Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions

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.

Goals and Challenges in Bacterial Phosphoproteomics

“…As stated above, phosphorylation in bacteria is dramatically lower than that in eukaryotes, making bacterial phosphoproteomics challenging, especially quantitative phosphoproteomics (i.e., analyses of the amount of specific phosphorylation sites and how they vary during development). To our knowledge, there are 15 reported quantitative phosphoproteomic studies on bacteria [6,15,17,25,29,31,34,35,36,37,38,40,41,42] (Table 4).…”

“…Label-free quantitative phosphoproteomic analyses were also performed in Bacillus subtilis , Acinetobacter baumannii , Mycobacterium smegmatis and Zymomonas mobilis [6,35,37,41]. Scheduled multiple reaction monitoring (sMRM), another label-free approach that consists in selecting the masses of the ions to be sequenced in the MS/MS, was used to analyse the E. coli and Saccharopolyspora erythraea phosphoprotoemes [30,34].…”

The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis

“…The Z. mobilis genome contains all genes necessary to perform N 2 -fixation, including an RpoN-like sigma factor (Sigma 54) [ 13 ]. All additional genes involved in this process are located in a single chromosomal region [ 13 ], which includes the gene encoding the NifA regulator, the operon nif HDKENX- fdx B- nif Q (which encodes the nitrogenase enzyme), two operons involved in nitrogenase maturation ( ni fB- fdx N and isc N- nif USVW- mod D), and the RNF operon ( rnf ABCDGEH), which encodes members of the RNF electron-transport complex, responsible for donating electrons to nitrogenase, during N 2 fixation. As observed in Figure 4 , most of these elements were identified as positively modulated, in response to the presence of AI-2.…”

Genome-Wide Analyses of Proteome and Acetylome in Zymomonas mobilis Under N2-Fixing Condition

“…Also, CitC and GltA (ZMO1963) from TCA cycle were found acetylated at site K57 and K311 under the N 2fixing condition, respectively. In comparison, for phosphorylatedbased PTM in Z. mobilis, only Mdh was reported phosphorylated among the TCA cycle proteins in the previous study (Tatli et al, 2019) under the nitrogen-fixing condition.…”