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We report the identification and cloning of an ntrA-like (glnF rpoN) gene of Rhizobium meliloti and show that the R. meliloi ntrA product (NtrA) is required for C4-dicarboxylate transport as well as for nitrate assimilation and symbiotic nitrogen fixation. DNA sequence analysis showed that R. meliloti NtrA is 38% homologous with KkebsieUla pneumoniae NtrA. Subcloning and complementation analysis suggested that the R. melUoti ntrA promoter lies within 125 base pairs of the initiation codon and may be constitutively expressed.

Transfer and integration of a defined region (T-DNA) of the tumor-inducing (Ti) plasmid of Agrobacteriwm tulnefaciens is essential for tumor formation. We used a physical assay to study structural changes induced in Agrobacteriun T-DNA by cocultivation with plant cells. We show that nicks are introduced at unique; identical locations in each of the 24-base-pair imperfect direct repeats which flank the T-DNA and present evidence that a linear, single-stranded molecule is generated. We propose that these changes result from processing of the T-DNA for transfer and that they occur by a mechanism similar to DNA processing during conjugative DNA transfer between bacteria.Agrobacterium tumefaciens causes crown gall tumors on a wide variety of dicotyledonous plants. Many of the genes responsible for tumorigenesis are located on a large, tumorinducing (Ti) plasmid (for reviews, see references 8, 30, and 41). All Ti plasmids contain a region of DNA, the T-DNA, which is transferred from Agrobacterium cells to the plant cell. There it is integrated colinearly into the plant genome (reviewed in reference 5).Transfer of the T-DNA depends on cis-acting border elements consisting of 24-base-pair (bp) imperfect direct repeats which bound and functionally define the T-DNA (3, 44). T-DNAs located on the Ti plasmid require a rightward 24-bp repeat in its native orientation and relative position (20,32,42), while orientation and position requirements are less stringent for T-DNAs on separate replicons (binary vectors [16,18,19]). In addition, trans-acting functions are provided by a 35-kilobase region outside the T-DNA, the vir region (13,17,21,37). No transfer functions are encoded by the T-DNA itself (for example, see references 10, 22, and 47). Most analyses of the transfer process have depended on expression of either the oncogenic functions of the T-DNA or drug resistance markers which are expressed in plant cells and therefore have demanded both transfer and integration. More recent experiments attempting to separate transfer from integration by using viroid genomes cloned on small binary vectors (12) have confirmed the roles of the 24-bp repeat and vir loci in the transfer step. This paper focuses on the mechanism of transfer of the T-DNA to the plant genome, specifically, the "processing" which prepares T-DNA for transfer across the bacterial membrane. Several groups (1,25,26) intermediates which is similar to models for processing of DNA during conjugative DNA transfer between bacteria. MATERIALS AND METHODSBacterial strains and plant ceil culture. A348(pVK225) is A.tumefaciens A136 containing pTiA6NC (13), is merodiploid for virG, virC, virD, and virE, and is kanamycin resistant (23, 37). Bacteria were maintained on AB minimal agar (9) with kanamycin at 100 ,ug/ml when needed. A 1:1 mixture of L broth (28) and mannitol-glutamate broth (4) was used for growth in liquid medium, with kanamycin at 10 ,ug/ml as needed.Nicotiana tabacum suspension culture cell lines were maintained in supplemented liquid Murashige and Skoog (2...

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Complementation of Rhizobium leguminosarum dct mutants with a cosmid bank yielded Rhizobium meliloti homologs of the dctA, dctB, and dctD genes. The genes dctB and dctD are thought to form a two-component system which responds to the presence of C4-dicarboxylates to regulate expression of a transport protein encoded by dctA. DNA sequence analysis showed that det coding and intergenic regions, including putative binding sites for the dctD protein and r54-RNA polymerase, were highly conserved between these two Rhizobium species. Mutation of R. meliloti dctD showed that it was not essential for symbiotic nitrogen fixation but was needed for growth on dctA promoter depends on an analog of the enteric alternative sigma factor r54 encoded by rpoN (also called ntrA and ginF) (60). Indeed, a DNA sequence very similar to the ur5 consensus promoter (28) was observed in the region between the R. leguminosarum dctA and dctB genes (56). Screening a transposon TnS insertion library for cells that were unable to express a dctA-IacZ fusion gene in the presence of succinate allowed the identification and subsequent characterization of rhizobial rpoN (60). However, this screen entailed a complex heterologous system in which R. leguminosarum dct genes were present in R. meliloti cells. The nature and organization of R. meliloti dct genes remain unknown.We have undertaken the isolation and characterization of R. meliloti dct genes, hoping that conservation between two closely related species would allow us to focus on key regulatory elements. Moreover, in R. meliloti the rhizobial rpoN gene and other two-component regulation systems (ntrB/ntrC [66] and fixLlfixJ [14]) have been characterized; thus, this work provides the basis for future comparative studies of the predicted signal-transducing domains and potential cross-talk interactions between the systems. We found that R. meliloti contains identically arranged homologs of the R. leguminosarum genes dctA, dctB, and dctD. Blocks of conserved DNA sequence that are separated from each other by nonconserved residues in the dctA-dctB intergenic region suggest elements that are probably involved in the regulation of transcription of these genes, among which are two DCTD-binding sites that precede a C54 consensus promoter for dctA (H. Ledebur, B. Gu, and B. T. Nixon, manuscript in preparation). We also found that in R. meliloti, dctD is required for basal and induced expression of dctA and that R. meliloti may contain dozens of genes that fall into the subclass of regulators that need rpoN to stimulate transcription.