Novel Isolate Of Pseudonocardia And Establishment Of Its Genetic Manipulation System

Pseudonocardia is a genus of rare actinomycetes with close evolutionary relationship and high G+C content(>70%).In the cell wall of Pseudonocardia,the peptidoglycan contains meso-diaminodiacid,and its hydrolytic solution possesses galactose and arabinose but not mycotic acid.The genus has received extensive attention for its unique ecological effects and applications in biotechnology,pharmaceuticals,agriculture and environmental remediation.However,due to the lack of genetic manipulation system,the study and application in this genus have been generally largely limited.In this study,taken a Pseudonocardia isolate sampled from the sediment of Yarlung Zangbo River,Tibet,China,as a model,we established a genetic manipulation system for this strain and other similar microorganisms.Comparison of the ANI(Average Nucleotide Identity)between the genome sequence of this isolate with those of known Pseudonocardia species in the NCBI database showed that this isolate belongs to Pseudonocardia autotrophica and was named as strain Yalu.The genome of P.autotrophica Yalu encodes at least 17 kinds of potential secondary metabolites synthesis pathways,and several antiviral defense systems such as the phage growth limitation system and the CRISPR-Cas system.These have made P.autotrophica Yalu an important strain theoretical and applied values.Further analyses of the genome reveal that a sequence motif almost identical to the ?C31 att/int site is present,indicative of being possible to introduce foreign DNAs via transconjugation.Therein,a genetic manipulation system for P.autotrophica Yalu was established based on transconjugation and introduced foreign DNAs into the host cells.Various transconjugation parameters were attempted,determined and further optimized.Eventually,using an ISP4 medium without Mg2+,the highest transconjugation efficiency of 5.5×10-6was achieved when 4.0×107recipient cells and 1.0×108donor cells,respectively,were in use.By using this system,a GFP-encoding gene was successfully integrated into genome at the putative ?C31 att/int site.The GFP signal was detected through flow cytometry,which can be thus be employed as a reporter system for P.autotrophica Yalu.In addition to the insertion of foreign DNA at the ?C31 att/int site,through homologous recombination,two nonessential genes were also deleted from the genome with an efficiency of 50%(5/10).Taken all these together,a genetic manipulation system was developed for P.autotrophica Yalu,providing an efficient toolkit for conducting fundamental research in this strain and facilitating strain development and application.