Impacting of cAMP receptor protein on the bile tolerance of Aeromonas hydrophila in early growth
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摘要:
目的 本研究拟确定cAMP 受体蛋白 对嗜水气单胞菌胆汁耐受性的影响。 方法 构建嗜水气单胞菌的luxS、crp的缺失株及crp回补株,通过胆汁生长实验确定嗜水气单胞菌及其构建株的胆汁耐受性。 结果 嗜水气单胞菌BJ018、BJ018ΔluxS和BJ018Δcrp在LB培养基中生长速度基本一致。 10%胆汁LB中,BJ018和缺失株ΔluxS前8 h生长被抑制,后期呈对数快速生长;其缺失株Δcrp生长速度领先于野生株,早期生长未被抑制。 嗜水气单胞菌BJ018Δcrp的回补株早期生长重新被抑制,与野生株BJ018基本一致。 其他两株嗜水气单胞菌BJ017、BJ054及其缺失株Δcrp胆汁生长结果与BJ018、BJ018Δcrp一致。 这些结果提示嗜水气单胞菌CRP影响菌株对胆汁的耐受。 结论 CRP的存在影响嗜水气单胞菌在胆汁中的生长,可能在胆汁耐受调控中起到重要作用。 Abstract:Objective Bile is an important signaling molecule that regulates the intestinal microflora and environment in both directions, playing an important role in viral and bacterial infections. CRP (cAMP receptor protein) is involved in the bile tolerance of Escherichia coli and Salmonella in infections. In this study, we evaluated the effect of CRP (cAMP receptor protein) on bile tolerance of Aeromonas hydrophila. Methods The luxS-deleted, crp-deleted and crp-complemented strains of A. hydrophila were constructed, and the bile tolerance of A. hydrophila and its constructed strains was detected by bile growth experiments. Results The growth rates of A. hydrophila strains BJ018, BJ018ΔluxS and BJ018Δcrp in Luria broth (LB ) were similar. However, in 10% bile LB, the growth of BJ018 and BJ018ΔluxS was inhibited in the first 8 hours, and the growth rate was logarithmic in the later stage; while the growth rate of BJ018Δcrp was higher than that of the wild strain, and the early growth was not inhibited. The early growth of the complemented strain BJ018Δcrp-pSRK-crp was re-inhibited in the early stage, which was similar to that of the wild strain BJ018 containing plasmid pSRK. The other A. hydrophila strains BJ017, BJ054 and their crp-deleted strains had the same result with BJ018 and BJ018Δcrp. The result suggested that CRP had influence on bile tolerance of A. hydrophila. Conclusion CRP negatively regulated the growth of A. hydrophila in bile and might play an important role in bile tolerance regulation. -
Key words:
- Aeromonas hydrophila /
- Bile /
- cAMP receptor protein
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表 1 嗜水气单胞菌缺失株及回补株构建相关引物
Table 1. Primers for deletion and complementation of A. hydrophila
编号 引物序列(5'~3') 酶切位点 产物(bp) crp112upF GCtctagaGCGATCGGCGATGACGAACTGA Xba I 565 crp112upR GCACTGCCACCGGCAAGACCATAGTGGTGT crp112dnF GGTCTTGCCGGTGGCAGTGCGACAAGAACC Kpn I 553 crp112dnL GGggtaccCCTGACGCGCCTTCTCCAGGAT luxSupF GCtctagaGCAGTGGGGAACCCCGATCCAGGTG Xba I 496 luxSupR CAGCTCGTCAGTAAAACTGTCCAATAACGGCAT luxSdnF ACAGTTTTACTGACGAGCTGGCCCTGCCGGAAG Kpn I 491 luxSdnR GGggtaccCCACATAGACCGCTTCAAGAGTTTCAAC crpF TGCtctagaTCAGCGGGTGCCGAACAC Xba I 639 crpR GGAATTCcatatgGTCATTGGCAAACCGCAAAGC Nde I crpinF GCGGAGATCAGCTCCTGC 576 crpinR TCCCACCCTGGAATGGTTCT 注:下划线部分为引入的酶切位点和保护性碱基,粗体字母为反向互补序列 -
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