Disruption of the glycine cleavage system enables Sinorhizobium fredii USDA257 to form nitrogen-fixing nodules on agronomically improved North American soybean cultivars

Abstract

The symbiosis between Sinorhizobium fredii USDA257 and soybean [Glycine max (L.) Merr.] exhibits a high degree of cultivar specificity. USDA257 nodulates primitive soybean cultivars but fails to nodulate agronomically improved cultivars such as McCall. In this study we provide evidence for the involvement of a new genetic locus that controls soybean cultivar specificity. This locus was identified in USDA257 by Tn5 transposon mutagenesis followed by nodulation screening on McCall soybean. We have cloned the region corresponding to the site of Tn5 insertion and found that it lies within a 1.5-kb EcoRI fragment. DNA sequence analysis of this fragment and an adjacent 4.4-kb region identified an operon made up of three open reading frames encoding proteins of deduced molecular masses of 41, 13, and 104 kDa, respectively. These proteins revealed significant amino acid homology to glycine cleavage (gcv) system T, H, and P proteins of Escherichia coli and other organisms. Southern blot analysis revealed the presence of similar sequences in diverse rhizobia. Measurement of beta-galactosidase activity of a USDA257 strain containing a transcriptional fusion of gcvT promoter sequences to the lacZ gene revealed that the USDA257 gcvTHP operon was inducible by glycine. Inactivation of either gcvT or gcvP of USDA257 enabled the mutant to nodulate several agronomically improved North American soybean cultivars. These nodules revealed anatomical features typical of determinate nodules, with numerous bacteroids within the infected cells. Unlike for the previously characterized soybean cultivar specificity locus nolBTUVW, inactivation of the gcv locus had no discernible effect on the secretion of nodulation outer proteins of USDA257.

FIG. 1.

Metabolic pathway of the glycine cleavage enzyme system. This system is a multimeric assembly of four loosely associated proteins known as P protein (GcvP pyridoxal phosphate-containing glycine decarboxylase), H protein (GcvH, lipoic acid-containing carrier), T protein (GcvT, tetrahydrofolate requiring aminomethyltransferase or glycine synthase), and L protein (GcvL, lipoamide dehydrogenase). 3-PGA, 3-phosphoglycerate; N⁵,N¹⁰-mTHF, N⁵,N¹⁰-methylene tetrahydrofolate; C₁ pool, pool of compounds containing only one carbon.

FIG. 2.

Coordinated physical and genetic maps of gcv region of S. fredii USDA257. The orientation of the three ORFs and the location of the Tn5 and omega cassette insertions are also shown.

FIG. 3.

Multiple amino acid sequence alignment of GcvT proteins from different organisms. (A) The accession numbers of the sequences are as follows: S. meliloti, AL591688; Agrobacterium, AE007869; E. coli, AP009048.1; Arabidopsis, AT1G11860; human, NM_000481; bovine, NM_177485. Sequences are aligned with the USDA257 GcvT sequence (GQ214396). Positions of amino acid identity are boxed. Multiple sequence alignment analysis was performed using the CLUSTAL W program from UniProt Knowledgebase (http://www.uniprot.org/). (B) Phylogenetic tree of GcvT. The scale shown in the phylogenetic tree represents the branch distance as the number of changes in character state between GcvT proteins from different organisms.

FIG. 4.

Southern blot analysis of gcvT in rhizobia. Genomic DNAs from B. liaoningense USDA3622 (lane 1), R. galegae USDA4128 (lane 2), R. hainanense USDA3588 (lane 3), R. huautlaense USDA4900 (lane 4), R. tropici USDA9030 (lane 5), M. amorphae USDA1001 (lane 6), S. fredii USDA205 (lane 7), S. meliloti USDA1002 (lane 8), S. terangae USDA4894 (lane 9), R. leguminosarum USDA2370 (lane 10), and S. saheli USDA4893 (lane 11) were restriction enzyme digested with EcoRI and separated electrophoretically in 0.8% agarose. The gel was blotted onto nitrocellulose and probed with the ³²P-labeled S. fredii USDA257 gcvT gene. Molecular weight markers in kilobases are shown on the left side of the figure.

FIG. 5.

Anatomy of soybean (G. max cv. McCall) nodules formed by the USDA257 gcvT mutant. (A) Light micrograph of a paraffin section of soybean nodule revealing a central infected zone (IZ). The arrows point to vascular bundles in the cortex. (B) Transmission electron micrograph of soybean nodule showing the presence of numerous bacteroids (B) which are surrounded by symbiosomes. Note the presence of numerous poly-β-hydroxybutyrate inclusions inside the bacteroids.

FIG. 6.

Extracellular protein profile and immunoblot analysis of USDA257 and the USDA257 gcvT mutant. Extracellular proteins were prepared from cells grown in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 1 μM apigenin. The proteins were resolved by 15% SDS-PAGE and silver stained (A) or transferred to a nitrocellulose membrane for immunological analysis with antibodies raised against Nops (B). Lanes 1 and 2, extracellular proteins from USDA257; lanes 3 and 4, extracellular proteins from the USDA257 gcvT mutant. The immunoreactive proteins are identified on the right side of the figure.