The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation. PRL1 encodes a regulatory WD protein that interacts with ATHKAP2, an alpha-importin nuclear import receptor, and is imported into the nucleus in Arabidopsis. Potential functional conservation of PRL1 homologs found in other eukaryotes is indicated by nuclear localization of PRL1 in monkey COS-1 cells and selective interaction of PRL1 with a nuclear protein kinase C-betaII isoenzyme involved in human insulin signaling.

SummaryThe fix-2 mutant of Rhizobium meliloti affected in the invasion of alfalfa root nodules (Inf ¹ /Fix ¹ ) is K þ sensitive and unable to adapt to alkaline pH in the presence of K þ . Using directed Tn5 mutagenesis, we delimited a 6 kb genomic region in which mutations resulted in both Inf ¹ /Fix ¹ and K þ -sensitive phenotypes. In this DNA region, seven open reading frames (ORFs) were identified and the corresponding genes were designated phaA, B, C, D, E, F and G. The putative PhaABC proteins exhibit homology to the subunits of a Na þ /H þ antiporter from an alkalophilic Bacillus strain. Moreover, PhaA and PhaD also show similarity to the ND5 and ND4 subunits of the proton-pumping NADH:ubiquinone oxidoreductase respectively. Computer analysis suggests that all seven proteins are highly hydrophobic with several possible transmembrane domains. Some of these domains were confirmed by generating active alkaline phosphatase fusions. Ion transport studies on phaA mutant cells revealed a defect in K þ efflux at alkaline pH after the addition of a membrane-permeable amine. These results suggest that the pha genes of R. meliloti encode for a novel type of K þ efflux system that is involved in pH adaptation and is required for the adaptation to the altered environment inside the plant.

Bacterial exopolysaccharide (EPS) and lipopolysaccharide (LPS) molecules have been shown to play important roles in plant-bacterium interactions. Here we have demonstrated that the fix-23 loci, which compensate for exo mutations during symbiotic nodule development, are involved in the production of a novel polysaccharide that is rich in 3-deoxy-D-manno-2-octulosonic acid (Kdo) but is not the classical LPS. This molecule is likely to be a surface antigen since antiserum to whole Rhizobium meliloti cells reacts strongly with it, and since mutations in fix-23 result in an inability to produce this polysaccharide and to bind bacteriophage 16-3. It is likely that this Kdo-rich polysaccharide is analogous to certain Escherichia coli K-antigens which are anchored to the membrane via a phospholipid moiety. DNA sequence analysis of one gene cluster of this region revealed that the predicted protein products of six genes exhibit a high degree of homology and similar organization to those of the rat fatty acid synthase multifunctional enzyme domains.

A fix region of Rhizobium meliloti 41 involved both in symbiotic nodule development and in the adsorption of bacteriophage 16-3 was delimited by directed TnS mutagenesis. Mutations in this DNA region were assigned to four complementation units and were mapped close to the pyr-2 and pyr-29 chromosomal markers. Phage inactivation studies with bacterial cell envelope preparations and crude lipopolysaccharides (LPS) as well as preliminary characterization of LPS in the mutants indicated that these genes are involved in the synthesis of a strain-specific LPS. Mutations in this DNA region resulted in a Fix-phenotype in AK631, an exopolysaccharide (EPS)-deficient derivative of R. meliloti 41; however, they did not influence the symbiotic efficiency of the parent strain. An exo region able to restore the EPS production of AK631 was isolated and shown to be homologous to the exoB region of R. meliloti SU47. By generating double mutants, we demonstrated that exo and Ips genes determine similar functions in the course of nodule development, suggesting that EPS and LPS may provide equivalent information for the host plant.Rhizobia are able to induce nitrogen-fixing nodules on the roots of their host leguminous plants. Products of the bacterial nodulation (nod) genes are responsible for the earliest events of the interaction with the plant, including hostspecific activation of meristematic cell division and root hair curling. When rhizobia enter the curled root hairs, a polysaccharide tube (the infection thread) forms, grows toward the root tissue, and branches out into the nodule cells; this process is followed by invasion of bacteria. Bacteria released from the infection thread are wrapped into a membrane of plant origin (peribacteroid membrane) and differentiate into nitrogen-fixing bacteroids. The development of symbiosis is directed also by signals from the environment and from the symbiotic partner. Constituents of the bacterial surface are known to be involved in this process, but their exact role has not been elucidated (for reviews, see references 14 and 34).The cell envelope of gram-negative bacteria (including Rhizobium and Bradyrhizobium species) consists of an inner cytoplasmic cell membrane and an outer membrane separated by the periplasmic space and the peptidoglycan layer (35). The extracellular surface of the outer membrane consists of abundant channel and specific minor proteins, excreted extracellular polysaccharides (EPS), and lipopolysaccharides (LPS). LPS molecules are anchored into the membrane by their lipid A subunits, which carry an oligosaccharide core and an antigenic polysaccharide chain (O antigen) synthesized from oligosaccharide units (35,44).In different Rhizobium species, involvement of either LPS or EPS in the formation of symbiosis has been reported. LPS mutants of Rhizobium leguminosarum bv. phaseoli and bv. viciae unable to synthesize the entire 0 antigen do not invade host cells; consequently, the nodules are 38,40). The pss genes involved in EPS synthesis of R. leguminosarum bv. phaseo...

