Sinorhizobium meliloti, a legume symbiont, and Brucella abortus, a phylogenetically related mammalian pathogen, both require the bacterial-encoded BacA protein to establish chronic intracellular infections in their respective hosts. We found that the bacterial BacA proteins share sequence similarity with a family of eukaryotic peroxisomal-membrane proteins, including the human adrenoleukodystrophy protein, required for the efficient transport of verylong-chain fatty acids out of the cytoplasm. This insight, along with the increased sensitivity of BacA-deficient mutants to detergents and cell envelope-disrupting agents, led us to discover that BacA affects the very-long-chain fatty acid (27-OHC28:0 and 29-OHC30:0) content of both Sinorhizobium and Brucella lipid A. We discuss models for how BacA function affects the lipid-A fatty-acid content and why this activity could be important for the establishment of chronic intracellular infections. Sinorhizobia and brucellae are Gram-negative ␣-proteobacteria that live intracellularly within their respective hosts. Sinorhizobia form a beneficial symbiosis with agriculturally important legumes that results in the conversion of N 2 to NH 3 (1). In contrast, brucellae are highly infectious pathogens that cause abortions and infertility in domestic and wild mammals and a severe and debilitating zoonotic disease in humans (2). Brucella melitensis, Brucella suis, and Brucella abortus are potential biological warfare agents, and they are a serious concern because there is presently no human vaccine (3). Despite the strikingly different outcomes that sinorhizobia and brucellae eventually have on their hosts, commonalties exist in the chronicinfection process because both are endocytosed into host cells, where they adapt and survive for extensive periods of time within acidic, membrane-bound compartments (1, 2, 4, 5). More importantly, the close phylogenetic relatedness of the sinorhizobia and the brucellae that was revealed initially by RNA homology studies has been confirmed recently by determination of the complete genome sequences of Sinorhizobium meliloti, B. melitensis, and B. suis (6-8).The BacA protein, initially found to be essential for S. meliloti to form a long-term infection within alfalfa-plant cells (9), was also shown subsequently to be essential for the establishment and maintenance of chronic spleen and liver infections by B. abortus in BALB͞c mice (10). BacA is predicted to span the inner membrane of S. meliloti and B. abortus seven times, and it is homologous to the SbmA protein of Escherichia coli, a putative transporter of peptide antibiotics (9, 11). Although an S. meliloti bacA null mutant displays altered sensitivity to peptide antibiotics (11), the increased sensitivity of this mutant to detergents and cell envelope-disrupting agents supports an alternative model wherein the function of BacA affects the integrity of the bacterial cell envelope (12). Recently, an S. meliloti lpsB mutant, altered dramatically in its lipopolysaccharide (LPS) carbohydrate co...

SummarySinorhizobium meliloti , a legume symbiont and Brucella abortus , a phylogenetically related mammalian pathogen, both require their BacA proteins to establish chronic intracellular infections in their respective hosts. The lipid A molecules of S. meliloti and B. abortus are unusually modified with a very-longchain fatty acid (VLCFA; C ≥ ≥ ≥ ≥ 28) and we discovered that BacA is involved in this unusual modification. This observation raised the possibility that the unusual lipid A modification could be crucial for the chronic infection of both S. meliloti and B. abortus . We investigated this by constructing and characterizing S. meliloti mutants in the lpxXL and acpXL genes, which encode an acyl transferase and acyl carrier protein directly involved in the biosynthesis of VLCFA-modified lipid A. Our analysis revealed that the unusually modified lipid A is important, but not crucial, for S. meliloti chronic infection and that BacA must have an additional function, which in combination with its observed effect on the lipid A in the freeliving form of S. meliloti , is essential for the chronic infection. Additionally, we discovered that in the absence of VLCFAs, S. meliloti produces novel pentaacylated lipid A species, modified with unhydroxylated fatty acids, which are important for stress resistance.

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