Identification of Sinorhizobium meliloti early symbiotic genes by use of a positive functional screen

Abstract

The soil bacterium Sinorhizobium meliloti establishes nitrogen-fixing symbiosis with its leguminous host plant, alfalfa, following a series of continuous signal exchanges. The complexity of the changes of alfalfa root structures during symbiosis and the amount of S. meliloti genes with unknown functions raised the possibility that more S. meliloti genes may be required for early stages of the symbiosis. A positive functional screen of the entire S. meliloti genome for symbiotic genes was carried out using a modified in vivo expression technology. A group of genes and putative genes were found to be expressed in early stages of the symbiosis, and 23 of them were alfalfa root exudate inducible. These 23 genes were further separated into two groups based on their responses to apigenin, a known nodulation (nod) gene inducer. The group of six genes not inducible by apigenin included the lsrA gene, which is essential for the symbiosis, and the dgkA gene, which is involved in the synthesis of cyclic beta-1,2-glucan required for the S. meliloti-alfalfa symbiosis. In the group of 17 apigenin-inducible genes, most have not been previously characterized in S. meliloti, and none of them belongs to the nod gene family. The identification of this large group of alfalfa root exudate-inducible S. meliloti genes suggests that the interactions in the early stages of the S. meliloti and alfalfa symbiosis could be complex and that further characterization of these genes will lead to a better understanding of the symbiosis.

FIG. 1.

(A) Schematic representation of promoter trap vector showing relative positions of key genes, including exoY, gfp, and parDE, and the Rm1021 genomic DNA in front of the exoY gene. (B) Schematic representation of the overall strategy used in functional screening for S. meliloti genes that function in the early stages of S. meliloti-alfalfa symbiosis. The numbers in parentheses indicate either the numbers of nodules screened or the numbers of symbiotic promoters isolated.

FIG. 2.

S. meliloti strains showing different intensities of calcofluor fluorescence, which reflect the levels of succinoglycan production by the strains. The average fluorescence intensities for the strains were determined using Kodak 1D analysis software. They are 105.5 ± 16.5 for Rm1021, 83.0 ± 5.8 for RmAR9007, 146.6 ± 33.8 for RmAR9007(pHC153), 193.7 ± 26.8 for RmAR9007(pHC169), and 80.0 ± 6.2 for RmAR9007(pHC170).

FIG. 3.

Colonies of strain RmAR9007 carrying the unsorted promoter library showing different intensities of GFP fluorescence. Colony C was formed by cells carrying plasmid pHC169, and colony A was formed by cells carrying plasmid pHC170. Colony B was formed by cells showing a low level of GFP fluorescence, which was not further analyzed.

FIG. 4.

Four-week-old alfalfa plants inoculated with or without different S. meliloti strains. (A) No bacteria (control); (B) Rm1021; (C) RmAR9007(pHC170); (D) RmAR9007(pHC169).

FIG. 5.

FACS profiles of GFP fluorescence intensities of RmAR9007(pHC153) and RmAR9007 carrying the entire promoter library. The activities of the exoY promoter in free-living RmAR9007(pHC153) cells, which are reflected in the levels of GFP fluorescence of the cells, were used as a guide to determine the levels of GFP fluorescence to separate the cells of RmAR9007 carrying the promoter library into dark, dim, and bright populations. Cells with fluorescence intensities of <6.31 were collected as the dark sublibrary; cells with fluorescence intensities between 6.31 and 20.62 were collected as the dim sublibrary; and cells with fluorescence intensities of about 20.62 were collected as the bright sublibrary.

FIG. 6.

Activities of promoters in the absence or presence of alfalfa root exudates or apigenin, as measured by specific GFP fluorescence intensities. (A) nodABC promoter; (B) exoY promoter; (C) SMb21651 promoter on plasmid pHC169; (D) genomic DNA containing no promoter from plasmid pHC170 (negative control); (E to J) promoters of the lsrA, dgkA, ppe, SMc02171, SMc02773R, and SMc03205R open reading frames, respectively.

FIG. 7.

Schematic representations of symbiotic promoters activated by alfalfa root exudates. The genomic locations of the trapped promoters are indicated by arrows and numbers above the diagrams of the open reading frames. The numbers reflect the genomic positions assigned by the genome sequencing project. *, positions of upstream ends of the promoter fragments were estimated based on the sizes of the inserts in the promoter trap vector. The names of the genes or putative open reading frames are indicated in the open arrows.