Genetic analysis and regulation of the Rhizobium meliloti genes controlling C4-dicarboxylic acid transport

The genes controlling the transport of C4-dicarboxylic acids from Rhizobium meliloti have been cloned and analysed. The nucleotide sequence of the control region of the structural dctA and the regulatory dctBD genes has been determined. Comparison with the Rhizobium leguminosarum dct genes revealed a high degree of homology. Gene fusions to the enteric lacZY reporter gene were constructed and the expression of the dctA and dctBD genes studied under various physiological conditions. In free-living cells, the regulatory dctBD genes are absolutely required for the expression of the dctA gene. In the root nodule environment, a dctA::lacZY gene fusion was found to be expressed in an R. meliloti strain mutated in both the dctB and dctD genes, but not in a strain mutated in the dctB gene alone. The presence of the conserved upstream NifA-binding sites on the dctA promoter sequence, coupled with the fact that the dctA::lacZY gene fusion is not expressed in root nodules formed by a nifA mutant strain of R. meliloti, supports the suggestion that NifA may be involved in the symbiotic expression of dctA in the absence of the regulatory dctBD genes. Under micro-aerobic conditions, however, NifA induction alone is not sufficient for expression of the dctA promoter, even though the NifA-dependent nifHDK promoter is highly expressed under these conditions.     

Identification and sequence analysis of the Rhizobium meliloti dctA gene encoding the C4-dicarboxylate carrier

Transposon Tn5-induced C4-dicarboxylate transport mutants of Rhizobium meliloti 2011 which could be complemented by cosmid pRmSC121 were subdivided into two classes. Class I mutants (RMS37 and RMS938) were defective in symbiotic C4-dicarboxylate transport and in nitrogen fixation. They were mutated in the structural gene dctA, which codes for the C4-dicarboxylate carrier. Class II mutants (RMS11, RMS16, RMS17, RMS24, and RMS31) expressed reduced activity in symbiotic C4-dicarboxylate transport and in nitrogen fixation. These mutants were mutated in regulatory dct genes which do not play an essential role in the symbiotic state. Thin sections of alfalfa nodules induced by the wild type and class I and class II mutants were analyzed by light microscopy. Class mutants induced typical Fix- nodules, showing a large senescent zone, whereas nodules induced by class II mutants only differed in an enhanced content of starch granules compared with wild-type nodules. Class I mutants could be complemented by a 2.1-kilobase SalI-HindIII subfragment of cosmid pRmSC121. DNA sequencing of this fragment resulted in the identification of an open reading frame, which was designated dctA because Tn5 insertion sites of the class I mutants mapped within this coding region. The dctA gene was preceded by a nif consensus promoter and an upstream NifA-binding element. Upstream of the dctA promoter, the 5' end of the R. meliloti dctB gene could be localized. The amino acid sequence of the N-terminal part of the R. meliloti DctB protein shared 49% homology with the corresponding part of the R. leguminosarum DctB protein. The DctA protein consisted of 441 or 453 amino acids due to two possible ATG start codons, with calculated molecular masses of 46.1 and 47.6 kilodaltons, respectively. The hydrophobicity plot suggests that DctA is a membrane protein with several membrane passages. The amino acid sequences of the R. meliloti and the R. leguminosarum DctA proteins were highly conserved (82%).     

Molecular cloning and genetic organization of C4-dicarboxylate transport genes from Rhizobium leguminosarum.

Cosmids containing C4-dicarboxylate transport (dct) genes were identified from a gene bank of Rhizobium leguminosarum DNA made in the broad-host-range vector pLAFR1 by their ability to complement R. trifolii dct mutants. The dct genes were further characterized by subcloning, restriction site mapping, and transposon Tn5 and Tn7 mutageneses. Three dct loci were identified within a 5.5-kilobase region of DNA, in the order dctA-dctB-dctC. The results suggested that dctA encoded a structural component necessary for C4-dicarboxylate transport, whereas dctB and dctC encoded positive regulatory elements, and that dctA was transcribed divergently from dctB and dctC. Expression of dctA and dctC was obtained from vector promoters in some pLAFR1- and pSUP106-based plasmids.     

Two C4-dicarboxylate transport systems in Rhizobium sp. NGR234: rhizobial dicarboxylate transport is essential for nitrogen fixation in tropical legume symbioses.

