Brucella abortus, a mammalian pathogen, and Rhizobium meliloti, a phylogenetically related plant symbiont, establish chronic infections in their respective hosts. Here a highly conserved B. abortus homolog of the R. meliloti bacA gene, which encodes a putative cytoplasmic membrane transport protein required for symbiosis, was identified. An isogenic B. abortus bacA mutant exhibited decreased survival in macrophages and greatly accelerated clearance from experimentally infected mice compared to the virulent parental strain. Thus, the bacA gene product is critical for the maintenance of two very diverse host-bacterial relationships.

BacA is an inner membrane protein associated with maintenance of chronic infections in several diverse host-pathogen interactions. To understand the function of the bacA gene in Mycobacterium tuberculosis (Rv1819c), we insertionally inactivated this gene and analyzed the resulting mutant for a variety of phenotypes. BacA deficiency in M. tuberculosis did not affect sensitivity to detergents, acidic pH, and zinc, indicating that there was no global compromise in membrane integrity, and a comprehensive evaluation of the major lipid constituents of the cell envelope failed to reveal any significant differences. Infection of mice with this mutant revealed no impact on establishment of infection but a profound effect on maintenance of extended chronic infection and ultimate outcome. As in alphaproteobacteria, deletion of BacA in M. tuberculosis led to increased bleomycin resistance, and heterologous expression of the M. tuberculosis BacA homolog in Escherichia coli conferred sensitivity to antimicrobial peptides. These results suggest a striking conservation of function for BacA-related proteins in transport of a critical molecule that determines the outcome of the host-pathogen interaction.

Sinorhizobium meliloti strains lacking BacA function are impaired in symbiosis with alfalfa host plants and display altered sensitivities to a number of compounds relative to wild-type strains. With the goal of finding clues to the currently unknown biological function(s) of BacA, we carried out a genetic analysis to determine which amino acids are critical for protein function and to attempt to ascertain whether the multiple phenotypes that result from a bacA-null allele were the result of a common cause or whether BacA has multiple functions. We have created a set of 20 site-directed mutants in which selected individual amino acids in bacA were replaced with glycine residues. The resulting mutants were characterized to determine how the various amino acid changes affected a number of phenotypes associated with loss of BacA function. Mutants H165G, W182G, D198G, and R284G had null phenotypes for all functions assayed, while mutants W57G, S83G, S231G, and K350G were indistinguishable from wild-type strains. The remaining 12 site-directed mutants demonstrate mixed phenotypic characteristics and fall into a number of distinctly different groups. These observations may be consistent with a role for BacA in multiple, nonoverlapping functions.The bacterial protein BacA is a putative transporter of unknown function that is unusually interesting because it is absolutely required for both the virulence of an animal pathogen (18) and the symbiotic capacity of an agriculturally beneficial plant endosymbiont (9). As part of our efforts to elucidate the biological role of this intriguing protein, we have initiated a genetic dissection of BacA in the gram-negative bacterial endosymbiont of alfalfa, Sinorhizobium meliloti, because of its genetic tractability and the ease with which one can monitor its ability to establish a persistent infection with its eukaryotic host.Nitrogen fixation is the end result of a complex symbiotic relationship between rhizobia and leguminous plants (23,26), in which the bacterial partner is harbored within plant root nodules and exchanges reduced atmospheric nitrogen, necessary for host plant growth, for photosynthetically derived carbon compounds. bacA, a key gene involved in nodule development in the S. meliloti-alfalfa symbiosis, was isolated in a screen that identified bacterial mutants with symbiotic deficiencies (21). Rhizobia invade the nodules they elicit on legumes via specialized plant-derived structures called "infection threads." Upon release from infection threads into plant membrane-bound compartments, wild-type rhizobia begin differentiating into nitrogen-fixing bacteroids. Electron microscopy has shown that S. meliloti mutants that lack bacA function invade nodules and are released properly from infection threads, but then appear to lyse and die at this intermediate developmental time point before they can differentiate and establish a functional symbiosis (9).bacA mutants from Brucella abortus, an animal pathogen and close phylogenetic relative of S. meliloti (5), are similarly u...

To identify possible iron sources for bacteroids in planta, soybean (Glycine max L. Merr.) symbiosomes (consisting of the bacteroidcontaining peribacteroid space enclosed by the peribacteroid membrane [PBMI) and bacteroids were assayed for the ability to transport iron supplied as various ferric [Fe(lll)l-chelates. lron presented as a number of Fe(lll)-chelates was transported at much higher rates across the PBM than across the bacteroid membranes, suggesting the presence of an iron storage pool in the peribacteroid space. Pulse-chase experiments confirmed the presence of such an iron storage pool. Because the PBM is derived from the plant plasma membrane, we reasoned that it may possess a ferric-chelate reductase activity similar to that present in plant plasma membrane. We detected ferric-chelate reductase activity associated with the PBM and suggest that reduction of Fe(lll) to ferrous [Fe(ll)l plays a role in the movement of iron into soybean symbiosomes.Rhizobia are totally dependent on their plant hosts for nutrients when living within the nodule. Bacteroids are surrounded by a PBM of plant origin (reviewed by Mellor, 1989), which may influence the transfer of a11 substances that pass between the plant and the bacteroids. The PBM possesses some of the properties of the plant PM from which it is derived but also has a set of PBM-specific proteins, some of which are presumed to be involved in the nitrogen fixation process (reviewed by Verma, 1992). Because the PBM is the interface between the plant host and the bacteroid, its role in controlling the flux of metabolites between the two compartments is likely to be important in the regulation of nitrogen fixation.Studies of the PBM have shown that this membrane has selective permeability to metabolites. Symbiosomes (consisting of the bacteroid-containing PBS enclosed by the PBM; Roth et al., 1988) prepared from soybean and French bean have dicarboxylate-transporting ability (Udvardi et al., 1988; Herrada et al., 1989), and dicarboxylates are thought to be the major carbon and energy source available for bacteroids. The form(s) of nitrogen transported to bacteroids is not currently known (Whitehead et al., 1995); however, the rescue of various amino acid auxotrophs in

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