Bacterial strains and culture conditions
M. gallisepticum strain S6 was grown in mycoplasma broth (MB) or on mycoplasma agar (MA; containing 1% agar (Oxoid) without phenol red) at 37°C . For selection of mycoplasma transformants, 16 μg of gentamicin/ml (Invitrogen) was added to the media.
E. coli DH5α cells were used as the host for genetic manipulation and cloning of plasmids. Clones were grown in Luria-Bertani broth (LB) or on LB agar plates (LB with 1% agar) containing 100 μg ampicillin/ml (Amresco) at 37°C. For detection of alkaline phosphatase activity in transformants grown on solid media, the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma) was added to the LB agar plates or MA to a final concentration of 40 μg/ml.
Amplification of DNA sequences by PCR
PCR was carried out using Platinum HiFi Taq DNA polymerase (Invitrogen) in a 25 μl volume containing 2.5 μl of 10 x buffer (Invitrogen), 2 mM MgSO4, 100 μM of each deoxynucleotide triphosphate (Bioline), 0.4 μM of each primer, 1.5 U of enzyme and 5 ng of each PCR product as template. The reaction was performed in an iCycler (BioRad) with an initial cycle of 95°C for 3 min, followed by 35 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 1 min/kb, with a final extension at 72°C for 7 min.
Development of alkaline phosphatase construct
The E. coli phoA gene lacking a promoter, signal sequence and the first 5 residues of the mature protein  was cloned under the control of the ltuf promoter and fused to the lipoprotein acylation signal sequence of vlh A1.1, and subsequently cloned into the Tn4001 transposon contained in pISM2062.2 to generate the plasmid, pISM2062.2ltuf acyphoA (pTAP) (Figure 1A). Another plasmid (pTP), lacking the signal and acylation sequence (pTP), was also produced (Figure 1B).
The pTAP and pTP constructs were introduced into E. coli DH5α by electroporation using a Gene Pulser (BioRad) with settings of 2.5 kV and 25 μF. Recombinants were selected for ampicillin resistance and clones were screened for the presence of the gentamicin resistance gene using the oligonucleotide primers GmF and GmR. Selected clones were cultured in larger volumes and plasmid DNA extracted using a Midi prep kit (Qiagen) according to the manufacturer’s instructions.
Transformation of M. gallisepticum
M. gallisepticum was transformed by electroporation as described previously [39, 40]. Following electroporation, cells were gently resuspended in 1 ml of ice-cold MB, incubated at 37°C to allow expression of the gentamicin resistance gene, then a 500 μl aliquot of the culture inoculated onto MA plates containing 16 μg of gentamicin/ml, which were allowed to dry and then incubated at 37°C for 4 days. The plates were examined for colony development and single colonies selected and subcultured in MB containing 16 μg of gentamicin/ml.
Detection of alkaline phosphatase activity on MA plates
To detect alkaline phosphatase activity in colonies of transformed M. gallisepticum on MA plates, a single tablet of BCIP/nitroblue tetrazolium (NBT) (Sigma Fast, Sigma) was dissolved in 3 ml distilled water and sprayed onto the colonies uniformly as a thin layer using a pump atomizer. After 10 min colonies were observed for the presence of a blue colour.
Genomic DNA sequencing
To determine the insertion site of the transposon, genomic DNA sequencing was carried out using the ABI Prism BigDye Terminator v3.1 (BDT) sequencing system (Perkin Elmer Applied Biosystems) and the UBR oligonucleotide primer (Table 1) according to the manufacturer's instructions, with minor modifications. Approximately 2 μg of genomic DNA was combined with 1 μM of the UBR oligonucleotide, 4 μl of the BDT enzyme mixture, 4 μl of 5 x BDT buffer and distilled water to 20 μl. The sequencing reaction mixture was incubated at 96°C for 5 min, then through 60 cycles of 96;°C for 30 s, 50°C for 10 s and 60°C for 4 min in an iCycler thermocycler (BioRad). The sequencing products were purified according to the manufacturer’s instructions using ethanol-EDTA-sodium acetate precipitation and analysed using an ABI3100 capillary sequencer.
