Rry expression was achieved using the ABI7000SDS (Applied Biosystems), SYBR Green chemistry, and 23388095 the standard curve method for relative quantification. The PCR reagents consisted of: 16 SYBR Green PCR Master Mix (Applied Biosystems), 400 nM of each primer, and 5 ml of sample cDNA, in a final volume of 25 ml. The thermocycling profile was: 10 min at 95uC followed by 40 cycles of 15 s at 95uC and 1 min at 60uC. qPCR primers for mCherry (34 and 35) citrine (36 and 37) and tetracycline (38 and 39) were designed using ABI7000SDS ?specific software, Primer Express (Applied Biosystems). Optical plates included plasmid standard curves for Citrine and mCherry, and duplicates of each cDNA sample. “No template” and “no RT” controls were also included in every qPCR Genz-644282 price assays. For each sample, the expression of Citrine or mCherry was determined from the respective standard curve by conversion of the mean threshold cycle values, and normalization was obtained by dividing the quantity of Citrine (or mCherry) cDNAs by the quantity of cDNA amplified within the gene encoding for the tetracycline resistance protein (used as the endogenous control), which is cloned in the same plasmid. The specificity of the amplified products was verified by analysis of the dissociation curves generated by the ABI 7000 software based on the specific melting temperature for each amplicon. The final qPCR results were based on two independent experiments.Figure 8. Applications of the developed tools for localization of S. pneumoniae proteins. (A) Localization of the cell division protein FtsZ as 1527786 a N-terminal fusion to CFP (CFP-FtsZ, strain BCSMH050) and to itagged CFP, (iCFP-FtsZ, strain BCSMH051). (B) Localization of the GLPG0187 membrane Wzd protein as a N-terminal fusion to CFP (CFP-Wzd, strain BCSJF004) and to improved i-tagged CFP, (iCFP-Wzd, strain BCSJF003). (C) Localization of the Wze tyrosine kinase as a N-terminal fusion to Citrine (Citrine-Wze, strain BCSJF002) and to improved i-tag Citrine (iCitrine-Wze, BCSJF001). The i-tagged versions of the fluorescent reporters allowed the visualization of each protein at the expected subcellular region of bacteria, the division septum. Exposure times: 5 sec. Scale bar: 2 mm. doi:10.1371/journal.pone.0055049.gGraphPad Prism 6 (GraphPad Software, Inc.). The nonparametric Kruskal-Wallis test, followed by Dunn’s multiple comparison, was used to avoid assuming a normal distribution of the data.Protein analysisBacterial cell aliquots of 1 ml of culture were harvested at midexponential growth phase. Cells were incubated at 37uC during 30 minutes in deoxicholate (0.25 mg/ml), RNase (10 mg/ml), DNase (10 mg/ml) and PMSF (1 mM). For the fluorescent protein analysis, proteins were incubated with solubilization buffer (200 mM Tris-HCl pH 8.8, 20 glycerol, 5 mM EDTA pH 8.0, 0.02 bromophenol blue, 4 SDS, 0.05M DDT) [27] at 37uC during 5 minutes and separated on SDS-PAGE. Gel images were acquired on a FUJI FLA 5100 laser scanner (Fuji Photo Film Co.) with 635 nm excitation and .665 nm band pass emission filter for protein molecular weight marker detection, 532 nm excitation and .575 nm band pass emission filter for mCherry detection and 473 nm excitation and .510 nm band pass emission filter for Citrine detection. For western-blot analysis, cells extracts were boiled during 5 minutes before being separated on SDS-PAGE. Proteins were transferred into a Hybond PVDF Membrane (Amersham) and probed with Living Colors H Av. Peptide Antibody (Clontech.Rry expression was achieved using the ABI7000SDS (Applied Biosystems), SYBR Green chemistry, and 23388095 the standard curve method for relative quantification. The PCR reagents consisted of: 16 SYBR Green PCR Master Mix (Applied Biosystems), 400 nM of each primer, and 5 ml of sample cDNA, in a final volume of 25 ml. The thermocycling profile was: 10 min at 95uC followed by 40 cycles of 15 s at 95uC and 1 min at 60uC. qPCR primers for mCherry (34 and 35) citrine (36 and 37) and tetracycline (38 and 39) were designed using ABI7000SDS ?specific software, Primer Express (Applied Biosystems). Optical plates included plasmid standard curves for Citrine and mCherry, and duplicates of each cDNA sample. “No template” and “no RT” controls were also included in every qPCR assays. For each sample, the expression of Citrine or mCherry was determined from the respective standard curve by conversion of the mean threshold cycle values, and normalization was obtained by dividing the quantity of Citrine (or mCherry) cDNAs by the quantity of cDNA amplified within the gene encoding for the tetracycline resistance protein (used as the endogenous control), which is cloned in the same plasmid. The specificity of the amplified products was verified by analysis of the dissociation curves generated by the ABI 7000 software based on the specific melting temperature for each amplicon. The final qPCR results were based on two independent experiments.Figure 8. Applications of the developed tools for localization of S. pneumoniae proteins. (A) Localization of the cell division protein FtsZ as 1527786 a N-terminal fusion to CFP (CFP-FtsZ, strain BCSMH050) and to itagged CFP, (iCFP-FtsZ, strain BCSMH051). (B) Localization of the membrane Wzd protein as a N-terminal fusion to CFP (CFP-Wzd, strain BCSJF004) and to improved i-tagged CFP, (iCFP-Wzd, strain BCSJF003). (C) Localization of the Wze tyrosine kinase as a N-terminal fusion to Citrine (Citrine-Wze, strain BCSJF002) and to improved i-tag Citrine (iCitrine-Wze, BCSJF001). The i-tagged versions of the fluorescent reporters allowed the visualization of each protein at the expected subcellular region of bacteria, the division septum. Exposure times: 5 sec. Scale bar: 2 mm. doi:10.1371/journal.pone.0055049.gGraphPad Prism 6 (GraphPad Software, Inc.). The nonparametric Kruskal-Wallis test, followed by Dunn’s multiple comparison, was used to avoid assuming a normal distribution of the data.Protein analysisBacterial cell aliquots of 1 ml of culture were harvested at midexponential growth phase. Cells were incubated at 37uC during 30 minutes in deoxicholate (0.25 mg/ml), RNase (10 mg/ml), DNase (10 mg/ml) and PMSF (1 mM). For the fluorescent protein analysis, proteins were incubated with solubilization buffer (200 mM Tris-HCl pH 8.8, 20 glycerol, 5 mM EDTA pH 8.0, 0.02 bromophenol blue, 4 SDS, 0.05M DDT) [27] at 37uC during 5 minutes and separated on SDS-PAGE. Gel images were acquired on a FUJI FLA 5100 laser scanner (Fuji Photo Film Co.) with 635 nm excitation and .665 nm band pass emission filter for protein molecular weight marker detection, 532 nm excitation and .575 nm band pass emission filter for mCherry detection and 473 nm excitation and .510 nm band pass emission filter for Citrine detection. For western-blot analysis, cells extracts were boiled during 5 minutes before being separated on SDS-PAGE. Proteins were transferred into a Hybond PVDF Membrane (Amersham) and probed with Living Colors H Av. Peptide Antibody (Clontech.
