Multiple groESL operons are not key targets of RpoH1 and RpoH2 in Sinorhizobium meliloti

Abstract

Among the rhizobia that establish nitrogen-fixing nodules on the roots of host plants, many contain multiple copies of genes encoding the sigma factor RpoH and the chaperone GroEL/GroES. In Sinorhizobium meliloti there are two rpoH genes, four groESL operons, and one groEL gene. rpoH1 mutants are defective for growth at high temperature and form ineffective nodules, rpoH1 rpoH2 double mutants are unable to form nodules, and groESL1 mutants form ineffective nodules. To explore the roles of RpoH1 and RpoH2, we identified mutants that suppress both the growth and nodulation defects. These mutants do not suppress the nitrogen fixation defect. This implies that the functions of RpoH1 during growth and RpoH1/RpoH2 during the initiation of symbiosis are similar but that there is a different function of RpoH1 needed later during symbiosis. We showed that, unlike in Escherichia coli, overexpression of groESL is not sufficient to bypass any of the RpoH defects. Under free-living conditions, we determined that RpoH2 does not control expression of the groE genes, and RpoH1 only controls expression of groESL5. Finally, we completed the series of groE mutants by constructing groESL3 and groEL4 mutants and demonstrated that they do not display symbiotic defects. Therefore, the only groESL operon required by itself for symbiosis is groESL1. Taken together, these results suggest that GroEL/GroES production alone cannot explain the requirements for RpoH1 and RpoH2 in S. meliloti and that there must be other crucial targets.

FIG. 1.

Nodulation by suppressor mutant strains. Alfalfa plants were inoculated with control and suppressor mutant strains, and the percentage of nodulated plants was determined after at least 3 weeks of incubation. The graph depicts the average percentage of nodulation over the indicated number of experiments, and error bars represent the sample standard deviation. At least 50 plants in total were inoculated with each bacterial strain. White bars indicate strains derived from the rpoH1 rpoH2 mutant strain VO3148, and gray bars indicate strains derived from the rpoH1 rpoH2 mutant strain VO3149. Significance was determined using the Student's t test. ***, P < 0.001; *, P < 0.05. The strains from left to right were Rm1021, VO3148-VO3151, AB35-AB44, VO3170, and VO3166.

FIG. 2.

Comparison of the growth of suppressor mutant cells with wild-type and rpoH1 rpoH2 double mutant cells, as measured using OD₅₉₅. Cells were grown in LB/MC medium plus streptomycin at 30°C (A) or 40°C (B). The control strains are Rm1021 (wild type; filled circles), VO3148 (rpoH1 rpoH2; filled triangles), and VO3149 (rpoH1 rpoH2; filled diamonds), and the suppressor mutant strains are VO3150 (NDS-1; open diamonds), AB35 (NDS-3; open triangles), VO3170 (GDS-1; crosses), and VO3166 (GDS-2; plus signs). The panels show representative data from one of four experiments.

FIG. 3.

Western analysis of heat shock proteins in suppressor mutant strains. Cells were grown to mid-log phase in LB/MC medium plus streptomycin at 30°C. Equal numbers of cells as measured by OD₅₉₅ were resuspended in buffer and sonicated. Equal volumes of cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting using primary antibodies generated to the following E. coli proteins: GroEL (A), DnaK (B), and DnaJ (C). A representative immunoblot is shown. Graphs depict the mean band intensity normalized to protein concentration and relative to the wild-type signal, with error bars representing the sample standard deviation (n = 3). Gray bars indicate strains with the rpoH1 mutant background, and white bars indicate strains with the rpoH1 rpoH2 double mutant background. The strains from left to right were Rm1021, VO3128, VO3148, VO3150, VO3151, AB35, AB36, VO3165, and VO3166.

FIG. 4.

groESL and rpoH expression in rpoH mutant cells grown in M9 sucrose minimal medium. Growth as measured by OD₅₉₅ and gene expression as monitored by β-glucuronidase (GUS) activity were determined in wild-type (filled circles), rpoH1 (open triangles), rpoH2 (open squares), and rpoH1 rpoH2 (open diamonds) backgrounds. (A) Representative growth curve of cells containing the groEL1-gus fusion. All of the strains in the experiment showed indistinguishable growth patterns. (B to H) GUS activity of cells containing groEL1-gus (B), groEL2-gus (C), groEL3-gus (D), groEL4-gus (E), groEL5-gus (F), rpoH1-gus (G), and rpoH2-gus (H). Each panel shows the data from one representative experiment.