Bacillus velezensis LG37: Functional verification of GlnL and analysis of the ammonia assimilation metabolic pathway

Abstract

High concentrations of ammonia have toxic effects on bred animals. In aquaculture, the possibility of using Bacillus preparations to remove ammonia nitrogen in aquaculture water through assimilation has been generally recognized. In this study, to analyze the metabolic pathways of ammonia assimilation in Bacillus, the characteristics and pathways of ammonia assimilation of Bacillus velezensis LG37 stored in the laboratory were analyzed. The results showed that the rate of ammonia assimilation by LG37 in a minimal medium could reach 94.2% at 20 h, and the highest assimilation rate was 95.4% within 60 h. In a minimal medium, the growth rate of LG37 in the glutamine group was significantly faster than that in the ammonia group, but the expression of glnL showed opposite results. The gene expression level of glnL in the ammonia group was significantly higher than in the glutamine group. glnL overexpression (OEglnL) and deletion (ΔglnL) strains were constructed by CRISPR/Cas9 technology and using the pHT1K vector, respectively. The expression levels of glnL in LG37, OEglnL, and ΔglnL strains were determined by RT-qPCR. The glnL gene expression levels were ordered as follows: OEglnL > LG37 > ΔglnL. In all three strains (LG37, ΔglnL, and OEglnL) and at different ammonia concentrations, the expression levels of glnL were consistent with glnA and glnB levels, showing a positive correlation. However, the expression levels of glnK and glnR in different strains hardly changed significantly.