BacA Is Essential for Bacteroid Development in Nodules of Galegoid,but not Phaseoloid,Legumes

BacA is an integral membrane protein, the mutation of which leads to increased resistance to the antimicrobial peptides bleomycin and Bac7_1-35 and a greater sensitivity to SDS and vancomycin in Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. phaseoli, and Rhizobium etli. The growth of Rhizobium strains on dicarboxylates as a sole carbon source was impaired in bacA mutants but was overcome by elevating the calcium level. While bacA mutants elicited indeterminate nodule formation on peas, which belong to the galegoid tribe of legumes, bacteria lysed after release from infection threads and mature bacteroids were not formed. Microarray analysis revealed almost no change in a bacA mutant of R. leguminosarum bv. viciae in free-living culture. In contrast, 45 genes were more-than 3-fold upregulated in a bacA mutant isolated from pea nodules. Almost half of these genes code for cell membrane components, suggesting that BacA is crucial to alterations that occur in the cell envelope during bacteroid development. In stark contrast, bacA mutants of R. leguminosarum bv. phaseoli and R. etli elicited the formation of normal determinate nodules on their bean host, which belongs to the phaseoloid tribe of legumes. Bacteroids from these nodules were indistinguishable from the wild type in morphology and nitrogen fixation. Thus, while bacA mutants of bacteria that infect galegoid or phaseoloid legumes have similar phenotypes in free-living culture, BacA is essential only for bacteroid development in indeterminate galegoid nodules.Bacteria of the family Rhizobiaceae are alphaproteobacteria, which form a species-specific symbiotic relationship with leguminous plants. Plants release flavonoids that typically induce the synthesis of lipochitooligosaccharides by rhizobia, which in turn initiate a signaling cascade in the plant, leading to nodule formation (34). Rhizobia become trapped by curling root hairs, which they enter via infection threads that grow and ramify into the root cortex, where newly induced meristematic cells form the nodule (34). Bacteria are released from infection threads and engulfed by a plant-derived symbiosome membrane. In galegoid legumes (a clade in the subfamily Papilionoideae, such as Medicago, Pisum, or Vicia), which form indeterminate nodules that have a persistent meristem, bacteria undergo the endoreduplication of their chromosome, resulting in dramatic increases in size, shape, and DNA content to become terminally differentiated bacteroids (32). However, in phaseoloid legumes (e.g., lotus, bean, and soybean), which form determinate nodules with a transient meristem, bacteria do not undergo endoreduplication and therefore do not enlarge substantially. These bacteroids retain a normal DNA content and can regrow after isolation from nodules (32). The endoreduplication of bacteroids is controlled by the plant, and it is believed that nodule-specific cysteine-rich (NCR) peptides, which are made in indeterminate, but not in determinate, nodules, may be responsible for inducing and maintaining bacteroid development (31, 32). Finally, mature bacteroids receive dicarboxylic acids from the plant, which they use as a carbon, reductant, and energy source for the reduction of N₂ to ammonia (38). The ammonia is secreted to the plant, where it is assimilated into amino acids or ureides, depending on the legume, for export to the shoot.Sinorhizobium meliloti BacA protein was the first bacterial factor identified to be essential for bacteroid development (15). More recently, it also has been shown to be essential for the Mesorhizobium-Astragalus symbiosis (42). S. meliloti elicits the formation of indeterminate nodules on alfalfa, and while S. meliloti bacA null mutants induce nodule formation, bacteria lyse soon after endocytosis but prior to bacteroid differentiation (15, 20). BacA is a cytoplasmic membrane protein that shares 64% identity with SbmA from Escherichia coli (15, 25). SbmA/BacA proteins belong to the ATP binding cassette (ABC) superfamily and share sequence similarity with a family of eukaryotic peroxisomal membrane proteins, including the human adrenoleukodystrophy protein, which is required for the efficient transport of very-long-chain fatty acids (VLCFAs) out of the cytoplasm (9). Consistent with this, S. meliloti BacA is required for the complete modification of lipid A with VLCFAs (9). However, since S. meliloti mutants, which are directly involved in the biosynthesis of VLCFA-modified lipid A, show bacteroid abnormalities but still can form a successful alfalfa symbiosis, the effect of BacA on lipid A VLCFA modification does not fully account for its essential role in bacteroid development (10, 11, 16). Strains mutated in bacA also have an increased resistance to the glycopeptide bleomycin, a low-level resistance to aminoglycoside antibiotics, and an increased sensitivity to ethanol, sodium dodecyl sulfate (SDS), and deoxycholate relative to the sensitivities of the parent strain (12, 18, 25). More recently it has been shown that an S. meliloti bacA null mutant has an increased resistance to a truncated form of a eukaryotic proline-rich peptide, Bac7_1-16, and was unable to accumulate a fluorescently labeled form of this peptide (28). This finding, combined with the increased resistance of an S. meliloti bacA null mutant to bleomycin, led to the hypothesis that BacA is itself a putative peptide transporter (BacA mediated) or able to alter the activity of such a transporter (BacA influenced) (11, 15, 18, 28).As the increased resistance of the S. meliloti bacA null mutant to bleomycin and Bac7_1-16 appears to be independent of the VLCFA modification of lipid A (11, 28), this suggested that either BacA-mediated or BacA-influenced peptide uptake into S. meliloti plays a role in bacteroid development. Since indeterminate galegoid nodules contain hundreds of NCR peptides, whereas determinate phaseoloid nodules lack these host peptides (31), we considered it important to assess the role of BacA in bacteroid development during the formation of both nodule types.Here, we show that bacA mutants of Rhizobium leguminosarum bv. viciae strains 3841 and A34 failed to develop bacteroids and did not fix nitrogen in indeterminate pea (Pisum sativum) nodules. However, bacA mutants of both R. leguminosarum bv. phaseoli 4292 and Rhizobium etli CE3 formed normal bacteroids and fixed nitrogen at wild-type rates in determinate bean (Phaseolus vulgaris) nodules. This is consistent with BacA being a key component of bacteroid development in indeterminate galegoid nodules that is not required for functional bacteroid formation in determinate phaseoloid nodules.