ABSTRACT
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.
ABSTRACT
Objective: To uncover new transcriptional regulators by screening putative transcriptional regulators in Salmonella enterica serovar Typhimurium (S. Typhimurium) through genome informatics and molecular biology analyses. Methods: S. Typhimurium genome informatics was analyzed and 30 putative transcriptional regulators were screened out. All candidate genes were deleted by λ-Red system. The log-phase acid tolerance response (ATR) ability was compared between knock-out (KO) strains and wild type (WT) strain. Cell model was used to detect the invasion ability and intracellular ability of KO strains that showed differences in acid stress compared to WT strain. The transcription level of phoP was detected by realtime-PCR in the mutants involved in both ATR and cell infection. Results: All 30 deletion mutants were successfully constructed. ΔSTM14_0739, ΔSTM14_2717 and ΔSTM14_1646 showed increased log-phase ATR ability, while ATR abilities of ΔSTM14_4338, ΔSTM14_1965 and ΔSTM14_1878 decreased, compared with WT. ΔSTM14_0739 and ΔSTM14_2717 mutants showed weaker invasion ability in HeLa cells than WT, and ΔSTM14_1878 and ΔSTM14_2717 mutants showed stronger replication ability in RAW264.7 cells than WT. Realtime-PCR suggested STM14_2717 deletion had no effect on phoP transcription. Conclusion: This work discovers unknown transcriptional regulators, and provides clues for future research in S. Typhimurium pathogenesis.
ABSTRACT
Objective: To construct a novel plasmid as Salmonella enterica serovar typhimurium (S. typhimurium) sipC gene knockouts candidate. Methods: In this research, 5' upstream and 3' downstream regions of S. typhimurium sipC gene and kanamycin gene were PCR amplified. Each of these DNA fragment was cloned into pGEM T-easy vector. The construct was confirmed by PCR and restriction digest. Results: PCR amplified 320, 206 and 835 bp DNA fragments were subcloned into pET-32 vector resulting with a plasmid called pET-32-sipC up-kan- sip C down. Conclusions: The new plasmid (pET-32-sipC up-kan- sip C down) is useful for genetic engineering and for future manipulation of S. typhimurium sipC gene.
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Aims: We investigated the antibacterial activity of three groups of phenolic compounds obtained from the chloroform (CHCl3) extract of the fleshy seed coat (sarcotestas) of Ginkgo biloba. Study Design: An experimental study. Methodology: Inhibition of microbial growth was measured by an agar diffusion method and susceptibility tests were performed by the broth microdilution method. Bactericidal effect of Ginkgo biloba compound 5-7 against Salmonella enterica serovar Typhimurium was assessed by time-kill assay. Results: Ginkgo biloba compounds 5-7 and 8-10 showed high antimicrobial activity against Gram-positive and Gram-negative bacteria, including several food-borne pathogens. In particular, compounds 5-7 and 8-10, containing phenolic acids and bilobols, respectively, were highly effective against Salmonella enteric serovar Typhimurium, Listeria monocytogenes, Listeria innocua, Streptococcus pyogenes, Escherichia coli, and Shigella dysenteriae. On the opposite, compounds 1-4, containing cardanols, showed little antibacterial activity. Compounds 5-7 exerted a bactericidal and bacteriolytic effect on Salmonella enteric serovar Typhimurium with a Minimal Inhibitory Concentration (MIC) and a Minimal Bactericidal Concentration (MBC) of 8.3 μg ml–1. Conclusion: The results of this study indicate that phenolic compounds derived from Ginkgo biloba sarcotestas, because of their strong inhibitory characteristics towards food pathogens, can be considered ideal candidates for possible application in food microbiology due to their natural origins.
ABSTRACT
Many Salmonella Typhimurium isolates produce type 1 fimbriae and exhibit fimbrial phase variation in vitro. Static broth culture favours the production of fimbriae, while solid agar medium inhibits the generation of these appendages. Little information is available regarding whether S. Typhimurium continues to produce type 1 fimbriae during in vivo growth. We used a type 1 fimbrial phase-variable strain S. Typhimurium LB5010 and its derivatives to infect RAW 264.7 macrophages. Following entry into macrophages, S. Typhimurium LB5010 gradually decreased the transcript levels of fimbrial subunit gene fimA, positive regulatory gene fimZ, and global regulatory gene lrp. A similar decrease in transcript levels was detected by RT-PCRwhen the pH of static brothmediumwas shifted frompH 7 to amore acidic pH 4. A fimA-deleted strain continued to multiply within macrophages as did the parental strain. An lrp deletion strain was unimpaired for in vitro growth at pH 7 or pH 4, while a strain harboring an lrp-containing plasmid exhibited impaired in vitro growth at pH 4. We propose that acidic medium, which resembles one aspect of the intracellular environment in a macrophage, inhibits type 1 fimbrial production by down-regulation of the expression of lrp, fimZ and fimA.
ABSTRACT
A total of 40 Salmolella enterica serovar Typhimurium (S. typhimurium) strains were isolated from clinical specimens of swine at 10 farms in Kyungpook province from 1998 to 2000. We investigated the clonal relationship of S. typhimurium isolates by antimicrobial susceptibility, plasmid profile, and Southern hybridization analysis with tetA, and pulsed-field gel electrophoresis (PFGE). All S. typhimurium isolates showed identical biochemical characteristics and were resistant to tetracycline, streptomycin, and sulfamehtoxazole. They were classified into 5 groups by antimicrobial resistance patterns. S. typhimurium isolates carried 3 to 5 plasmids and were classified into 5 groups by plasmid profiles. Southern hybridization showed that tetA gene was located in 21 kb of plasmid. S. typhimurium isolates from 9 different farms showed identical or similar PFGE patterns, which indicates clonal origin of the strains. All S. typhimurium isolates, except one isolate from 1998, seemed to belong to be one clone by the combination of three epidemiological typing methods. These data demonstrated that a specific clone of Salmolella enterica serovar Typhimurium was widely spread in swine farms in Kyungpook province.