Role of methylation in regulating PhoP activity in Salmonella enterica serovar Typhimurium / 上海交通大学学报（医学版）

Objective :To investigate the correlation between methylation and the activity of transcriptional regulator PhoP in Salmonella enterica serovar Typhimurium (S. Typhimurium), and screen for the methyltransferase of PhoP. Methods :The methylation level of PhoP in S. Typhimurium under different culture conditions was determined by Western blotting. The transcription levels of phoP and the genes regulated by phoP were examined to screen for the methyltransferase of PhoP after overexpressing methyltransferase candidates predicted by bioinformatics. And then methyltransferase assay was verified in vitro. The transcription levels of phoP and its methyltransferase were determined by real-time PCR in high concentration of Mg2+ or weak acid conditions. Results :As the concentration of Mg2+increased in the medium, the methylation level of PhoP increased, and its methylation level decreased after S. Typhimurium was stimulated by weak acid. In the screening of 9 methyltransferases predicted by bioinformatics, overexpression of STM14_0023 reduced the transcription level of phoP and its downstream genes and the protein level of PhoP in vivo, but knockout of STM14_0023 had no effect on the expression of phoP and its downstream genes. STM14_0023 was capable of methylating PhoP in vitro and could also increase the methylation level of PhoP after overexpression of STM14_0023 in vivo. Under the condition of high concentration of Mg2+ when the expression of phoP was inhibited, the transcriptional level of STM14_0023 was reduced. Conclusion :STM14_0023 is the methyltransferase of PhoP, and methylation inhibits PhoP activity.

Autoregulation of PhoP/PhoQ in Yersinia pestis under different conditions / 军事医学

Objective To investigate the transcriptional autoregulation of PhoP/PhoQ under different growth conditions in Yersinia pestis.Methods The entire promoter region of YPO1635 was amplified and cloned into the pRW50 vector containing a promoterless lacZ reporter gene.The recombinant LacZ reporter plasmid was transformed into the wild-type strain (WT) and the phoP mutant strain (ΔphoP),respectively,to measure the promoter activity (the β-galactosidase activity) of the target gene in WT and ΔphoP by using the β-galactosidase enzyme assay system.Total RNAs were extracted from WT and ΔphoP strains,and primer extension assay was employed to detect the promoter activity by examining the amount of primer extension products of YPO1635 in WT and ΔphoP.Results The LacZ fusion results showed that the transcription of YPO1635 was positively regulated by PhoP under L-TMH and brain-heart infusion(BHI) conditions,but it was not regulated in H-TMH medium.The primer extension assay detected two transcriptional start sites located at 90 and 118 bp upstream of the translation initiation site of phoP,named P1 and P2,respectively.Under low Mg2+ TMH conditions,the promoter activity of P1 rather than P2 was positively regulated by PhoP.Under high Mg2+ TMH conditions,the promoter activities of both P1 and P2 showed no obvious difference in the WT and ΔphoP strains.Under rich BHI conditions,both promoters were under negative control of PhoP.Conclusion Different autoregualtion patterns of PhoP/PhoQ under different growth conditions would help Y.pestis to quickly adapt to the changing living environment.

Deletion of phoQ gene decreases the resistance of Pseudomonas aeruginosa to aminoglycoside antibiot-ics / 中华微生物学和免疫学杂志

Objective To analyze the sequences of two component signaling system PhoP/PhoQ encoding genes of Pseudomonas aeruginosa strains sensitive or resistant to aminoglycoside antibiotics and to determine the correlation between the PhoQ/PhoP and the resistance. Methods The segments of entire pimQ and phoP genes of P. aeruginosa were obtained by PCR and then sequenced after T-A cloning. Two prokaryotic expression systems of phoQ and phoP genes were constructed and the target recombinant expres-sion products rPhoQ and rPhoP were extracted by Ni-NTA chromatography. Rabbits were intracutaneoualy immunized with rPhoQ and rPhoP to obtain antisera and double immunodiffusion test was used to detect the titers of antisera. The phoQ genes of aminloglycoside antibiotics-resistant P. aeruginosa strains were knocked out by using Red recombination system, and phoQ mutants were identified by PCR plus sequencing and Western blot assay. Tube dilution method was applied to determine MIC values of wild and mutant strains of P. aeruginosa to four different aminoglycoside antibiotics. Results In comparison with the corresponding sequences in GenBank, the similarities of nueleotide and putative amino acid sequences of the cloned phop and phoQ genes were 98.7%-99.6% and 98.7%-100% , and 98.4%-99.8% and 99.1%-100%, respec-tively. Both rPhoQ and rPhoP were successfully expressed using pET-42a and E. coil BL21 DE3 system, and their rabbit antisera with 1 : 4 and 1 : 8 double immunodiffusion titers were also obtained. The deletion of phoQ genes and absence of the products in the two phoQ mutants were confirmed by PCR, sequencing and West-ern blot assay. MIC values of the four different aminoglycoside antibiotics to the two mutants were 1/512-1/2048 as those of their wild strains. Conclusion PboQ/PhoP is a sequence conserved two component sig-naling system of P. aeruginosa, and this system mediates resistance of the microbe to aminoglycoside antibiotics.

Analysis of genes involved in the pathogenesis of intracellularly survival bacteria / 영남의대학술지

Eight bacterial strains were examined whether they have phoP/phoQ genes which were known to be involved in the intracellular survival of Salmonella typhimurium. The phoP/phoQ operon were known to sense the stimuli of the genes involved in the adaptation of the environment. Using 514-basepairs EcoRV DNA fragment of phoP region of Salmonella typhimurium as a probe, dot blot hybridization were performed. Chromosomal DNAs of Klebsiella pneumonia, Pseudomonas aeruginosa, Serratia marscescens, Enterobacter cloacae, Salmonella typhimurium, Escherichia coli, Shigella dysenteriae, and Listeria monocytogenes were examined by DNA hybridization assay. Against our expectation, intracellular pathogen, L. monocytogenes, did not have similar DNA sequences to phoP/phoQ of S. typhimurium, while E, coli, S. dysenteriae, and E. cloacae showed the positive signal even though they were not intracellular pathogens. This result suggested that the phoP/phoQ operon was absent in intracellular pathogenic bacterias other than S. typhimurium. Rather it was found in phylogenetically closer bacterias to S. typhimurium, which were not able to survive in intracellular environment. Some different mechanism, which is not dependent on phoP/phoQ operon, could be involved in the intracellular survival of L. monocytogenes.