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.

Key words: Klebsiella pneumoniae; NarXL; nitrate reductase; anaerobic growth; intestinal environment