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Sinorhizobium meliloti is an alpha-proteobacterium that alternates between a free-living phase in bulk soil or in the rhizosphere of plants and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids. As a step toward understanding the physiology of S. meliloti in its free-living and symbiotic forms and the transition between the two, gene expression profiles were determined under two sets of biological conditions: growth under oxic versus microoxic conditions, and in free-living versus symbiotic state. Data acquisition was based on both macro- and microarrays. Transcriptome profiles highlighted a profound modification of gene expression during bacteroid differentiation, with 16% of genes being altered. The data are consistent with an overall slow down of bacteroid metabolism during adaptation to symbiotic life and acquisition of nitrogen fixation capability. A large number of genes of unknown function, including potential regulators, that may play a role in symbiosis were identified. Transcriptome profiling in response to oxygen limitation indicated that up to 5% of the genes were oxygen regulated. However, the microoxic and bacteroid transcriptomes only partially overlap, implying that oxygen contributes to a limited extent to the control of symbiotic gene expression.

Rhizobium tropici strain CIAT899 displays a high intrinsic thermal tolerance, and had been used in this work to study the molecular basis of bacterial responses to high temperature. We generated a collection of R. tropici CIAT899 mutants affected in thermal tolerance using TnS-luxAB mutagenesis and described the characterization of a mutant strain, CIAT899-10T, that fails to grow under conditions of high temperature. Strain CIAT899-10T carries a single transposon insertion in a gene showing a high degree of similarity with the guaB gene of Escherichia coli and other organisms, encoding the enzyme inosine monophosphate dehydrogenase. The guaB strain CIAT899-10T does not require guanine for growth due to an alternative pathway via xanthine dehydrogenase and, phenotypically, in addition to the thermal sensitivity, the mutant is also defective in symbiosis with beans, forming nodules that lack rhizobial content. Guanine and its precursors restore wild-type tolerance to grow at high temperature. Our data show that, in R. tropici, the production of guanine via inosine monophosphate dehydrogenase is essential for growth at extreme temperatures and for effective nodulation.

Upon infection of the tropical legume Sesbania rostrata with Azorhizobium caulinodans ORS571, nodules are formed on the roots as well as on the stems. Stem nodules appear at multiple predetermined sites consisting of dormant root primordia, which are positioned in vertical rows along the stem of the plant. We used the differential display method to isolate and characterize three cDNA clones (differential display; didi-2, didi-13, and didi-20), corresponding to genes whose expression is enhanced in the dormant root primordia after inoculation. Database searches revealed that the deduced (partial) didi-2 gene product shares significant similarity with hydroxyproline-rich cell wall proteins. The (partial) didi-13 and didi-20 products are similar to chitinases and chalcone reductases, respectively. Transcripts corresponding to the cDNA clones didi-2 and didi-13 were first detectable 1 day after inoculation. In contrast, didi-20 transcripts were found at low levels in uninfected root primordia and were enhanced significantly around 3 days after inoculation. In addition, a cDNA was isolated (didi-42) that corresponds to the previously identified leghemoglobin gene Srlb6. These studies show that differential display is a useful method for the isolation of infection-related genes.

Rhizopine (L-3-O-methyl-scyllo-inosamine; 3-O-MSI) synthesis (mos) and catabolism (moc) genes were originally isolated from Rhizobium meliloti strain L5-30 (Murphy et al., Proc. Natl. Acad. Sci. U.S.A., 84:493, 1987). These genes have been postulated to give a competitive advantage to this strain in the rhizosphere, since the ability to utilize the unusual nutritional mediator rhizopine as nitrogen and carbon source appears to be correlated with the ability of Moc+ bacteria to efficiently infect alfalfa plants. This study examines the distribution of rhizopine catabolism (moc) genes among different soil bacteria. By using oligonucleotide primers homologous to the moc genes and the polymerase chain reaction (PCR), moc genes were shown to be absent from a random collection of 100 different soil isolates. However, screening 50 different electrophoretic type strains of a worldwide R. meliloti collection (Eardly et al., Appl. Environ. Microbiol. 56:187, 1990) revealed the presence of moc genes in three additional strains, S33, 102F51, and 74B3. These three strains were found to be able to synthesize rhizopine in planta (Mos+) and to catabolize it (Moc+). To determine the relatedness of the Mos+/Moc+ strains to each other and to other R. meliloti strains, we used the rep-PCR method to generate genomic fingerprints, and to create a phylogenetic tree with the help of an optical imaging system and data analysis program (AMBIS). Because of the apparent infrequent occurrence of moc genes among soil bacteria, we suggest that the use of moc genes as a selectable marker trait for tracking genetically manipulated organisms is feasible.

Using site-directed mutagenesis, mutations were introduced in the -24/-12 promoter element of the Azorhizobium caulinodans nifA gene, and chimeric nifA-lacZ reporter gene fusions were constructed. Single base-pair mutations in the conserved -25 or -13 G residues were found to reduce or abolish nifA promoter activity, respectively, demonstrating that the -24/-12 promoter element is important for nifA gene expression and suggesting the involvement of a sigma 54 (NtrA/RpoN)-type transcription factor in nifA gene regulation. A 2-bp mutation at positions -25 and -16 was found to create a relatively nitrogen-control-independent, highly expressed nifA promoter. Using a heterologous ntrA(rpoN) gene probe, an A. caulinodans ntrA(rpoN)-like gene was cloned and the DNA sequence of this gene and flanking regions was determined. The presence of three open reading frames (ORF1-3) was demonstrated. ORF2 was found to contain regions sharing a high degree of homology with all characterized bacterial ntrA(rpoN) genes. ORF1 was found to share homology with ORFs found upstream of other bacterial ntrA(rpoN) genes, which have been postulated to encode members of a superfamily of ATP-binding proteins. Transposon Tn5 insertion mutations were introduced into the cloned ntrA(rpoN) gene, and chromosomal ntrA(rpoN)::Tn5 A. caulinodans mutants were created. The resulting mutants were found to be unable to fix nitrogen in the free-living state (Nif- in culture) or in stem or root nodules induced on Sesbania rostrata (Fix- in planta), and to be unable to grow aerobically in the presence of nitrate as sole nitrogen source (Ntr-). A nifH-lacZ gene fusion was found to be silent in ntrA(rpoN)::Tn5 mutant strains, but a nifA-lacZ gene fusion was found to be expressed at a wild-type level, suggesting that the ntrA(rpoN) gene identified here controls the expression of some of the A. caulinodans nif genes, but not the central nif regulatory gene nifA. Based on these results, a new model for the regulation of nif/fix gene expression in A. caulinodans is proposed.