The Sinorhizobium meliloti LpxXL and AcpXL Proteins Play Important Roles in Bacteroid Development within Alfalfa

Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of S. meliloti bacteroid lipid A with VLCFAs.Sinorhizobium meliloti and Brucella abortus form chronic intracellular infections within legumes and mammalian hosts, respectively (3, 20), and their BacA proteins play essential roles in these processes (8, 12). The precise function(s) of the BacA proteins has not been resolved, but free-living S. meliloti and B. abortus mutants lacking BacA have increased resistance to the glycopeptide bleomycin (9, 12) and there are ∼50% decreases in their lipid A very-long-chain fatty acid (VLCFA) contents (4, 7). It has also been determined that the increased resistance of an S. meliloti bacA null mutant to bleomycin and a truncated eukaryotic peptide, Bac7_1-16, is independent of its lipid A VLCFA alteration (6, 15). Together, these findings support a model in which BacA could have multiple nonoverlapping functions which lead to lipid A VLCFA modification and peptide uptake. The fact that two symbiotically defective S. meliloti BacA site-directed mutants (Q193G and R389G) (13) show defects in BacA-mediated lipid A VLCFA modification (4) but are still capable of peptide uptake (15) suggests that the S. meliloti lipid A VLCFA modification could play a key role in the symbiosis of this organism with alfalfa.Since the mechanism by which BacA leads to the lipid A VLCFA modification has not been resolved (4), S. meliloti mutants were constructed with mutations in the lpxXL and acpXL genes, which encode a lipid A VLCFA acyl transferase and a VLCFA acyl carrier protein directly involved in the biosynthesis of VLCFA-modified lipid A (5, 23). The S. meliloti lpxXL and acpXL mutants completely lack the lipid A VLCFA modification in their free-living states, but, unlike the S. meliloti bacA null mutant, these mutants can still form a successful symbiosis with alfalfa (5, 8, 23). However, the fact that the S. meliloti acpXL and lpxXL mutants are substantially less competitive in the alfalfa symbiosis than the parent strain (5, 23) indicates that the AcpXL and LpxXL proteins play important roles in at least one of the stages of the alfalfa symbiosis. Although the free-living S. meliloti acpXL and lpxXL mutants completely lack the lipid A VLCFA, they produce different species of lipid A (5). For example, in the absence of AcpXL, S. meliloti is able to modify lipid A with either C16:0 or C18:0 in the position normally modified with the VLCFA in the parent strain lipid A. This process is LpxXL dependent, as it does not occur in either an S. meliloti lpxXL single mutant or an S. meliloti acpXL lpxXL double mutant. In addition, since a Rhizobium leguminosarum acpXL mutant completely lacks the lipid A VLCFA modification in its free-living state but its lipid A is partially modified with the VLCFA to ∼58% of the amount in the parent strain lipid A during passage through peas (25), it is also possible that the S. meliloti acpXL mutant and possibly the S. meliloti lpxXL mutant undergo further lipid A changes during the interaction with alfalfa.In this study, we found that LpxXL and AcpXL play important but distinct roles in S. meliloti bacteroid development during alfalfa symbiosis. Additionally, we demonstrated that there is a minor host-induced AcpXL-independent mechanism by which S. meliloti bacteroid lipopolysaccharide (LPS) can be modified with the VLCFA. In contrast, we found that the LpxXL protein plays an essential role in the modification of S. meliloti bacteroids with VLCFAs.