Search

A unique nuclease is released by Fusarium solani f. spp. pisi and phaseoli macroconidia during germination on pea pod endocarp tissue. This Fsp nuclease is produced in greater amounts by macroconidia germinating on pea pods than in Vogel's rich medium or in water. We have characterized the enzymatic activities, and physical properties of this enzyme. Fsp nuclease makes randomly situated single stranded nicks in single- or double-stranded DNA, leaving a 5' phosphate group and a 3' hydroxyl group on the nicked DNA strand. This nicking activity is stimulated by divalent cations over a range of 2 10 mM, with Mn²⁺ > Ca²⁺ > Co²⁺ > Mg²⁺. Zinc inhibited the nuclease, even in the presence of stimulatory divalent cations. Fsp nuclease is susceptible to treatment with proteinase K or SDS, indicating a proteinaceous identity, but surprisingly, is resistant to boiling. A 22 kDa molecular weight was estimated by renaturing the enzyme following denaturing electrophoresis. The possible role of this nuclease in virulence is being investigated. Copyright 1993, 1999 Academic Press

Several soybean genotypes have been identified which specifically exclude nodulation by members of Bradyrhizobium japonicum serocluster 123. We have identified and sequenced a DNA region from B. japonicum strain USDA 110 which is involved in genotype-specific nodulation of soybeans. This 2.3-kilobase region, cloned in pMJS12, allows B. japonicum serocluster 123 isolates to form nodules on plants of serogroup 123-restricting genotypes. The nodules, however, were ineffective for symbiotic nitrogen fixation. The nodulation-complementing region is located approximately 590 base pairs transcriptionally downstream from nodD2. The 5' end of pMJS12 contains a putative open reading frame (ORF) of 710 base pairs, termed nolA. Transposon Tn3-HoHo mutations only within the ORF abolished nodulation complementation. The N terminus of the predicted nolA gene product has strong similarity with the N terminus of MerR, the regulator of mercury resistance genes. Translational lacZ fusion experiments indicated that nolA was moderately induced by soybean seed extract and the isoflavone genistein. Restriction fragments that hybridize to pMJS12 were detected in genomic DNAs from both nodulation-restricted and -unrestricted strains.

By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).

The cell surface polysaccharides of wild-type Bradyrhizobium japonicum USDA 110 and a nonnodulating mutant, strain HS123, were analyzed. The capsular polysaccharide (CPS) and exopolysaccharide (EPS) of the wild type and the mutant strain do not differ in their sugar composition. CPS and EPS are composed of mannose, 4-O-methylgalactose/galactose, glucose, and galacturonic acid in a ratio of 1:1:2:1, respectively. H nuclear magnetic resonance spectra of the EPS and CPS of the wild type and mutant strain are very similar, but not identical, suggesting minor structural variation in these polysaccharides. The lipopolysaccharides (LPS) of the above two strains were purified, and their compositions were determined. Gross differences in the chemical compositions of the two LPS were observed. Chemical and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that strain HS123 is a rough-type mutant lacking a complete LPS. The LPS of mutant strain HS123 is composed of mannose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, and lipid A. The wild-type LPS is composed of fucose, xylose, arabinose, mannose, glucose, fucosamine, quinovosamine, glucosamine, uronic acid, 2-keto-3-deoxyoctulosonic acid, and lipid A. Preliminary sugar analysis of lipid A from B. japonicum identified mannose, while traces of glucosamine were detected. 3-Hydroxydodecanoic and 3-hydroxytetradecanoic acids formed a major portion of the fatty acids in lipid A. Lesser quantities of nonhydroxylated 16:0, 18:0, 22:0, and 24:0 acids also were detected.