Abstract
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.
Highlights
The soil bacterium Bradyrhizobium japonicum symbiotically infects its host plant soybean
We identified a 5.5-kb gene region involved in LPS synthesis of B. japonicum
Wzy_C superfamily domain whose sequence is located between 200th and 354th amino acid positions. This domain is known to be involved in the synthesis of O-antigen and representing O-antigen ligases such as enteric WaaL; none of the top 100 hits is from enteric bacteria including E. coli and Salmonella
Summary
The soil bacterium Bradyrhizobium japonicum symbiotically infects its host plant soybean Lipopolysaccharide (LPS) is a major component found in rhizobial cell envelope and has been known to be involved in establishing a symbiotic relationship with leguminous plants. 5.5 kb LPS-gene region previously identified in B. japonicum 61A101C showed that mutation of either rfaD, rfaF, lpcC, or galE encoding heptose epimerase, heptosyl transferase, mannosyl transferase, or glucose epimerase, respectively, affects initiation and maintenance of the nodulation process [14,15,16,17,18]. WaaL proteins of Escherichia coli K-12 and Salmonella enterica serovar Typhimurium do not share substantial primary-sequence similarity, but they have strikingly similar hydropathy plot patterns [20]. Little is known about the role of waaL in the symbiotic relationship between leguminous plants and nitrogen-fixing rhizobia, especially soybean endosymbiont B. japonicum. We conclude that waaL is another essential gene responsible for the successful symbiotic nitrogen fixation
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