In both the legume symbiont Sinorhizobium meliloti and the mammalian pathogen Brucella abortus, the inner membrane BacA protein is essential for host persistence. In free-living S. meliloti and B. abortus loss of the BacA protein also results in an increased resistance to the glycopeptide bleomycin and a ~ 50% decrease in the lipopolysaccharide (LPS) very-long-chain-fatty-acid (VLCFA) content. Consequently, it was proposed that BacA may be involved in transport of peptides into the cell and/or that BacA may be involved in the VLCFA modification of the LPS. During this work it was determined that the increased resistance observed in an S. meliloti DbacA mutant to bleomycin and to the truncated eukaryotic peptide Bac7(1-16), is independent of the VLCFA modification. These data support a model for BacA having multiple non-overlapping functions. Using flow cytometry studies with fluorescently labelled forms of bleomycin and Bac7(1-16) it was found that the BacA protein plays a role in the uptake of bleomycin. However, BacA was shown to be essential for the uptake of Bac7(1-16). Additionally, it was determined that two symbiotically defective bacA site directed mutants with known reductions in their VLCFA could still take up Bac7, suggesting that the BacA function that leads to the VLCFA modification could also play a key role in host persistence. To investigate further the role of BacA in the VLCFA modification and where in the cell envelope the lipid A is modified with the VLCFA, the role of the putative lipid trafficking protein MsbA2 was investigated. Interestingly, it was discovered that S. meliloti lacking the MsbA2 protein, is unable to enter host cells and induces a plant defence response more characteristic of a pathogen. To investigate the importance of the VLCFA modification during the symbiosis S. meliloti mutants lacking either the AcpXL (VLCFA acyl carrier protein) or LpxXL (VLCFA acyl transferase protein) were characterized in the host. Although not essential for host persistence, loss of each of the proteins did result in distinct defects, suggesting the VLCFA modification is important during the symbiosis. Since there are hundreds of nodule specific cysteine-rich peptides produced by the host plant Medicago truncatula, the BacA mediated uptake of one of these peptides combined with the VLCFA modification may account for the essential role of the BacA protein in the legume symbiosis.

Abstract Very long chain fatty acids (VLCFA) are essential molecules that take part in many different cellular processes such as membrane pore stabilization, membrane trafficking and signaling pathways. The fatty acid elongation pathway in yeast has been studied for about a decade. As part of our work on cellular VLCFA elongation, we identified and characterized the condensing enzyme as well as ketoacyl reductases of the elongation pathway in cotton. In order to identify the yeast 3-hydroxyacyl-CoA dehydratase, we introduced a redundancy in this function by engineering a chimera consisting of the two first predicted transmembrane domains of Elo3p and the hydratase2 domain of Candida tropicalis Mfe2p. Yeast harboring the chimeric construct were subjected to random mutagenesis, and screened for mutants whose survival was dependent on the chimera. The mutants isolated contained RFT1 mutations and exhibited a defect in protein glycosylation, but no VLCFA deficiencies. The N-linked glycosylation pathway is well conserved in eukaryotes. Glycan synthesis occurs on the ER membrane; first on the cytoplasmic side up to Dol-PP-GlcNAc2Man5, which is then translocated to the ER luminal side in an Rft1p-dependent flipping process. The core glycan is further extended to Dol-PP-GlcNAc2Glc3Man9, and then transferred to an asparagine side chain of the nascent polypeptide to be glycosylated. It was found that the Elo3'-hydratase2 chimera acts as a multicopy suppressor of the Rft1p deficiency. The subsequent studies elucidated new aspects of Rft1p function, as well as a hitherto under-appreciated role of the ER associated protein degradation process in the maintenance of ER integral membrane complexes and the physical integrity of the membrane. The functionality of the human Rft1p homologue was demonstrated using a yeast complementation assay. A mutant variant from a patient was analyzed, aiding in the identification and characterization of the first reported case of a glycosylation deficiency in humans caused by a defective RFT1 allele.

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