The phosphoproteome of the minimal bacterium Mycoplasma pneumoniae: analysis of the complete known Ser/Thr kinome suggests the existence of novel kinases

Abstract

Mycoplasma pneumoniae belongs to the Mollicutes, the group of organisms with the smallest genomes that are capable of host-independent life. These bacteria show little regulation in gene expression, suggesting an important role for the control of protein activities. We have studied protein phosphorylation in M. pneumoniae to identify phosphorylated proteins. Two-dimensional gel electrophoresis and mass spectrometry allowed the detection of 63 phosphorylated proteins, many of them enzymes of central carbon metabolism and proteins related to host cell adhesion. We identified 16 phosphorylation sites, among them 8 serine and 8 threonine residues, respectively. A phosphoproteome analysis with mutants affected in the two annotated protein kinase genes or in the single known protein phosphatase gene suggested that only one protein (HPr) is phosphorylated by the HPr kinase, HPrK, whereas four adhesion-related or surface proteins were targets of the protein kinase C, PrkC. A comparison with the phosphoproteomes of other bacteria revealed that protein phosphorylation is evolutionarily only poorly conserved. Only one single protein with an identified phosphorylation site, a phosphosugar mutase (ManB in M. pneumoniae), is phosphorylated on a conserved serine residue in all studied organisms from archaea and bacteria to man. We demonstrate that this protein undergoes autophosphorylation. This explains the strong conservation of this phosphorylation event. For most other proteins, even if they are phosphorylated in different species, the actual phosphorylation sites are different. This suggests that protein phosphorylation is a form of adaptation of the bacteria to the specific needs of their particular ecological niche.

Fig. 1.

Dual channel image of total protein amount (Flamingo fluorescent dye-stained; green) and phosphorylated proteins (Pro-Q Diamond-stained; red) of stationary phase M. pneumoniae cells. About 125 μg of a total protein extract of M. pneumoniae grown in modified Hayflick medium supplemented with glucose were subjected to two-dimensional gel electrophoresis using either an 18-cm IPG strip with a linear pH gradient of pI 6–11 (A) or pI 4–7 (B) in the first dimension. Dual channel images were obtained using the software DECODON Delta2D 3.6. Spots with high Pro-Q Diamond/Flamingo log ratios representing phosphorylated proteins (9) are indicated on the gel. Multiple protein spots corresponding to the same original protein are distinguished by appended digits (e.g. Tuf-1 and Tuf-2). Protein spots were cut from the gel and identified by MS/MS (see Table I and supplemental Table S3).

Fig. 2.

PrpC/PrkC-dependent modification of cytadherence proteins in M. pneumoniae Shown are sections of dual channel images of Flamingo fluorescent dye- (green) and Pro-Q Diamond-stained (red) two-dimensional gels representing selected proteins in the M. pneumoniae wild type, prpC::Tn mutant, and prkC::Tn mutant. Proteins were separated on an 18-cm IPG strip with a linear pH gradient of pI 4.5–5.5 in the first dimension. The proteins HMW3, MPN474, P65 (A), MPN256, RpoE (B), and P41 (C) are shown. Putative phosphorylated proteins in the M. pneumoniae wild type as well as proteins with increased or new phosphorylation spots in the prpC::Tn mutant are indicated. Missing phosphorylation spots in the M. pneumoniae wild type and the prkC::Tn mutant are highlighted by a box. Protein amounts of HMW3, MPN256, and P41 (all represented by a triple protein spot) seem to be reduced as well as for the P65 protein (underlined) in the prkC::Tn mutant. Note that the P41 spot “2*” is only putative because of a low Pro-Q Diamond/Flamingo log ratio in the analyzed strains. Protein spots were cut from the gel and identified by MS/MS (see Table I and supplemental Table S3).

Fig. 3.

Comparison of HPr phosphorylation pattern between different M. pneumoniae strains. Section of dual channel images of Flamingo fluorescent dye- (green) and Pro-Q Diamond-stained (red) two-dimensional gels displaying the phosphorylated spot of HPr in the M. pneumoniae wild type and different mutant strains. Proteins were separated on an 18-cm IPG strip with a linear pH gradient of pI 4–7 in the first dimension. Note that the HPr spot represents a multiprotein spot containing HPr and UlaB, whereas only UlaB could be detected in the hprK::Tn mutant (much lesser intensity of the phosphorylation spot).

Fig. 4.

Schematic illustration of phosphorylation events in central metabolic pathways of M. pneumoniae. Of the central metabolic pathways, only glycolysis is operative in M. pneumoniae, and a few carbon sources such as glucose, fructose, and glycerol can be utilized. Pyruvate can be oxidized to acetate or reduced to lactate. An incomplete pentose phosphate pathway serves to generate phosphoribosyl pyrophosphate for nucleotide biosynthesis. Phosphorylated enzymes are highlighted in gray. DHAP, dihydroxyacetone phosphate; GPC, glycerophosphorylcholine; PEP, phosphoenolpyruvate; PRPP, phosphoribosyl pyrophosphate; P, phosphate.

Fig. 5.

MS/MS spectrum of serine-phosphorylated peptide from phosphomannomutase/phosphoglucomutase ManB (MPN066). The peptide was measured on line by ESI-mass spectrometry using a nanoACQUITY UPLC system coupled to an LTQ Orbitrap mass spectrometer (see “Experimental Procedures”). The phosphorylation site (Ser-149) is located in the conserved phosphoserine signature of phosphosugar mutases. Detection of the same phosphorylated peptide in all 10 spots of ManB (enhanced image section) suggests further post-translational modifications that affect the isoelectric point of the ManB protein.

Fig. 6.

Autophosphorylation on universally conserved serine residue in active site of phosphosugar mutases. A, multiple alignment of the conserved phosphoserine signature of phosphosugar mutases from all domains of life. Amino acids with similarities in at least two of the sequences are highlighted in gray, whereas amino acids that are identical in at least two of the sequences are depicted in a black background. The UniProtKB entry names of the aligned sequences are MANB_MYCPN (ManB, M. pneumoniae), GLMM_BACSU (GlmM; B. subtilis), GLMM_LACLA (GlmM; L. lactis), ALGC_PSEAE (AlgC; P. aeruginosa), MANB_ECOLI (ManB; E. coli), Q0P8K7_CAMJE (ManB; C. jejuni), B0R7B7_HALS3 (PMM1; H. salinarum), AGM1_YEAST (AGM1; Saccharomyces cerevisiae), PGM_DROME (PGM; D. melanogaster), AGM1_MOUSE (AGM1; Mus musculus), and PGM1_HUMAN (PGM1; Homo sapiens). The conserved active site phosphoserine is indicated by an arrow. B, M. pneumoniae ManB and B. subtilis GlmM autophosphorylation assay in the presence of various divalent cations. About 5 μg of purified His-tagged ManB (lanes 1–4) or GlmM (lanes 4–8) were incubated in the presence of [γ³²P]ATP in an autophosphorylation assay (see “Experimental Procedures”). Each reaction mixture was analyzed by SDS-PAGE and phosphorimaging analysis. Divalent cations (5 mm) used in the assays are indicated below the lanes. Bsu, B. subtilis; Mpn, M. pneumoniae. C, autophosphorylation assay of M. pneumoniae ManB (wild type (wt) and S149A) and B. subtilis GlmM (wild type and S100A) recombinant proteins. The four recombinant proteins were checked for autophosphorylation as described in B. Autophosphorylation reactions were conducted in the presence of Mn²⁺ (5 mm) as divalent cation as it showed the strongest signal in B.