which we identified as a substrate of CtpA through comparative proteomics. Supporting this, the overexpression of MepM in the wild-type enhanced salt resilience. We also discovered that the peptidoglycan of Z. mobilis is O-acetylated at Mur N Ac residues, a modification usually associated with virulence in pathogenic bacteria. Interestingly, O-acetylation was crucial for salt resilience, supporting a role in PG growth regulation under the stress condition. Overall, this study highlights the importance of investigating cell envelope biology in Z. mobilis as a foundation for engineering strains with improved resilience to environmental stress and, more general, studying the cell envelopes of non-model bacteria to expand our fundamental understanding of cell function Importance Fossil fuels have negative impacts on the environment and will become limited in the next decades. Hence, alternative, sustainable energy sources need to be urgently established. Microbial fermentation of biomass for biofuel production presents a promising avenue. The Gram-negative alpha-proteobacterium Z. mobilis exhibits a superior capacity to convert sugars into ethanol, a clean, renewable and widely-used fuel. However, Z. mobilis has not been used as a first choice as a bio-fuel producer. The ethanol producer, baker’s yeast Saccharomyces cerevisiae serves as a model species in cell biology, but we lack fundamental understanding of the cell envelope biology of Z. mobilis , which would be critical to engineer strain with increased resilience. Here, we demonstrate that knowledge about cell envelope biogenesis factors in Z. mobilis can help engineering optimised strains that grow under conditions of bio-fuel production.