Mapping phosphoproteins in Mycoplasma genitalium and Mycoplasma pneumoniae.
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.


CONCLUSIONS
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.
Su
Hsun-Cheng
HC
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. seansu@email.unc.edu <seansu@email.unc.edu>
Hutchison
Clyde A
CA
Giddings
Morgan C
MC
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004734:Energy Metabolism; D045704:Mycoplasma genitalium / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011019:Pneumonia, Mycoplasma / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D010750:Phosphoproteins; D030481:Pyruvate Dehydrogenase (Lipoamide); C111305:pyruvate dehydrogenase E1 beta subunit; C065640:pyruvate dehydrogenase E1alpha subunit; D010444:Peptide Elongation Factor Tu; D010751:Phosphopyruvate Hydratase