Figure 1.
Schematic illustration of the machinery for uptake and conversion of carbohydrates leading to the formation of glycerol-3-phosphate in M. pneumoniae.
UgpC (MPN134), UgpA (MPN135), and UgpE (MPN136) form a putative ABC transport system for glycerol-3-phosphate, whereas GlpF (MPN043) is the glycerol uptake facilitator. The glycerol kinase GlpK (MPN050) and the glycerol-3-phosphate oxidase GlpD (MPN051) metabolize glycerol to the glycolytic intermediate dihydroxyacetone phosphate. Hydrogen peroxide formation by GlpD is crucial for the cytotoxic effects of M. pneumoniae. GlpQ (MPN420) and MPN566 encode two paralogous glycerophosphodiesterases that may be able to metabolize glycerophosphocholine to glycerol-3-phosphate and choline. The uptake system for glycerophosphocholine is so far unknown. Proteins highlighted in grey seem not to fulfill the predicted function (this work). CM, cell membrane; DHAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; GPC, glycerophosphocholine; Gly, glycerol; Cho, choline.
Figure 2.
Effect of divalent cations on GlpQ activity.
GlpQ activity was measured in the presence of 0.5 mM glycerophosphocholine and various concentrations of divalent cations. Error bars indicate the standard deviation (based on three independent experiments).
Figure 3.
Isolation of M. pneumoniae glycerophosphodiesterases transposon insertion mutants.
(A, C) Schematic representation of the genomic region surrounding the glpQ and mpn566 gene (both designated as glycerophosphodiesterases) in M. pneumoniae and site of the transposon insertion in the knockout strains GPM81 and GPM82, respectively. The annealing sites of oligonucleotides used for the determination of the transposon insertion site are indicated by small arrows. Probes A and B hybridizing to internal fragments of the glycerophosphodiesterases and the aac-ahpD genes are depicted as dotted lines. (B, D) Southern blot analysis to confirm the single insertion of the mini-transposon into the glpQ and mpn566 gene of the strains GPM81 and GPM82, respectively. Chromosomal DNAs of the wild type and both glycerophosphodiesterase mutants were digested as indicated. Blots were hybridized with the respective glycerophosphodiesterase-specific probe (left) and a probe hybridizing to the aac-ahpD gene of the mini-transposon (right).
Figure 4.
Growth of M. pneumoniae in modified Hayflick medium containing different carbon sources.
One hundred milliliters of medium were inoculated with 5 mg of the relevant M. pneumoniae cells and incubated for up to six days at 37°C in 150-cm2 cell culture flasks. The following strains were used: wild type (wt), glpQ::Tn, mpn566::Tn, and glpD::Tn, glpQ::Tn + glpQ (complemented mutant) and glpQ::Tn + cv (control strain carrying the empty vector used for complementation). Glucose (A) and glycerol (B) were added to a final concentration of 1% (wt/vol). Attached cells were collected by scraping and growth was monitored by determination of the wet weight of the cell pellets. All measurements were done three times. Results are from a representative experiment.
Figure 5.
Examination of M. pneumoniae hydrogen peroxide release.
Hydrogen peroxide production of M. pneumoniae mutant strains was measured in the presence of different carbon sources (100 µM) after 2 h. The following strains were used: wild type (wt), glpQ::Tn, mpn566::Tn, and glpD::Tn, glpQ::Tn + glpQ (complemented mutant) and glpQ::Tn + cv (control strain carrying the empty vector used for complementation). Error bars indicate standard deviation (based on three independent experiments). G3P, glycerol-3-phosphate; GPC, glycerophosphocholine; Glc, glucose; Gly, glycerol; w/o, without addition of any carbon source.
Figure 6.
Cytotoxicity of M. pneumoniae toward HeLa cell cultures.
(A) Infection assay to verify cytotoxic effects of M. pneumoniae glpQ::Tn and mpn566::Tn mutant strains. HeLa cells were infected with M. pneumoniae wild type (wt), glpQ::Tn, and mpn566::Tn mutant cells. As control served two HeLa cell cultures: One without addition of any M. pneumoniae cells and another after infection with glpD::Tn mutant strain [10]. Moreover, cytotoxicity of the complemented mutant (glpQ::Tn + glpQ) and of the mutant carrying the empty vector (glpQ::Tn + cv) was tested. After four days, HeLa cell cultures were stained with crystal violet and photographed. All pictures are shown at the same magnification. Scale bar, 0.1 mm. (B) Quantification of cytotoxicity caused by M. pneumoniae in HeLa cell cultures. HeLa cells were infected with different M. pneumoniae strains or left uninfected (negative control). LDH release of HeLa cell cultures after 2 h of infection was used as an index of cytotoxicity. Cytotoxicity was calculated as the percentage of total LDH release after complete cell lysis. Error bars indicate standard deviation (based on three independent experiments).
Table 1.
Proteins with GlpQ-dependent expression pattern.
Figure 7.
Transcription analysis of GlpQ-dependent genes in M. pneumoniae.
Slot blots were performed with whole RNA extracts of M. pneumoniae wild type (wt), glpQ::Tn, mpn566::Tn, and glpD::Tn (control) mutant strains grown in modified Hayflick medium containing either glucose or glycerol as sole carbon source (1% [wt/vol]). A dilution series of RNA extracts was blotted onto a positively charged nylon membrane and probed with a DIG-labeled riboprobe specific for an internal part of a particular open reading frame. Names of riboprobes are given next to each blot. Signals obtained with 1 µg of RNA are shown. Yeast tRNA and M. pneumoniae chromosomal DNA served as controls. For detailed information on differences of transcript levels see Table 1, S3, and S4.
Figure 8.
Transcriptional organization of GlpQ-dependent genes in M. pneumoniae.
(A) Nucleotide sequence of promoter regions of M. pneumoniae glpF (mpn043), mpn162, cbiO (mpn433), mpn284, and mpn506 genes. Promoter sequences are numbered relative to the 5’ end. Directions of the open reading frames (ORF) are indicated by dotted arrows and predicted ATG/TTG start codons are highlighted by bold type. The respective -10 motifs are underlined. GlpQ-dependent cis-acting elements (palindromic DNA motifs) are indicated by grey shading. (B) Consensus sequence of the GlpQ-dependent palindromic DNA motif in M. pneumoniae. The sequence logo was created using WebLogo v2.8.2 [50] based on all five palindromic DNA motifs mentioned in A.