D Guan1, G S Pettis. 1. Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
Abstract
AIMS: To develop an effective gene transfer system for Streptomyces ipomoeae, the causative agent of soil rot disease of sweet potatoes. METHODS AND RESULTS: Of the several methods investigated, introduction of genes into S. ipomoeae could only be achieved using intergeneric conjugation from Escherichia coli. However, even results for that method varied greatly and were dependent on using particular media for S. ipomoeae spore preparation and conjugation. Transconjugant to recipient ratios as great as 4.1 x 10(-5) were achieved when International Streptomyces Project Medium 4 was used for both sporulation and conjugation protocols. Both site-specifically integrating and autonomously replicating plasmids could be introduced and maintained in S. ipomoeae, and plasmids could be introduced with approximate equivalent frequencies from either methyl-proficient or methyl- deficient E. coli donors; the latter result indicates a likely absence of relevant methyl-specific restriction in S. ipomoeae. CONCLUSIONS: Efficient transfer of genes into S. ipomoeae was achieved here by using an optimized intergeneric mating procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: The described protocol will facilitate further genetic manipulation of this agriculturally important pathogen.
AIMS: To develop an effective gene transfer system for Streptomyces ipomoeae, the causative agent of soil rot disease of sweet potatoes. METHODS AND RESULTS: Of the several methods investigated, introduction of genes into S. ipomoeae could only be achieved using intergeneric conjugation from Escherichia coli. However, even results for that method varied greatly and were dependent on using particular media for S. ipomoeae spore preparation and conjugation. Transconjugant to recipient ratios as great as 4.1 x 10(-5) were achieved when International Streptomyces Project Medium 4 was used for both sporulation and conjugation protocols. Both site-specifically integrating and autonomously replicating plasmids could be introduced and maintained in S. ipomoeae, and plasmids could be introduced with approximate equivalent frequencies from either methyl-proficient or methyl- deficient E. coli donors; the latter result indicates a likely absence of relevant methyl-specific restriction in S. ipomoeae. CONCLUSIONS: Efficient transfer of genes into S. ipomoeae was achieved here by using an optimized intergeneric mating procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: The described protocol will facilitate further genetic manipulation of this agriculturally important pathogen.
Authors: Tina Netzker; Volker Schroeckh; Matthew A Gregory; Michal Flak; Mario K C Krespach; Peter F Leadlay; Axel A Brakhage Journal: Appl Environ Microbiol Date: 2016-05-31 Impact factor: 4.792