To investigate the role of dicarboxylate transport in nitrogen-fixing symbioses between Rhizobium and tropical legumes, we made a molecular genetic analysis of the bacterial transport system in Rhizobium sp. NGR234. This braod host range strain fixes nitrogen in association with evolutionarily divergent legumes. Two dicarboxylate transport systems were cloned from Rhizobium NGR234. One locus was chromosomally located, whereas the other was carried on the symbiotic plasmid (pSym) and contained a dctA carrier protein gene, which was analyzed in detail. Although the DNA and derived amino acid sequences of the structural gene were substantially homologous to that of R. meliloti, its promoter sequences was quite distinct, and the upstream sequence also exhibited no homology to dctB, which is found at this position in R. meliloti. A site-directed internal deletion mutant in dctA of NGR234 exhibited a (unique) exclusively symbiotic phenotype that could grow on dicarboxylates ex planta, but could not fix nitrogen in planta. This phenotype was found for tested host plants of NGR234 with either determinate- or indeterminate-type nodules, confirming for the first time that symbiosis-specific uptake of dicarboxylates is a prerequisite for nitrogen fixation in tropical legume symbioses.     

Rhizobium meliloti genes required for C4-dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid.

A mutant of Rhizobium meliloti unable to transport C4 dicarboxylates (dct) was isolated after Tn5 mutagenesis. The mutant, 4F6, could not grow on aspartate or the tricarboxylic acid cycle intermediates succinate, fumarate, or malate. It produced symbiotically ineffective nodules on Medicago sativa in which bacteroids appeared normal, but the symbiotic zone was reduced and the plant cells contained numerous starch granules at their peripheries. Cosmids containing the dct region were obtained by selecting those which restored the ability of 4F6 to grow on succinate. The Tn5 insertion in 4F6 was found to be within a 5.9-kilobase (kb) EcoRI fragment common to the complementing cosmids. Site-specific Tn5-mutagenesis revealed dct genes in a segment of DNA about 4 kb in size extending from within the 5.9-kb EcoRI fragment into an adjacent 2.9-kb EcoRI fragment. The 4F6 mutation was found to be in a complementation group in which mutations yielded a Fix- phenotype, whereas other dct mutations in the region resulted in mutants which produced effective nodules in most, although not all, plant tests (partially Fix-). The dct region was found to be located on a megaplasmid known to carry genes required for exopolysaccharide production.     

Overexpression of the dctA gene in Rhizobium meliloti: effect on transport of C4 dicarboxylates and symbiotic nitrogen fixation.

Symbiotic nitrogen fixation may be limited by the transport of C4 dicarboxylates into bacteroids in the nodule for use as a carbon and energy source. In an attempt to increase dicarboxylate transport, a plasmid was constructed in which the Rhizobium meliloti structural transport gene dctA was fused to a tryptophan operon promoter from Salmonella typhimurium, trpPO. This resulted in a functional dctA gene that was no longer under the control of the dctBD regulatory genes, but the recombinant plasmid was found to be unstable in R. meliloti. To stably integrate the trpPO-dctA fusion, it was recloned into pBR325 and recombined into the R. meliloti exo megaplasmid in the dctABD region. The resultant strain showed constitutive dctA-specific mRNA synthesis which was about 5-fold higher than that found in fully induced wild-type cells. Uptake assays showed that [14C]succinate transport by the trpPO-dctA fusion strain was constitutive, and the transport rate was the same as that of induced control cells. Acetylene reduction assays indicated a significantly higher rate of nitrogen fixation in plants inoculated with the trpPO-dctA fusion strain compared with the control. Despite this apparent increase, the plants had the same top dry weights as those inoculated with control cells.     

Sinorhizobium meliloti dctA mutants with partial ability to transport dicarboxylic acids

Sinorhizobium meliloti dctA encodes a transport protein needed for a successful nitrogen-fixing symbiosis between the bacteria and alfalfa. Using the toxicity of the DctA substrate fluoroorotic acid as a selective agent in an iterated selection procedure, four independent S. meliloti dctA mutants were isolated that retained some ability to transport dicarboxylates. Two mutations were located in a region called motif B located in a predicted transmembrane helix of the protein that has been shown in other members of the glutamate transporter family to be involved in cation binding. A G114D mutation was located in the third transmembrane helix, which had not previously been directly implicated in transport. Multiple sequence alignment of more than 60 members of the glutamate transporter family revealed a glycine at this position in nearly all members of the family. The fourth mutant was able to transport succinate at almost wild-type levels but was impaired in malate and fumarate transport. It contains two mutations: one in a periplasmic domain and the other predicted to be in the cytoplasm. Separation of the mutations showed that each contributed to the altered substrate preference. dctA deletion mutants that contain the mutant dctA alleles on a plasmid can proceed further in symbiotic development than null mutants of dctA, but none of the plasmids could support symbiotic nitrogen fixation, although they can transport dicarboxylates, some at relatively high levels.     

