Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in Sinorhizobium meliloti increases its ability to nodulate alfalfa

Abstract

1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant growth-promoting rhizobacteria, including rhizobia. This enzyme degrades ACC, the immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene in higher plants. The ACC deaminase of Rhizobium leguminosarum bv. viciae 128C53K was previously reported to be able to enhance nodulation of peas. The ACC deaminase structural gene (acdS) and its upstream regulatory gene, a leucine-responsive regulatory protein (LRP)-like gene (lrpL), from R. leguminosarum bv. viciae 128C53K were introduced into Sinorhizobium meliloti, which does not produce this enzyme, in two different ways: through a plasmid vector and by in situ transposon replacement. The resulting ACC deaminase-producing S. meliloti strains showed 35 to 40% greater efficiency in nodulating Medicago sativa (alfalfa), likely by reducing ethylene production in the host plants. Furthermore, the ACC deaminase-producing S. meliloti strain was more competitive in nodulation than the wild-type strain. We postulate that the increased competitiveness might be related to utilization of ACC as a nutrient within the infection threads.

FIG. 1.

Construction of S. meliloti Rm11466 by transposon replacement. A 4-kb DNA fragment containing the lrpL and acdS genes from R. leguminosarum bv. viciae 128C53K was inserted into the BamHI site of transposon Tn5 in pGS220 to construct pWM3. pWM3 is suicidal after being introduced into S. meliloti Rm5356. After double crossover between the IS50 regions of the two transposons, Tn5-233 and Tn5-acd, the neomycin resistance gene, leucine-responsive regulatory-like gene (lrpL), and the ACC deaminase structural gene (acdS) were inserted into megaplasmid pRmeSU47b in S. meliloti Rm5356. The resulting variant was named Rm11466. Restriction sites: B, BamHI; E, EcoRI; H, HindIII. There are multiple HindIII sites in Tn5-233 and Tn5-acd, but only the locations of the sites inside the IS50 regions are shown on the restriction map.

FIG. 2.

ACC deaminase activities of the S. meliloti strains induced by 5 mM ACC. R. leguminosarum bv. viciae 128C53K, which is the source of the lrpL and acdS genes, was used as a positive control. Rm1021 and Rm5356 are wild-type strains that were used as negative controls. Rm1021 containing the vector pSP329 was also used as a negative control for Rm1021(pWM2). N/D, no activity detected. The error bars represent the standard error of the mean of two independent assays.

FIG. 3.

Nodule numbers (panel A) and shoot dry weights (panel B) of alfalfa inoculated with wild-type S. meliloti strains Rm1021 and Rm5356 and their ACC deaminase-producing variants, Rm1021(pWM2) and Rm11466. Plants not inoculated with a bacterium (0.85% NaCl only) were used as negative controls. Two independent nodulation assays were performed, and each had 18 plants per treatment. The error bars represent the standard error of the mean of a pool of 36 plants.