ABSTRACT
Objective To construct a mutant strain of Streptococcus pneumoniae ( S.pneumoniae) with ciaH gene-knockout (ΔciaH) and to analyze the correlation between the ciaH gene and the bacterial re-sistance against β-lactam antibiotics.Methods The ciaH gene segament of S.pneumoniae strain ATCC6306 was amplified by PCR.The PCR product was sequenced after T-A cloning.A suicide plasmid pEVP3ciaH was constructed for the deletion of ciaH gene and then transformed into the ATCC 6306 strain by using the CaCl2 method .The mutant strain of S.pneumonia strain ATCC6306 with ciaH gene-knockout (ΔciaH) was genera-ted through homologous recombination , insertion inactivation and amphemycin screening , which was further identified by PCR , sequencing analysis and laser confocal microscopy .Double agar dilution method was used to detect the minimal inhibitory concentrations ( MICs ) of penicillin G ( PCN ) and cefotaxime ( CTX ) against theΔciaH mutant strain and the wild type strain .The differences between the MICs were further ana-lyzed.The changes of ciaH gene expression at mRNA level after treatment with 1/4 MIC of PCN or CTX were detected by real-time fluorescent quantitative RT-PCR ( qRT-PCR ) .Results The ciaH gene in the genomic DNA of the generated ΔciaH mutant strain was inactivated by insertion as indicated by PCR and se-quencing analysis .Results of the immunofluorescence assay showed that the ΔciaH mutant strain did not ex-press the CiaH protein .The MICs of PCN and CTX against the ΔciaH mutant strain were 32 μg/ml and 64μg/ml, respectively, which were significantly higher than that of the wild type strain (0.06 μg/ml and 1μg/ml) (P<0.01).The expression of ciaH gene at mRNA level was significantly elevated after treatment S.pneumoniae ATCC6306 strain with 1/4 MIC PCN or CTX (P<0.01).Conclusion The CiaH protein in the CiaH/CiaR two-component signaling system is involved in the resistance of S.pneumoniae against β-lac-tam antibiotics.
ABSTRACT
Objective To construct a prokaryotic expression system for expressing the phosphatase-encoding gene phpP in StkP/PhpP signaling couple in Streptococcus pneumonia ( S.pneumoniae) strains, and to further understand the phosphatase activity of the recombinant protein rPhpP.Methods The entire phpP gene of S.pneumoniae strain ATCC6306 was amplified by PCR.The PCR products were sequenced.A prokaryotic ex-pression system for expressing the phpP gene was constructed by the genetic engineering technique.The ex-pressed protein rPhpP and the solubility of rPhpP were assessed by SDS-PAGE and gel image analyzer.Ni-NTA affinity chromatography was performed to purify rPhpP.The changes of phpP gene transcription after the treat-ment with sublethal dosages of penicillin and cefotaxime were determined by real-time fluorescent quantitative RT-PCR.The functional domain in the sequence of the phpP gene and its type was analyzed by bioinformatic softwares.The activity of rPhpP in hydrolyzing the substrate of PP2C phosphotase was measured with Serine/Threonine Phosphotase Assay Kit.The enzyme kinetic parameters of rPhpP were calculated.Results The se-quence of the cloned phpP gene was identical with that reported in GenBank.The rPhpP in soluble form was ex-pressed in the constructed prokaryotic expression system.An increased expression of phpP gene at mRNA level was induced by sublethal dosage of penicillin or cefotaxime.The domain of PP2Cc type phosphatase was detec-ted in the sequence of phpP gene.The purified rPhpP protein could hydrolyze phosphopeptides [ RRA ( pT) VA], a substrate of PP2C type phosphatase, in a dose-dependent manner with Km and Kcat values of 277.35μmol/L and 0.71 S-1 ,respectively.Conclusion The protein encoded by phpP gene of S.pneumoniae was a PP2C type phosphatase.The expression of phpP gene could be enhanced by sublethal dosage of penicillin or ce-fotaxime.