The amidases potentially responsible for the transformation of TFNG-AM to TFNG were identified using a protein homology analysis strategy. The genome of P. salicylatoxidans CGMCC 1.17248 was sequenced and all annotated proteins were screened as possible amidases. Because the amidases AmiA and AmiB of the N 2 -fixing bacterium M. flocculans CGMCC 1.16731 were previously demonstrated to convert TFNG-AM to TFNG, all putative amidase protein sequences were aligned with AmiA and AmiB. A phylogenetic tree was constructed for potential amidases based on sequence similarity using the neighbor-joining algorithm in MEGA 8.0 software (Figure S1). Then, amidase sequences with high homology to the amidases AmiA and AmiB of M. flocculans were chosen for gene cloning.
The above-selected amidase-encoding genes were cloned using primers synthesized by Springen Biotechnology Co., Ltd. (Nanjing, China) (Table 1). A Sangon Bacterial Genomic DNA Extraction Kit (Shanghai, China) was used to extract genomic DNA from P. salicylatoxidans CGMCC 1.17248. For DNA amplification operations, PrimeSTAR Max DNA Polymerase Premix (Takara, Dalian, China) was employed. The DNAMAN software version 8.0 was used to analyze protein sequence similarity. The PsmiA and PsmiB sequences characterized in the present study have been submitted to GenBank under the indicated accession numbers WHP70861 and WHP70862, respectively.
The above-selected amidase-encoding genes were cloned using primers synthesized by Springen Biotechnology Co., Ltd. (Nanjing, China) (Table 1). A Sangon Bacterial Genomic DNA Extraction Kit (Shanghai, China) was used to extract genomic DNA from P. salicylatoxidans CGMCC 1.17248. For DNA amplification operations, PrimeSTAR Max DNA Polymerase Premix (Takara, Dalian, China) was employed. The DNAMAN software version 8.0 was used to analyze protein sequence similarity. The PsmiA and PsmiB sequences characterized in the present study have been submitted to GenBank under the indicated accession numbers WHP70861 and WHP70862, respectively.