The molecular mechanisms of dicarboxylic acid transport in Escherichia coli K12. The role and orientation of the two membrane-bound dicarboxylate binding proteins.

Previous communications from this laboratory have indicated that dicarboxylic acid transport in Escherichia coli is an active process, and that at least three genes are responsible for this transport system. In attempts to identify the transport components, one periplasmic binding protein and two membrane integral proteins (SBP 1 and SBP 2) were implicated to participate in the transport system in vivo. In the present communication, we demonstrate, through biochemical analysis of the transport mutants, that the two membrane transport genes, dctA and dctB, are responsible for the two membrane-bound dicarboxylate binding proteins, SBP 2 and SBP 1, respectively. We also find that the substrate recognition sites of SBP 1 and SBP 2 are exposed to the inner and outer surfaces of the membrane, respectively. This may have important implications for the role of SBP 1 and SBP 2 in the translocation process.     

Purification, characterization and nucleotide sequence of the periplasmic C4-dicarboxylate-binding protein (DctP) from Rhodobacter capsulatus

A periplasmic binding protein essential for high-affinity transport of the C4-dicarboxylates malate, succinate and fumarate across the cytoplasmic membrane of the purple photosynthetic bacterium Rhodobacter capsulatus has been purified to homogeneity and some of its ligand-binding properties characterized. The protein was not produced in a Tn5 insertion mutant unable to transport C4-dicarboxylates under aerobic conditions in the dark. Wild-type DNA corresponding to the location of the transposon insertion site was subcloned and a 1.5 kb section sequenced. A complete open reading frame of 999 bp was identified that encoded a 333-residue protein (DctP) with a molecular weight of 36,128 with a 26-residue amino-terminal signal peptide. The identify of this protein with the purified dicarboxylate-binding protein and the position of the predicted signal peptide cleavage site was confirmed by N-terminal sequencing. No significant homology with other proteins was detected in database searches. A GC-rich region of dyad symmetry was located 7 bp downstream of the dctP translational stop codon. This structure may be of significance in regulating the relative abundance of DctP and other dct gene products which comprise the high-affinity dicarboxylate transport system in this bacterium.     

Structural and functional analysis of the fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512

The fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512 were identified by DNA hybridization of a genomic library with an internal fragment of the Rhizobium meliloti fixJ gene. The nucleotide sequence was determined and the corresponding amino acid sequence was aligned with the amino acid sequences of the FixL proteins of R. meliloti, Bradyrhizobium japonicum and Azorhizobium caulinodans. While the FixJ protein and the carboxy-terminal part of the FixL protein are highly homologous to the other FixL and FixJ proteins, the homology in the central heme-binding, oxygen-sensing domain and in the amino-terminal domain of FixL is very low. The R. leguminosarum bv. phaseoli FixL protein does not contain the heme-binding motif defined for the previously described FixL proteins. R. leguminosarum bv. phaseoli fixLJ and fixJ mutants were constructed. These mutants can still fix nitrogen, albeit at a reduced level. Expression analysis of nifA-gusA and nifH-gusA fusions in the constructed mutants revealed that the R. leguminosarum bv. phaseoli fixLJ genes are involved in microaerobic nifH expression but not in nifA expression.     

Detection and separation of Rhizobium and Bradyrhizobium Nod metabolites using thin-layer chromatography.

Using radioactive acetate as a precursor, it was shown that the common nodABC genes of Rhizobium and Bradyrhizobium strains are involved in the production of one or more metabolites that are excreted into the growth medium. A rapid thin-layer chromatography (TLC) system has been developed to separate these so-called Nod metabolites that can then be visualized by autoradiography. Different patterns of Nod metabolites were observed in the tested strains of the cross-inoculation groups of R. leguminosarum bv. viceae, R. l. bv. trifolii, R. meliloti, and B. japonicum. Only Nod metabolites of R. meliloti became labeled when radioactive sulphate was present in the medium. The role of the other nodulation genes of R. l. bv. viceae in the production of the detected Nod metabolites was tested in further detail. In addition to the common nodABC genes, the nodFE and nodL genes are involved in the production of Nod metabolites. In contrast, the chromosomal background did not influence the number of detected Nod metabolites or their mobilities on TLC plates. Nod metabolites could also be produced and excreted in Escherichia coli cells in which the appropriate nodulation genes were expressed.     

