Abstract
Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N2-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N2-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the PII regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in Z. mobilis. This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.
Highlights
Zymomonas mobilis, a facultatively anaerobic alphaproteobacterium, possesses several desirable characteristics for industrial biofuel production
We categorized the phosphoproteins that we identified according to their biological functions and found them to be distributed across essential cellular and metabolic processes such as glycolysis, TCA cycle, amino acid, nucleotide, and protein biosynthesis, nitrogen fixation, and ammonia assimilation (Figure 1)
The serine/threonine/tyrosine (STY) distribution of phosphorylation sites across all phosphoproteins was 73% serine, 21% threonine, and 6% tyrosine (Table 1). This was very similar to the STY distributions found in E. coli, B. subtilis, and K. pneumoniae (Ravikumar et al, 2014; Lin et al, 2015; Potel et al, 2018), but somewhat different than R. palustris (STY: 63.5/16/19.5) and S. coelicolor (STY: 46.8/48/5.2) (Manteca et al, 2011; Hu et al, 2012; Table 1)
Summary
A facultatively anaerobic alphaproteobacterium, possesses several desirable characteristics for industrial biofuel production. Recent studies have indicated that during aerobic growth, oxidative damage to iron-sulfur (FeS) clusters constitutes a major factor influencing Z. mobilis metabolism and that respiratory enzymes and the ability to form multicellular aggregates are important for its survival (Jones-Burrage et al, 2019; Martien et al, 2019). Despite these and other recent advances (Yang et al, 2009; Rutkis et al, 2016; Strazdina et al, 2018), much remains to be learned about the regulation of Z. mobilis physiology during aerobic growth
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