We report the genetic and biochemical analysis of Rhizobium meliloti mutants defective in symbiotic nitrogen fixation (Fix-) and "respiratory" nitrate reduction (Rnr-). The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus proved to be identical to the previously described fix-14 locus. By directed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited in which all mutations resulted in Rnr- and Fix- phenotypes. Nucleotide sequence analysis of this region revealed the presence of four open reading frames coding for integral membrane and membrane-anchored proteins. Biochemical analysis of the mutants showed that the four proteins were necessary for the biogenesis of all cellular c-type cytochromes. In agreement with the nomenclature proposed for rhizobial genes involved in the formation of c-type cytochromes, the four genes were designated cycH, cycJ, cycK, and cycL, respectively. The predicted protein product of cycH exhibited a high degree of similarity to the Bradyrhizobium japonicum counterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatus Cc11 and Cc12 proteins, respectively. cycJ encodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes for (parts of) a multisubunit cytochrome c haem lyase. Moreover, our results indicate that in R. meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nitrogen fixation in root nodules.

Abstract.To identify bacterial genes involved in symbiotic nodule development, ineffective nodules of alfalfa (Medicago sativa) induced by 64 different Fixmutants of Rhizobium meliloti were characterized by assaying for symbiotic gene expression and by morphological studies. The expression of leghemoglobin and nodulin-25 genes from alfalfa and of the nifHD genes from R. meliloti were monitored by hybridizing the appropriate DNA probes to RNA samples prepared from nodules. The mutants were accordingly divided into three groups. In group I none of the genes were expressed, in group II only the plant genes were expressed and in group III all three genes were transcribed. Light and electron microscopical analysis of nodules revealed that nodule development was halted at different stages in nodules induced by different group I mutants. In most cases nodules were empty lacking infection threads and bacteroids or nodules contained infection threads and a few released bacteroids. In nodules induced by a third mutant class bacteria were released into the host cells, however the formation of the peribacteroid membrane was not normal. On this basis we suggest that peribacteroid membrane formation precedes leghemoglobin and nodulin-25 induction, moreover, after induction of nodulation by the nod genes at least two communication steps between the bacteria and the host plants are necessary for the development of the mature nodule. By complementing each mutant of group I with a genomic R. meliloti library made in pLAFR1, four new fix loci were identified, indicating that several bacterial genes are involved in late nodule development.HIZOBIA are able to cooperate with leguminous plants to fix atmospheric nitrogen. Nitrogen fixation takes place in symbiotic nodules, new plant organs developed for this purpose. Nodule cells harbor unique cell organelles, bacteroids, derived from Rhizobium bacteria which reduce dinitrogen to ammonia. The differentiation and function of nodules involves symbiosis-specific expression of both bacterial and plant genes (nodulin genes) in a highly coordinated manner, as well as communication between the two partners. This communication process seems to consist of signal exchange during nodule development. The molecular basis of these events are, at present, mostly unknown.Recently, plant flavones were reported to act as signal molecules inducing the expression of bacterial nodulation (nod)

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The rkp-3 region is indispensable for capsular polysaccharide (K antigen) synthesis in Sinorhizobium meliloti Rm41. Strain Rm41 produces a K antigen of strain-specific structure, designated as the KR5 antigen. The data in this report show that the rkp-3 gene region comprises 10 open reading frames involved in bacterial polysaccharide synthesis and export. The predicted amino acid sequences for the rkpL-Q gene products are homologous to enzymes involved in the production of specific sugar moieties, while the putative products of the rkpRST genes show a high degree of similarity to proteins required for transporting polysaccharides to the cell surface. Southern analysis experiments using gene-specific probes suggest that genes involved in the synthesis of the precursor sugars are unique in strain Rm41, whereas sequences coding for export proteins are widely distributed among Sinorhizobium species. Mutations in the rkpL-Q genes result in a modified K antigen pattern and impaired symbiotic capabilities. On this basis, we suggest that these genes are required for the production of the KR5 antigen that is necessary for S. meliloti Rm41 exoB (AK631)-alfalfa (Medicago sativa) symbiosis.