Plasmid pSym1-32 from Rhizobium leguminosarum bv. viceae, controlling nitrogen-fixing activity, effectiveness of symbiosis, competitiveness, and acid tolerance

The symbiotic plasmid (pSym1-32) of the highly effective Rhizobium leguminosarum bv. viceae 1-32 strain was identified after the conjugal transfer of replicons carrying Tn5-mob into the plasmidless Agrobacterium tumefaciens Gm1-9023 strain. Plasmid pSym1-32 was transferred into R. leguminosarum bv. viceae strains Y14 (showing low effectiveness of symbiosis with Vicia villosa) and Y57 (unable to fix nitrogen). Transconjugants formed Fix+ nodules on roots of V. villosa and had a highly enhanced nitrogen fixing ability, increased plant weight, and increased nitrogen accumulation compared to the recipient strains. Variation of transconjugants in symbiotic properties (accompanied by alterations in plasmid composition in some of the conjugants) was detected. Moreover, the donor strain R. leguminosarum bv. viceae 1-32 was shown to be more efficient in the competitiveness and acid tolerance than the recipient Y14 strain. Both these properties were transmitted upon transfer of pSym1-32 into the recipient. Thus, plasmid pSym1-32 was shown to carry genes involved in the control of the nitrogen fixing ability, symbiotic effectiveness, competitiveness, and acid tolerance in R. leguminosarum bv. viceae.     

Structural and functional analysis of the fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512

The fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512 were identified by DNA hybridization of a genomic library with an internal fragment of the Rhizobium meliloti fixJ gene. The nucleotide sequence was determined and the corresponding amino acid sequence was aligned with the amino acid sequences of the FixL proteins of R. meliloti, Bradyrhizobium japonicum and Azorhizobium caulinodans. While the FixJ protein and the carboxy-terminal part of the FixL protein are highly homologous to the other FixL and FixJ proteins, the homology in the central heme-binding, oxygen-sensing domain and in the amino-terminal domain of FixL is very low. The R. leguminosarum bv. phaseoli FixL protein does not contain the heme-binding motif defined for the previously described FixL proteins. R. leguminosarum bv. phaseoli fixLJ and fixJ mutants were constructed. These mutants can still fix nitrogen, albeit at a reduced level. Expression analysis of nifA-gusA and nifH-gusA fusions in the constructed mutants revealed that the R. leguminosarum bv. phaseoli fixLJ genes are involved in microaerobic nifH expression but not in nifA expression.The nucleotide sequence data reported will appear in the EMBL, Genbank and DDBJ Nucleotide Sequence Databases under the accession number U27314     

Symbiotic loci of Rhizobium meliloti identified by random TnphoA mutagenesis.

We have developed a system for using TnphoA (TnphoA is Tn5 IS50L::phoA), which generates fusions to alkaline phosphatase (C. Manoil and J. Beckwith, Proc. Natl. Acad. Sci. USA 82:8129-8133, 1985), in Rhizobium meliloti. Active fusions expressing alkaline phosphatase can arise only when this transposon inserts in genes encoding secreted or membrane-spanning proteins. By confining our screening to 1,250 TnphoA-generated mutants of R. meliloti that expressed alkaline phosphatase, we efficiently identified 25 symbiotically defective mutants, all of which formed ineffective (Fix-) nodules on alfalfa. Thirteen of the mutants were unable to synthesize an acidic exopolysaccharide (exo::TnphoA) that is required for nodule invasion. Twelve of the mutations created blocked at later stages of nodule development (fix::TnphoA) and were assigned to nine symbiotic loci. One of these appeared to be a previously undescribed locus located on the pRmeSU47a megaplasmid and to encode a membrane protein. Two others were located on the pRmeSU47b megaplasmid: one was a new locus which was induced by luteolin and encoded a membrane protein, and the other was dctA, the structural gene for dicarboxylic acid transport. The remaining six loci were located on the R. meliloti chromosome. One of these was inducible by luteolin and encoded a membrane protein which determined lipopolysaccharide structure. Three additional chromosomal loci also appeared to encode membrane proteins necessary for symbiosis. The remaining two chromosomal loci encoded periplasmic proteins required for symbiosis.