Construction of a Genetic Linkage Map of the Rhizobium meliloti 1600 Kilobase Megaplasmid pRmeSU47b, Generation of Defined Megaplasmid Deletions, and Study of Megaplasmid-Borne Genes

Abstract

<p>A significant amount of the Rhizobium meliloti strain SU47 genome is located on two megaplasmids, both of which carry genes that are involved in the formation of N₂- fixing alfalfa root nodules. The function of most of the DNA carried by these replicons is undefined. A genetic linkage map was made of the larger of the two megaplasmids, pRmeSU47b (pEXO). The map consists of an ordered collection of transposon Tn5 and Tn5-derivative insertions encoding alternating antibiotic resistances, sequentially linked by phage ΦM12 transduction. By converting cotransduction frequencies to physical distance, pRmeSU47b was estimated to be 1600 kb in size. The positions of all known pRmeSU47b loci were determined on the linkage map: two clusters of exopolysaccharide synthetic genes, exo and exp; two separate thiamine biosynthetic loci, thi-502 and thi- 504; a locus involved in symbiosis, fix-386; genes for dicarboxylic acid transport, dct; and lactose utilization, lac.</p> <p>Deletions with defined endpoints were created by recombination between the IS50 elements of transposon insertions. One additional deletion was isolated after selection for loss of the sucrose sensitivity encoded by a Tn5-B12S insertion. In all 1400 kb of pRmeSU47b was deleted. Phenotypic analysis of deletion carrying strains resulted in the discovery of several novel loci: a locus required for the formation of symbiotically effective nodules, fix114, and loci required for utilization of several carbon sources (dulcitol, melibiose, raffinose, β-hydroxybutyrate, acetoacetate, protocatechuate and quinate).</p> <p>Analysis of the fix114 locus by cloning, site-directed Tn5 mutagenesis, and the isolation of active translational fusions to the E. coli reporter gene phoA revealed the size of this locus to be ca. 5 kb and suggested that it encoded extracytoplasmic protein(s). On low osmolarity solid media, fix114 mutants exhibited a mucoid phenotype which appeared to be dependent on expression of genes for synthesis of exopolysaccharide II but not exopolysaccharide I.</p>