Effects of the Bradyrhizobium japonicum waaL (rfaL) Gene on Hydrophobicity, Motility, Stress Tolerance, and Symbiotic Relationship with Soybeans

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.

Keywords: O-antigen ligase; lipopolysaccharide (LPS); rfaL; soybean symbiont Bradyrhizobium japonicum; stress responses; symbiotic nitrogen fixation; waaL.

Figure 1

Comparison of hydropathy plots of WaaL proteins from E. coli (a); S. enterica serovar Typhimurium (b); and B. japonicum 61A101C (c). The x-axis represents the amino acid residue position, while the y-axis represents the relative hydrophobicity score. Peaks above the red line indicate potential membrane-spanning domains. Each protein contains 12 potential membrane-spanning domains.

Figure 2

SDS-PAGE of LPS from wild-type, waaL knock-out mutant, and complemented strains. LPS I is the high molecular weight form of the LPS, which contains the O-antigen. LPS II lacks the O-antigen. Lane 1, LPS standard from E. coli serotype 055:B5 (Invitrogen, Carlsbad, CA, USA); Lane 2, 61A101C; Lane 3, JS015; Lane 4, CS015.

Figure 3

Cell surface hydrophobicity (A) and cell pellet pattern (B) of three B. japonicum strains: 1, 61A101C; 2, JS015; 3, CS015. (A) The x-axis represents removal rate per min as a function of the hexadecane-to-water volume ratio (V_H/V_W), while the y-axis represents hexadecane-to-water volume ratios (V_H/V_W).

Figure 4

Motility of three B. japonicum strains 61A101C, JS015, and CS015: (A) 1% solid agar medium; and (B) 0.3% soft agar medium.

Figure 5

Electron microscopic analysis of flagella from the wild type (A); the waaL knock-out mutant (B); and its complemented strain (C). Bars = 5 µm.