PrkA Protein Kinase Activity Determination And Phosphoproteome Analysis Of Sinorhizobium Meliloti

Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. The kinase PrkA is widely distributed in prokaryote and strongly induced in stationary phase and various stress conditions. But the impact of PrkA remains elusive due to undetectable phenotype for the prkA mutant.In this study, we cloned, overexpressed and purified the Sinorhizobium meliloti prkA gene product, whose amino acid sequence displays homology to prokaryotic serine protein kinases. The gene coding for PrkA was generated by amplifying the prkA gene from Sinorhizobium meliloti CCBAU01290 by polymerase chain reaction. It was inserted into the expression plasmid pET-28a (+), under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21 (DE3) E. coli strain transformed with the PrkA-expression vector pET-28a (+)-prkA accumulates large amounts of a soluble protein with a molecular mass of 75 kDa in SDS-PAGE, which matches the expected PrkA molecular weight of 74.2 kDa. The PrkA protein lacking AAA domain sequence was also purified and the protein kinase activity of PrkA and AAAA_PrkA in the presence of Mn2+ or Mg2+ were demonstrated with in-vitro phosphorylation assay. The purified protein displays autokinase and casein kinase activities which are optimal in the presence of 2 mM Mn2+ whereas its kinase activity inhibited in the presence of Mg2+ Meanwhile, we found AAA domain is necessary for PrkA’s catalytic activity.To uncover the potential PrkA-dependent phosphorylation events in S. meliloti, TiO2 enrichment and subsequent LC-MS/MS determination were used to investigate the stationary-phase (OD600=1.886) phosphoproteomes of the prkA mutant and the wild type (WT) cultured in minimum medium. There are a total of 154 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 97:51:6, in 142 unique phosphopeptides identified from 123 proteins.45 and 40 phosphoproteins were specifically found in WT and prkA, respectively. Even within the 38 phosphoproteins shared by two strains, the phosphosites were not always conserved. Phosphoproteins identified in prkA showed a wide distribution pattern, similar to those of WT, in terms of biological processes, cellular component and molecular function. The observed coordinated regulation of phosphorylation events might be used by S. meliloti prkA to adapt to the tested culture condition where prkA behaving indistinguishably from WT. However, prkA was outcompeted by WT when co-cultured in the minimum medium with reduced phosphate supply (2 mM). Further phosphoproteomic analysis of the stationary-phase(OD600=0.889) prkA and WT in this medium identified 8 and 12 phosphoproteins in prkA and WT, respectively. The implication of distinct phosphoproteins and differentially phosphorylated protein sites were discussed.This work not only provides the first phosphoproteomic analysis of rhizobia but also unravels a distinct phosphoproteome of the subhealthy S. meliloti lacking the stress kinase PrkA.