Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1(T) identified the role of protein phosphorylation in methanogenesis and osmoregulation

Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1(T), a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change

Avenaciolides: Potential MurA-Targeted Inhibitors Against Peptidoglycan Biosynthesis in Methicillin-Resistant Staphylococcus aureus (MRSA)

Discovery of new antibiotics for combating methicillin-resistant Staphylococcus aureus (MRSA) is of vital importance in the post-antibiotic era. Here, we report four avenaciolide derivatives (<b>1</b>–<b>4</b>) isolated from Neosartorya fischeri, three of which had significant antimicrobial activity against MRSA. The morphology of avenaciolide-treated cells was protoplast-like, which indicated that cell wall biosynthesis was interrupted. Comparing the structures and minimum inhibitory concentrations of <b>1</b>–<b>4</b>, the α,β-unsaturated carbonyl group seems to be an indispensable moiety for antimicrobial activity. Based on a structural similarity survey of other inhibitors with the same moiety, we revealed that MurA was the drug target. This conclusion was validated by ³¹P NMR spectroscopy and MS/MS analysis. Although fosfomycin, which is the only clinically used MurA-targeted antibiotic, is ineffective for treating bacteria harboring the catalytically important Cys-to-Asp mutation, avenaciolides <b>1</b> and <b>2</b> inhibited not only wild-type but also fosfomycin-resistant MurA in an unprecedented way. Molecular simulation revealed that <b>2</b> competitively perturbs the formation of the tetrahedral intermediate in MurA. Our findings demonstrated that <b>2</b> is a potent inhibitor of MRSA and fosfomycin-resistant MurA, laying the foundation for the development of new scaffolds for MurA-targeted antibiotics

Avenaciolides: Potential MurA-Targeted Inhibitors Against Peptidoglycan Biosynthesis in Methicillin-Resistant Staphylococcus aureus (MRSA)

Discovery of new antibiotics for combating methicillin-resistant Staphylococcus aureus (MRSA) is of vital importance in the post-antibiotic era. Here, we report four avenaciolide derivatives (<b>1</b>–<b>4</b>) isolated from Neosartorya fischeri, three of which had significant antimicrobial activity against MRSA. The morphology of avenaciolide-treated cells was protoplast-like, which indicated that cell wall biosynthesis was interrupted. Comparing the structures and minimum inhibitory concentrations of <b>1</b>–<b>4</b>, the α,β-unsaturated carbonyl group seems to be an indispensable moiety for antimicrobial activity. Based on a structural similarity survey of other inhibitors with the same moiety, we revealed that MurA was the drug target. This conclusion was validated by ³¹P NMR spectroscopy and MS/MS analysis. Although fosfomycin, which is the only clinically used MurA-targeted antibiotic, is ineffective for treating bacteria harboring the catalytically important Cys-to-Asp mutation, avenaciolides <b>1</b> and <b>2</b> inhibited not only wild-type but also fosfomycin-resistant MurA in an unprecedented way. Molecular simulation revealed that <b>2</b> competitively perturbs the formation of the tetrahedral intermediate in MurA. Our findings demonstrated that <b>2</b> is a potent inhibitor of MRSA and fosfomycin-resistant MurA, laying the foundation for the development of new scaffolds for MurA-targeted antibiotics