Quantitative RT-PCR (qRT-PCR) was used to determine the level of transcription of the phoA gene in each of the transformants. To achieve this, total RNA from 6 ml of transformant cells was extracted using an RNA purification kit (Qiagen), following the manufacturer’s instructions. The total amount of RNA was determined using an ND-1000 spectrophotometer (NanoDrop). To remove any contaminating DNA, 2 μg of extracted RNA was treated with 2 U of DNase I (Invitrogen) in a buffer containing 2 μl of 10 x DNase I buffer and RNase-free water in a total volume of 20 μl for 15 min at room temperature. To produce cDNA, 1 μg of DNAse I-treated RNA was used. For each 1 μg of DNAse I-treated RNA, 50 ng of random hexamers (Invitrogen) and 10 nM of each deoxynucleoside triphosphate (dNTPs, Bioline) were added and the mixture incubated at 65°C for 5 min, then immediately cooled on ice. To this, 4 μl of 5 x first strand reaction buffer (Invitrogen) and dithiothreitol (Invitrogen) to a final concentration of 0.1 M were added and the mixture incubated at 25°C for 2 min, then 1 μl (200 U) of Superscript II reverse transcriptase (RT) (Invitrogen) was added and the reaction incubated for 10 min. A negative control (no RT) was also included, with 1 μl of RNase-free water substituted for the Superscript II reverse transcriptase. The reverse transcription reactions were incubated at 42°C for 50 min. The reaction was stopped by incubation at 70°C for 15 min and the total volume made up to 600 μl with nuclease-free water and aliquots stored at −20°C. Each qRT-PCR reaction was conducted in a 20 μl volume and contained 5 μl template cDNA, 10 μl of 2 x Platinum SYBR Green qPCR Supermix containing Rox Dye (Invitrogen) and 100 nM each of the PRTF and PRTR primers (Table 1). Reactions were run using a Stratagene MX3000P. Each assay included test cDNA, the no-RT control reaction previously described and a no template control, to which only water was added. The cycling conditions were an initial incubation for 2 min at 50°C, followed by 5 min at 95°C, then 40 cycles of 95°C for 30 s and 60°C for 30 s. Reactions were carried out in triplicate for each sample. Relative quantification of phoA transcription was normalised against transcription from the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH, GeneID: 1090024) using the HLF and HMR primers (Table 1) and the relative level of expression calculated using the delta-delta Ct method .
Detection of alkaline phosphatase activity in cultured cells
Mycoplasma transformants were grown in 10 ml MB supplemented with gentamicin at 16 μg/ml until an approximate pH of 7.2 was reached, then pelleted by centrifugation at 20,000 x g for 20 min at 4°C. The cells were resuspended and washed twice in ice-cold 0.05 M Tris, pH 8.0 (T buffer) and again centrifuged and washed as before. The cells were finally resuspended in T buffer with 1% Triton X-100 (ICN) added and incubated for 15 min at 4°C. The total protein concentration of the cell lysate was determined in triplicate using a BCA kit (Pierce) following the manufacturer’s instructions. To determine the AP activity of each transformant in triplicate, 10 μl of the cell lysate was added to reaction buffer (1 M Tris, pH 8.0, 1 mM MgCl2) to which 50 μl of 2 mM disodium p-nitrophenyl phosphate (pNPP, Calbiochem) in reaction buffer was added and the mixture incubated at 37°C for 30 min. The reaction was terminated by addition of 100 μl 2 M NaOH and the absorbance read at 410 nm using a spectrophotometer (Labsystems Multiskan MS). In each assay doubling dilutions in triplicate, starting at 12 U of bacterial alkaline phosphatase (BAP, Invitrogen), were made in T buffer and similarly treated, enabling a standard curve to be constructed. The alkaline phosphatase activity in the BAP dilution series was plotted against absorbance and this used to determine the alkaline phosphatase activity in each sample, which was expressed as BAP U/mg of total cell protein.
SDS-PAGE, Western blotting and immunostaining
Mycoplasma cell proteins were separated by SDS-PAGE as described previously . The protein concentrations of mycoplasma cells were determined using the Pierce BCA protein assay kit (Thermo Scientific), using bovine serum albumin as the standard, and 10 μg of total cell protein was loaded into each well of a polyacrylamide gel. After separation in a 10% polyacrylamide gel, proteins were transferred onto PVDF membranes and incubated in blocking solution containing 5% (w/v) skim milk (Devondale) in PBS with 0.1% (v/v) Tween 20 (PBS-T) for 2 h at room temperature on a rocking platform. Following blocking, membranes were washed three times for 5 min each in PBS-T. Membranes were then incubated for 1 h with mouse monoclonal antibody (MAb) to alkaline phosphatase (Chemicon) at a 1:5000 dilution in blocking solution. The membranes were washed thrice for 5 min with PBS-T and incubated with rabbit anti-mouse-horseradish peroxidase (HRPO) conjugate (Dako) for 1 h at a 1:5000 dilution in blocking solution. This was followed by washing thrice for 5 min each with PBS-T and bound conjugate was then detected by chemiluminiscence using an ECL Plus kit (GE Healthcare) according to the manufacturer's recommendations. As molecular weight marker, 10 μl of biotinylated protein ladder (Cell Signaling Technology) was loaded, and for detection in Western blots, HRP-linked anti-biotin antibody was used.
