Open AccessDevelopment of a Functional Genomics Platform for Sinorhizobium meliloti : Construction of an ORFeome
Author(s) -
Brenda K. Schroeder,
Brent House,
Michael W. Mortimer,
Svetla. Yurgel,
Scott C. Maloney,
Kristel L. Ward,
Michael L. Kahn
Publication year - 2005
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.71.10.5858-5864.2005
Subject(s) - sinorhizobium meliloti , orfs , biology , plasmid , functional genomics , genetics , genome , context (archaeology) , computational biology , gene , genomics , open reading frame , mutant , peptide sequence , paleontology
The nitrogen-fixing, symbiotic bacteriumSinorhizobium meliloti reduces molecular dinitrogen to ammonia in a specific symbiotic context, supporting the nitrogen requirements of various forage legumes, including alfalfa. Determining the DNA sequence of theS. meliloti genome was an important step in plant-microbe interaction research, adding to the considerable information already available about this bacterium by suggesting possible functions for many of the >6,200 annotated open reading frames (ORFs). However, the predictive power of bioinformatic analysis is limited, and putting the role of these genes into a biological context will require more definitive functional approaches. We present here a strategy for genetic analysis ofS. meliloti on a genomic scale and report the successful implementation of the first step of this strategy by constructing a set of plasmids representing 100% of the 6,317 annotated ORFs cloned into a mobilizable plasmid by using efficient PCR and recombination protocols. By using integrase recombination to insert these ORFs into other plasmids in vitro or in vivo (B. L. House et al., Appl. Environ. Microbiol. 70:2806-2815, 2004), this ORFeome can be used to generate various specialized genetic materials for functional analysis ofS. meliloti , such as operon fusions, mutants, and protein expression plasmids. The strategy can be generalized to many other genome projects, and theS. meliloti clones should be useful for investigators wanting an accessible source of cloned genes encoding specific enzymes.