A dominant-negative fur mutation in Bradyrhizobium japonicum

Abstract

In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified a fur mutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of the fur gene repress iron uptake systems and starve for iron, whereas fur mutants fail to repress iron uptake systems and survive. The B. japonicum fur mutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron. Unexpectedly, a wild-type copy of the fur gene cannot complement the fur mutant. Expression of the fur mutant allele in wild-type cells leads to a fur phenotype. Unlike a B. japonicum fur-null mutant, the strain carrying the dominant-negative fur mutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.

FIG. 1.

Amino acid alignment of Fur proteins from B. japonicum USDA I110, 61A152, and the Mn^r 61A152 fur mutant. Amino acids that are different in the three strains are highlighted. Asterisks denote amino acid differences between the fur mutant and the two wild-type strains. The proposed iron-binding domain is underlined.

FIG. 2.

Iron-regulated OMPs prepared from B. japonicum 61A152. Coomassie-stained 8.6% polyacrylamde SDS-PAGE gel of OMPs prepped from cells grown under iron-deficient and iron-sufficient conditions. The three OMPs that are overexpressed under iron-deficient conditions are labeled 61A1 (unknown), 61A2 (FegA, ferrichrome receptor), and 61A3 (putative heme receptor, homolog of hmuR). The wild-type strain is 61A152, the fur mutant is MLG100, and the pmrfur wild type is 61A152, with the Mn^r fur gene in trans. M (marker) is the protein molecular mass standard, and the sizes (in kilodaltons) are indicated to the right.

FIG. 3.

Soybean plants inoculated with 61A152 or the 61A152 fur mutant or mock inoculated without bacteria. Plants were harvested six weeks post germination and assayed for the total nodule weight per soybean plant (A), the total number of nodules per plant (B), and the total chlorophyll extracted from soybean leaves (C). The standard error is shown.