Mapping phosphoproteins in Mycoplasma genitalium and Mycoplasma pneumoniae

Abstract

Background: Little is known regarding the extent or targets of phosphorylation in mycoplasmas, yet in many other bacterial species phosphorylation is known to play an important role in signaling and regulation of cellular processes. To determine the prevalence of phosphorylation in mycoplasmas, we examined the CHAPS-soluble protein fractions of Mycoplasma genitalium and Mycoplasma pneumoniae by two-dimensional gel electrophoresis (2-DE), using a combination of Pro-Q Diamond phosphoprotein stain and 33P labeling. Protein spots that were positive for phosphorylation were identified by peptide mass fingerprinting using MALDI-TOF-TOF mass spectrometry.

Results: We identified a total of 24 distinct phosphoproteins, about 3% and 5% of the total protein complement in M. pneumoniae and M. genitalium, respectively, indicating that phosphorylation occurs with prevalence similar to many other bacterial species. Identified phosphoproteins include pyruvate dehydrogenase E1 alpha and beta subunits, enolase, heat shock proteins DnaK and GroEL, elongation factor Tu, cytadherence accessory protein HMW3, P65, and several hypothetical proteins. These proteins are involved in energy metabolism, carbohydrate metabolism, translation/transcription and cytadherence. Interestingly, fourteen of the 24 phosphoproteins we identified (58%) were previously reported as putatively associated with a cytoskeleton-like structure that is present in the mycoplasmas, indicating a potential regulatory role for phosphorylation in this structure.

Conclusion: This study has shown that phosphorylation in mycoplasmas is comparable to that of other bacterial species. Our evidence supports a link between phosphorylation and cytadherence and/or a cytoskeleton-like structure, since over half of the proteins identified as phosphorylated have been previously associated with these functions. This opens the door to further research into the purposes and mechanisms of phosphorylation for mycoplasmas.

Figure 1

CHAPS-Soluble Phosphoprotein map for Mycoplasma pneumoniae. 250 μg CHAPS-soluble cellular extract of stationary phase M. pneumoniae proteins were separated by 2D PAGE using 12.5%T SDS-polyacrylamide gels. (A) Shows a gel separated in the first dimension using a linear pH gradient pI3–10 (18 cm IPG strip). Identified phosphorylated proteins are numbered (see Table 3 for details). Molecular weight markers are indicated at the left and pI values at the top. The gel was stained with Pro-Q Diamond for detection of phosphoproteins (shown as blue/white), scanned, and the same gel was then stained with SYPRO Ruby protein stain (shown as red). The overlay images were generated using ImageMaster 2D software. [56] (B) Shows a partial image of an IPG 6–11 gel stained by Pro-Q Diamond, and (C) shows a partial image from an IPG 4–7 gel stained by Pro-Q Diamond.

Figure 2

CHAPS-Soluble Phosphoprotein map for Mycoplasma genitalium. 250 μg CHAPS-soluble cellular extract of stationary phase M. genitalium proteins were separated by 2-D gel electrophoresis with immobilized linear pH gradients from 3 to 10 and 12.5%T SDS-polyacrylamide gels. Panel (A) shows the 2-D gel with the total protein complement stained by SYPRO Ruby. Molecular weight markers are indicated at the left, and pI values at the top. Panel (B) shows the proteins stained by Pro-Q Diamond, and (C) shows the autoradiographic gel obtained by [³³P]-phosphoric acid-labeling. Identified phosphorylated proteins are numbered (see Table 2 for details).

Figure 3

Comparison of protein phosphorylation between different growth states. Selected sections of the overlay image from the 2D-gel phosphoprotein profile of (A) exponential growth phase and (B) stationary growth phase of M. pneumoniae are shown, with growth cycle determined by wet cell weight as described in the methods. Phosphoproteins were detected by Pro-Q Diamond stain (shown as blue/white) and then the total proteins were labeled by SYPRO Ruby protein stain (shown as red). See Figure 1 and Table 3 for protein numbering and details. A putative structural protein (spots N1 and N2), and heat shock protein GroEL (spot N9) displayed a significant rise in protein phosphorylation when M. pneumoniae was harvested for analysis in the stationary phase.

Figure 4

Schematic of phosphorylated proteins within Mycoplasma pneumoniae overlaid on a diagram of the hypothesized complex terminal organelle and cytoskeleton-like structure. "Phos (+)" means the protein has been identified as phosphorylated in our data or previous studies. "Phos (+?)" means there is weak experimental evidence published supporting phosphorylation, and "(?)" means there is no evidence regarding phosphorylation, but that the protein was localized by immunofluorescence studies to the cytoskeleton-like structure. All protein locations are represented based upon the presently available literature data, but may be subject to change based upon further studies. For proteins labeled with "(~)", conflicting evidence indicates that they may have multiple locations in the cell. The localization of proteins were determined as follows: HMW1, HMW2 and HMW3 were shown essential in the electron-dense core of the terminal organelle in M. pneumoniae, though HMW3 may also have other cellular locations; P30 and P65 are localized at the surface of the distal end of the terminal organelle [38, 39]; P1, P90 and P40 are known to interact, with P1 penetrating the cell membrane, anchored to cytoskeletal structures by P90 and P40 [41]; and in a cross-linked protein complex with the P1 adhesin of M. pneumoniae, it appears that DnaK might be involved in translocation of proteins from cytoplasm to the membrane. Pyruvate dehydrogenase is implicated as a structural protein in the attachment organelle [41]. EF-Tu is a putative cytoskeletal element in E. coli [45], but has also been associated with translation so it may have multiple functions and hence locations. It, along with DnaK and pyruvate dehydrogenase, have previously been shown involved in the filamentous network. [41, 45]