title: Acidic conditions permit effective nodulation of alfalfa by invasion-deficient Rhizobium meliloti exoD mutants.
abstract: Rhizobium meliloti exoD mutants are deficient in invasion of alfalfa nodules and, as a consequence, the nodules that exoD strains induce fail to fix nitrogen. These nodules appear to be arrested at the same stage as nodules induced by other exo mutants, which do not make an acidic exopolysaccharide called EPS I, or by ndv mutants, which do not produce a periplasmic cyclic beta(1,2) glucan. However previous genetic and biochemical evidence suggested that the nodule invasion defect of exoD mutants arose from a biochemical deficiency distinct from those of both EPS I-deficient exo mutants and ndv mutants. In this study, we characterize mutant phenotypes of exoD strains in both free-living and symbiotic states. Nodules induced by exoD mutants are generally small and empty of bacteria, and exhibit the same structural features as nodules induced by other invasion-deficient mutants. Putative incipient infection threads were visible in outer cortical cells of these nodules but not in the plant cells in the interior of the nodule. We show that exoD mutants are sensitive to alkaline conditions, ceasing to grow at elevated pH in liquid yeast extract cultures and exhibiting decreased viability in alkaline medium. Interestingly, we find that buffering the plant growth medium at slightly acidic pH (6.0-6.5) restores the ability of exoD mutants to invade alfalfa nodules. exoD mutants are thus alkali sensitive for both free-living and symbiotic phenotypes. This result implies that the nodule invasion defect of exoD mutants arises from their sensitivity to alkaline conditions and, furthermore, that alkaline conditions may obtain in the developing infection thread. The deduced amino acid sequence of ExoD is extremely hydrophobic, suggesting that the protein is membrane associated. We propose models whereby absence of a putative membrane protein might lead to sensitivity to alkaline conditions and consequent arrest of nodule invasion.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
authors.forename: J W
authors.initials: JW
authors.lastname: Reed
authors.forename: G C
authors.initials: GC
authors.lastname: Walker
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D000455:Medicago sativa / Q000382:microbiology*; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009586:Nitrogen Fixation / Q000235:genetics*; D016962:Sinorhizobium meliloti / Q000235:genetics* / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008565:Membrane Proteins; C072006:ExoD protein, Rhizobium meliloti