Sinorhizobium meliloti dctA Mutants with Partial Ability To Transport Dicarboxylic Acids

Abstract

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.In order for rhizobia to be able to establish an effective nitrogen-fixing (Fix ϩ ) symbiosis with a legume plant, the plant must provide the bacteria with energy, which is ultimately derived from plant photosynthesis. Dicarboxylic acids, such as malate, fumarate, and succinate, are thought to be a major carbon and energy source for the nitrogen-fixing bacteria (25). Importing these compounds requires the activity of the bacterial dicarboxylate transport protein, DctA. DctA belongs to the glutamate transporter family which, in addition to the prokaryotic dicarboxylate transporters, includes glutamate, aspartate, and neutral amino acid transporters found in bacteria and eukaryotes. Sinorhizobium meliloti DctA is typical of the bacterial C 4 -dicarboxylate transporters. Although it was originally identified by its ability to transport dicarboxylic acids, recent studies revealed that DctA exhibits a much broader substrate specificity (27). In addition to succinate, malate, fumarate, oxaloacetate, and aspartate, DctA can transport D-malate and compounds that are not dicarboxylates, such as orotate and succinamic acid. It also does not appear to transport some dicarboxylates, such as maleate, the cis-isomer of fumarate.Glutamate and aspartate transporters are found in neurons and are involved in the postsynaptic reuptake of glutamate, an excitatory neurotransmitter. Defects in these transporters have been implicated in several neurological disorders and, as a result, they have attracted much attention. Several studies predicted six hydrophobic segments in the...

Pleiotropic effects of mutations that alter the Sinorhizobium meliloti cytochrome c respiratory system

“…The correctness of the cloned fragment was confirmed by sequencing. The constructed deletion was transferred to the chromosome by recombination, as described by Yurgel & Kahn (2005). The S. meliloti strain missing SMc00010 was named Rm1021DSMc00010.…”

A mutant GlnD nitrogen sensor protein leads to a nitrogen-fixing but ineffective Sinorhizobium meliloti symbiosis with alfalfa

“…Transposon insertions were located using an arbitrary PCR (14) to determine DNA sequences adjacent to the Tn5. Deletion mutants were constructed using an insertion/excision strategy (47). Deletion of the entire glnB (SMc00947) and glnD (SMc01124) gene, as well as a 210-bp deletion of glnD gene (Fig.…”

Nutrient Sharing between Symbionts

“…The system is made up of three genes: dctA encoding a transport protein, and dctB and dctD, which are divergently transcribed and encode a twocomponent sensor regulator system that activates transcription of dctA in response to C 4 dicarboxylates (Watson, 1990;Reid and Poole, 1998;Yurgel et al, 2000;Yurgel and Kahn, 2005). DctA of Rhizobium strains is typical of most bacterial DctA carriers in being a cation (H 1 ) symporter of around 440 amino acids (Janausch et al, 2002).…”