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Enhanced symbiotic performance by Rhizobium tropici glycogen synthase mutants.

Journal of bacteriology (2001-02-24)
S Marroquí, A Zorreguieta, C Santamaría, F Temprano, M Soberón, M Megías, J A Downie
ABSTRACT

We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c(1) and CycM and a small increase in the amount of cytochrome aa(3). In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
N,N-Dimethyl-p-phenylenediamine, 97%
Sigma-Aldrich
N,N-Dimethyl-1,4-phenylenediamine oxalate, 98%
Sigma-Aldrich
N,N-Dimethyl-p-phenylenediamine sulfate salt, 98%
Millipore
N,N-Dimethyl-p-phenylenediamine dihydrochloride, suitable for microbiology, ≥99.0%
Sigma-Aldrich
N,N-Dimethyl-p-phenylenediamine dihydrochloride, suitable for peroxidase test, ≥99.0% (titration)
Supelco
N,N-Dimethyl-p-phenylenediamine, for spectrophotometric det. of SO42-, S2-, ≥97.0%