[Effect of intestinal nitrate on growth of Klebsiella pneumoniae and its regulatory mechanism].

Q3 Medicine Nan fang yi ke da xue xue bao = Journal of Southern Medical University Pub Date : 2024-04-20 DOI:10.12122/j.issn.1673-4254.2024.04.18

J Xie, R Ma, M Li, B Li, L Xiong

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Abstract

Objective: To explore the effect of intestinal nitrates on the growth of Klebsiella pneumoniae and its regulatory mechanisms.

Methods: K. pneumoniae strains with nitrate reductase narG and narZ single or double gene knockout or with NarXL gene knockout were constructed and observed for both aerobic and anaerobic growth in the presence of KNO₃ using an automated bacterial growth analyzer and a spectrophotometer, respectively. The mRNA expressions of narG and narZ in K. pneumoniae in anaerobic cultures in the presence of KNO₃ and the effect of the binary regulatory system NarXL on their expresisons were detected using qRT-PCR. Electrophoretic mobility shift assays (EMSA) and MST analysis were performed to explore the specific regulatory mechanisms of NarXL in sensing and utilizing nitrates. Competitive experiments were conducted to examine anaerobic growth advantages of narG and narZ gene knockout strains of K. pneumoniae in the presence of KNO₃.

Results: The presence of KNO₃ in anaerobic conditions, but not in aerobic conditions, promoted bacterial growth more effectively in the wild-type K. pneumoniae strain than in the narXL gene knockout strain. In anaerobic conditions, the narXL gene knockout strain showed significantly lowered mRNA expressions of narG and narZ (P < 0.0001). EMSA and MST experiments demonstrated that the NarXL regulator could directly bind to narG and narZ promoter regions. The wild-type K. pneumoniae strain in anaerobic cultures showed significantly increased expressions of narG and narZ mRNAs in the presence of KNO₃ (P < 0.01), and narG gene knockout resulted in significantly attenuated anaerobic growth and competitive growth abilities of K. pneumoniae in the presence of KNO₃ (P < 0.01).

Conclusion: The binary regulatory system NarXL of K. pneumoniae can sense changes in intestinal nitrate concentration and directly regulate the expression of nitrate reductase genes narG and narZ to promote bacterial growth.

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[肠道硝酸盐对肺炎克雷伯氏菌生长的影响及其调节机制]。

目的：探讨肠道硝酸盐对肺炎克雷伯氏菌生长的影响及其调控机制：探讨肠道硝酸盐对肺炎克雷伯氏菌生长的影响及其调控机制：方法：构建了硝酸盐还原酶narG和narZ单、双基因敲除或NarXL基因敲除的肺炎克雷伯菌株，并分别用自动细菌生长分析仪和分光光度计观察其在KNO3存在下的需氧和厌氧生长情况。利用 qRT-PCR 技术检测了 KNO3 存在下厌氧培养的肺炎双球菌中 narG 和 narZ 的 mRNA 表达量，以及二元调控系统 NarXL 对其表达量的影响。进行了电泳迁移实验（EMSA）和MST分析，以探索NarXL感知和利用硝酸盐的特定调控机制。在 KNO3 存在的情况下，进行了竞争性实验以检验 NarG 和 narZ 基因敲除菌株的厌氧生长优势：结果：与 narXL 基因敲除菌株相比，在厌氧条件下，KNO3 的存在能更有效地促进肺炎双球菌野生型菌株的细菌生长，而在有氧条件下则不然。在厌氧条件下，narXL 基因敲除菌株的 narG 和 narZ 的 mRNA 表达量明显降低（P < 0.0001）。EMSA和MST实验证明，NarXL调节因子可直接与narG和narZ启动子区域结合。在厌氧培养中，野生型肺炎双球菌菌株在KNO3存在下的narG和narZ mRNA表达量显著增加（P＜0.01），narG基因敲除导致肺炎双球菌在KNO3存在下的厌氧生长和竞争生长能力显著减弱（P＜0.01）：结论：肺炎双球菌的二元调控系统 NarXL 能感知肠道硝酸盐浓度的变化，直接调控硝酸盐还原酶基因 narG 和 narZ 的表达，从而促进细菌生长。

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