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Genomic resources for identification of the minimal N 2 ‐fixing symbiotic genome
Author(s) -
diCenzo George C.,
Zamani Maryam,
Milunovic Branislava,
Finan Turlough M.
Publication year - 2016
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/1462-2920.13221
Subject(s) - biology , replicon , gene , genome , genetics , sinorhizobium meliloti , symbiosis , computational biology , mutant , bacteria
Summary The lack of an appropriate genomic platform has precluded the use of gain‐of‐function approaches to study the rhizobium–legume symbiosis, preventing the establishment of the genes necessary and sufficient for symbiotic nitrogen fixation ( SNF ) and potentially hindering synthetic biology approaches aimed at engineering this process. Here, we describe the development of an appropriate system by reverse engineering S inorhizobium meliloti . Using a novel in vivo cloning procedure, the engA ‐tRNA‐ rmlC ( ETR ) region, essential for cell viability and symbiosis, was transferred from S inorhizobium fredii to the ancestral location on the S . meliloti chromosome, rendering the ETR region on pSymB redundant. A derivative of this strain lacking both the large symbiotic replicons ( pSymA and pSymB ) was constructed. Transfer of pSymA and pSymB back into this strain restored symbiotic capabilities with alfalfa. To delineate the location of the single‐copy genes essential for SNF on these replicons, we screened a S . meliloti deletion library, representing > 95% of the 2900 genes of the symbiotic replicons, for their phenotypes with alfalfa. Only four loci, accounting for < 12% of pSymA and pSymB , were essential for SNF . These regions will serve as our preliminary target of the minimal set of horizontally acquired genes necessary and sufficient for SNF .