We cloned and sequenced the waaL (rfaL) gene from Bradyrhizobium japonicum, which infects soybean and forms nitrogen-fixing nodules on soybean roots. waaL has been extensively studied in the lipopolysaccharide (LPS) biosynthesis of enteric bacteria, but little is known about its function in (brady)rhizobial LPS architecture. To characterize its role as O-antigen ligase in the LPS biosynthesis pathway, we constructed a waaL knock-out mutant and its complemented strain named JS015 and CS015, respectively. LPS analysis showed that an LPS structure of JS015 is deficient in O-antigen as compared to that of the wild type and complemented strain CS015, suggesting that WaaL ligates the O-antigen to lipid A-core oligosaccharide to form a complete LPS. JS015 also revealed increased cell surface hydrophobicity, but it showed decreased motility in soft agar plates. In addition to the alteration in cell surface properties, disruption of the waaL gene caused increased sensitivity of JS015 to hydrogen peroxide, osmotic pressure, and novobiocin. Specifically, plant tests revealed that JS015 failed to nodulate the host plant soybean, indicating that the rhizobial waaL gene is responsible for the establishment of a symbiotic relationship between soybean and B. japonicum.

Significance and Impact of the Study: This study demonstrates the role of the B. japonicum lpcC in nodulation with soybean and importance of cell surface hydrophobicity. The results also highlight that intact LPS is required for successful symbiosis between B. japonicum and soybeans. Our findings not only support previous studies emphasizing the necessity of LPS on the interaction between the two symbiotic partners, but also contribute to a better understanding of the symbiotic mechanisms. AbstractWe investigated the role of the Bradyrhizobium japonicum lpcC gene, encoding a mannosyl transferase, involved in the lipopolysaccharide (LPS) biosynthesis. The inactivation of the lpcC gene considerably altered the LPS structure and the cell surface properties. LPS analysis showed that the lpcC mutant JS715 had an abnormal LPS structure deficient in O-antigen. The cell surface hydrophobicity increased approximately threefold in JS715 compared to the wild type. The increased cell surface hydrophobicity is likely to be related with cell aggregation in the mutant culture. For the growth comparison, JS715 showed slower growth rate than the wild type. The motility of JS715 decreased in soft agar plates, but it showed enhanced biofilm-forming ability. Interestingly, JS715 was not able to nodulate the host legume soybean (Glycine max). This study shows not only that lpcC is involved in the biosynthesis of O-antigen in the B. japonicum LPS, but also that inactivation of the lpcC gene affects symbiotic capability of B. japonicum and surface-related properties such as cell hydrophobicity, biofilm formation and motility.

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