Abstract
Many early molecular events in symbiotic infection have been documented, although factors enabling Rhizobium to progress within the plant-derived infection thread and ultimately survive within the intracellular symbiosome compartment as mature nitrogen-fixing bacteroids are poorly understood. Rhizobial surface polysaccharides (SPS), including the capsular polysaccharides (K-antigens), exist in close proximity to plant-derived membranes throughout the infection process. SPSs are essential for bacterial survival, adaptation, and as potential determinants of nodulation and/or host specificity. Relatively few studies have examined the role of K-antigens in these events. However, we constructed a mutant that lacks genes essential for the production of the K-antigen strain-specific sugar precursor, pseudaminic acid, in the broad host range Rhizobium sp. NGR234. The complete structure of the K-antigen of strain NGR234 was established, and it consists of disaccharide repeating units of glucuronic and pseudaminic acid having the structure -->4)-beta-d-glucuronic acid-(1-->4)-beta-5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid-(2-->. Deletion of three genes located in the rkp-3 gene cluster, rkpM, rkpN, and part of rkpO, abolished pseudaminic acid synthesis, yielding a mutant in which the strain-specific K-antigen was totally absent: other surface glycoconjugates, including the lipopolysaccharides, exopolysaccharides, and flagellin glycoprotein appeared unaffected. The NGRDeltarkpMNO mutant was symbiotically defective, showing reduced nodulation efficiency on several legumes. K-antigen production was found to decline after rhizobia were exposed to plant flavonoids, and the decrease coincided with induction of a symbiotically active (bacteroid-specific) rhamnan-LPS, suggesting an exchange of SPS occurs during bacterial differentiation in the developing nodule.
Highlights
The rkpY gene is identified in pathogenic bacteria including Aeromonas spp. that are required for normal infection alter cell motility [49]
Genes involved in the synthesis of the K-antigens (KPS) have been well characterized in R. meliloti strain Rm41 [29, 60, 61]
The present study demonstrates that selective inhibition of KPS synthesis in strain NGR234 reduced its symbiotic efficiency on several legume hosts, including V. unguiculata, a preferred host of the parent strain (Fig. 3)
Summary
Growth of Bacteria—E. coli recombinants were grown at 37 °C in Luria-Bertani medium [25]. Water layer extracts containing KPS and LPS were dialyzed and treated sequentially with ribonuclease, deoxyribonuclease, and proteinase K [38] subjected to size exclusion chromatography under dissociative conditions (0.25% sodium deoxycholate, 0.2 M NaCl, 1.0 mM EDTA, 10 mM Tris pH 9.2) [39] on a column of Sephadex G-150 (1.1 ϫ 100 cm). This procedure separates the major K-antigen polysaccharides from the rough and smooth LPS [40]. For NOE analysis [48], phase-sensitive 1H-1H ROESY was collected with a 300-ms mixing time, with a matrix size identical to that used for COSY and TOCSY experiments, with 48 scans per increment
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