Doris Jording

MOLECULAR ANALYSIS OF THE RHIZOBIUM-MELILOTI MUCR GENE REGULATING THE BIOSYNTHESIS OF THE EXOPOLYSACCHARIDES SUCCINOGLYCAN AND GALACTOGLUCAN

The Rhizobium meliloti Tn5 mutant Rm3131, producing galactoglucan (EPS II) instead of succinoglycan (EPS I), was complemented by a 3.6-kb EcoRI-fragment of the Rhizobium meliloti genome. Sequencing of this fragment revealed six open reading frames (ORFs). The ORF found to be affected in the mutant Rm3131 codes for a putative protein of 15.7 kDa and forms a monocistronic transcriptional unit. Further genetic analysis revealed that the gene mutated in Rm3131 is identical to the previously described R. meliloti mucR gene (H. Zhan, S.B. Levery, C. C. Lee, and J.A. Leigh, 1989, Proc. Natl. Acad. Sci. USA 86:3055-3059). By hybridization it was shown that a mucR homologous gene is present in several rhizobacteria. The deduced amino acid sequence of MucR showed nearly 80% identity to the Agrobacterium tumefaciens Ros protein, a negative regulator of vir genes and necessary for succinoglycan production. MucR contains like Ros a putative zinc finger sequence of the C2H2 type. Transcriptional fusions of genes for EPS I and EPS II synthesis, the so-called exo and exp genes, with the marker gene lacZ were used to delineate the role of mucR for exo and exp gene expression. It was found that exp genes are negatively regulated by MucR on the transcriptional level, whereas a posttranscriptional regulation by MucR is assumed for exo genes. Furthermore, mucR is negatively regulating its own transcription.

THE MEMBRANE TOPOLOGY OF THE RHIZOBIUM-MELILOTI C4-DICARBOXYLATE PERMEASE (DCTA) AS DERIVED FROM PROTEIN FUSIONS WITH ESCHERICHIA-COLI K12 ALKALINE-PHOSPHATASE (PHOA) AND BETA-GALACTOSIDASE (LACZ)

The Rhizobium melilotidctA gene encodes the C4-dicarboxylate permease which mediates uptake of C4-dicarboxylates, both in free-living and symbiotic cells. Based on the hydrophobicity of the DctA protein, 12 putative membrane spanning regions were predicted. The membrane topology was further analysed by isolating in vivo fusions of DctA to Escherichia coli alkaline phosphatase (PhoA) and E. coli β-galactosidase (LacZ). Of 10 different fusions 7 indicated a periplasmic and 3 a cytoplasmic location of the corresponding region of the DctA protein. From these data a two-dimensional model of DctA was constructed which comprised twelve transmembrane α-helices with the amino-terminus and the carboxy-terminus located in the cytoplasm. In addition, four conserved amino acid motifs present in many eukaryotic and prokaryotic transport proteins were observed.

Regulatory aspects of the C4-dicarboxylate transport in Rhizobium meliloti: Transcriptional activation and dependence on effectave symbiosis

Summary The transcriptional regulation of the Rhizobium meliloti gene region coding for C 4 -dicarboxylate transport was analyzed by constructing transcriptional lac Z and translational pho A fusions. In the case of the regulatory genes dct B and dct D, it was found that both genes were constitutively expressed when grown in minimal medium supplemented with succinate or with glucose. The transcription of the dct B gene was always lower than that of the dct D gene. Since a transposon-induced mutation in dct B did not abolish the transcription of dct D, it was concluded that both genes were expressed from their own promoters. In contrast to the regulatory dct genes, the expresion of the dct A gene coding for the C 4 -dicarboxylate permease, was regulated by C 4 -dicarboxylates. It was f ound that a dct A + background was necessary for the induction of the dct A expresion. In a dct A − background, the dct A promoter was no longer regulated by C 4 -dicarboxylates and was constitutively expressed at its highest level. Bacteroids isolated from alfalfa nodules gave similar results with respect to the regulation of the dct gene region; the dct B gene was transcribed at a lower level than dct D and a dct A + background was necessary for the transcriptional regulation of the dct A gene. In addition, it was demonstrated that in bacteroids, the NifA protein was not involved in the expresion of the dct A gene. In general, the dct A expresion rate was approximately equal in effectave and ineffective bacteroids. In contrast, the C 4 -dicarboxylate transport rate was reduced to 50% in ineffective bacteroids, indicating that the remaining 50% was necessary for the nitrogen fixation proces.

The C4-dicarboxylate transport system ofRhizobium meliloti and its role in nitrogen fixation during symbiosis with alfalfa (Medicago sativa)

TheRhizobium meliloti C4-dicarboxylate transport (Dct) system is essential for an effective symbiosis with alfalfa plants. C4-dicarboxylates are the major carbon source taken up by bacteroids. Genetic analysis of Dct− mutant strains led to the isolation of thedct carrier genedctA and the regulatory genesdctB anddctD. The carrier genedctA is regulated in free-living cells by the alternative sigma factor RpoN and the two-component regulatory system DctB/D. In addition, DctA is involved in its own regulation, possibly by interacting with DctB. In bacteroids, besides the DctB/DctD system an additional symbiotic activator is thought to be involved indctA expression. Further regulation ofdctA in the free-living state is reflected by diauxic growth of rhizobia, with succinate being the preferred carbon source. The tight coupling of C4-dicarboxylate transport and nitrogen fixation is revealed by a reduced level of C4-dicarboxylate transport in nitrogenase negative bacteroids.

Regulation of the C4-dicarboxylate transport in free-living and symbiotic Rhizobium meliloti

Nitrogen fixation in the Rhizobium-legume symbiosis is an energy demanding process. The plant supplies energy to the bacteroid and receives in return, fixed nitrogen. C4-dicarboxylates are considered to be the compounds which are provided by the plant. These C4-dicarboxylates have to pass two barriers, the peribacteroid and the bacteroid membrane. In recent years, the investigations were focussed on the transport process through the bacteroid membrane. This transport process is particularly well investigated in Rhizobium leguminosarum (4,5,6) and Rhizobium meliloti (1,7,8,9).