Partitioning of mycoplasma cell proteins into hydrophobic and aqueous fractions using Triton X-114
Mycoplasma cell proteins from a 20 ml overnight culture were separated into hydrophobic and aqueous fractions using the detergent Triton X-114 (Sigma) [43, 44]. The urea solubilised protein fractions were then analysed by SDS-PAGE.
Membrane and cytoplasmic separation
Membrane and cytoplasmic fractions of M. gallisepticum were purified essentially as previously described for M. pneumoniae . The cytosolic and membrane fractions were then analysed by SDS-PAGE and immunoblotting.
Trypsin treatment of intact M. gallisepticum transformant cells
M. gallisepticum cells were cultured and the cell pellet washed in 50 mM Tris, 0.145 M NaCl, pH 7.4 (TS buffer). This was repeated twice and the cells finally resuspended in 600 μl TS buffer, then divided into 6 equal aliquots. A dilution series of trypsin (Sigma) at 250, 125, 62, 31 and 15 μg/ml was made in TS buffer and 100 μl of each dilution, as well as a control without any trypsin, added to a separate aliquot of cells and these incubated at 37°C for 30 min. Digestion was stopped by the addition of 200 μl of 0.125% (w/v) trypsin inhibitor (Sigma). The trypsin-treated cells were collected by centrifugation, resuspended in TS buffer and proteins in the sample separated by SDS-PAGE and either stained with Coomassie brilliant blue or immunoblotted.
Globomycin is a peptide antibiotic that inhibits the processing of prolipoprotein to mature lipoprotein by signal peptidase II [46, 47]. Mycoplasma cells were grown in the presence or absence of globomycin (a gift from Dr. M. Inukai, IUHW, Japan), dissolved in methanol. Cells were grown in MB with 25 μg globomycin/ml and the cells were harvested by centrifugation at 20,000 x g for 20 min at 4°C, washed thrice in PBS and proteins in the sample separated by SDS-PAGE and either stained with Coomassie brilliant blue or immunoblotted.
Radiolabelling of M. gallisepticum lipoproteins
M. gallisepticum transformants were cultured in 20 ml MB to pH 7.2 and cells harvested and resuspended in 2 ml of fresh MB containing 10 μCi [14 C]palmitate/ml (Perkin Elmer), then incubated at 37°C for 18 h. The cells were centrifuged at 8000 g for 20 min at 4°C and washed in 2 ml PBS. The washing step was repeated three times. The cells were resuspended in 100 μl PBS and SDS-PAGE lysis buffer added. Mycoplasma proteins, together with [14 C] methylated molecular weight markers (Sigma), were separated by SDS-PAGE in a 10% polyacrylamide gel and fixed in a solution of 10% (v/v) glacial acetic acid and 30% (v/v) methanol for 30 min. The gel was incubated in EN3HANCE (Life Science Products) according to the manufacturer's instructions, vacuum dried and then exposed to X-ray film (Kodak).
Two-dimensional gel electrophoresis of fractionated mycoplasma cell proteins
M. gallisepticum cells were harvested and fractionated with Triton X-114 as described above, and the hydrophobic fraction was resuspended in 8 M urea, 2% CHAPS, 0.5% IPG buffer (3–10) and 18 mM dithiothreitol (DTT, GE Healthcare). A 125–150 μg sample of protein, as estimated using the 2-D-Quant kit (Amersham Biosciences), was subjected to isoelectric focusing (IEF) on 7 cm strips over the pH range of 3–10 (GE Healthcare) using the following parameters: rehydration at 30 V for 6 h, 60 V for 6 h; running at 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 1000–8000 V for 1 h and 8000 V for 1.5 h. After isoelectric focusing the gel strips were equilibrated twice in 6 M urea, 75 mM Tris–HCl, pH 8.8, 2% SDS and 30% glycerol (65 mM DTT, 0.135 M iodoacetamide) for 15 min each. Immediately following equilibration and fixing, the IEF strips were transferred onto a 10% SDS-polyacrylamide gel and fixed in place with 0.5% agarose containing bromophenol blue. Electrophoresis was carried out at 200 V for 1 h. The gels were stained with Coomassie brilliant blue.
Mass spectrometry of PhoA
Following 2-D gel electrophoresis of fractionated cellular proteins of untransformed and TAP- transformed M. gallisepticum , the gel images were compared in order to locate the gel spot likely to correspond to PhoA. This spot was excised and subjected to in-gel digestion and peptide mass fingerprint analysis as described previously  using an Ultraflex III MALDI TOF/TOF instrument (Bruker Daltonics, Bremen). Spectra were acquired in reflectron mode and calibrated externally using a standard peptide mix (Bruker Daltonics). Proteins were identified using Mascot v 2.2 (Matrix Science) with the following search parameters: database = NCBI, taxonomy = bacteria, enzyme = trypsin, mass tolerance = 30 ppm, missed cleavages = 1, fixed modifications = carbamidomethyl (Cys) and optional modifications = oxidation (Met).