Rhizobium leguminosarum nodulation gene (nod) expression is lowered by an allele-specific mutation in the dicarboxylate transport gene dctB.
To identify host genes that might influence nod (nodulation) gene expression in Rhizobium leguminosarum, a nodC-phoA reporter plasmid (carrying nodD) was introduced into a chemically mutagenized population of a R. leguminosarum strain lacking a symbiotic plasmid. The transconjugants were screened for expression of alkaline phosphatase (PhoA) on plates containing hesperetin, an inducer of nod genes, and a mutant with reduced expression was identified. When the nodC-phoA plasmid was cured from the mutant and the symbiotic plasmid pRL1Jl introduced, the mutant formed nodules, but symbiotic nitrogen fixation was less than 20% of normal. When the nodC-phoA allele was introduced on pRL1Jl a low level of nod gene induction was found. The reduced nodC expression appeared to be caused by a decrease in expression of the regulatory gene nodD, since expression of a nodD-lacZ fusion was also lower in the mutant than in the control. These mutant phenotypes and the low nitrogen fixation were complemented with a plasmid (plJ1848) from a R. leguminosarum cosmid library. DNA hybridization confirmed that plJ1848 was not from the symbiotic plasmid and showed that a DNA insertion was present in the mutant. The complementing region of plJ1848 was defined by transposon mutagenesis; DNA sequencing revealed that it carried the dicarboxylic acid transport (dct) genes. However, the mutant grew well with succinate as sole C-source. Genetic analysis revealed that the mutant appeared to contain IS50 in the regulatory gene dctB and that this mutation caused the reduction in nod gene expression. The effect was allele-specific since other mutations in dctB did not influence nod gene expression. Surprisingly, the mutant had a constitutive high level of succinate transport, indicating that the mutation caused unregulated expression of dctA the structural gene for dicarboxylic acid transport. This in some way appears to have lowered the expression of nodD, indicating that the nodD promoter may be influenced by the metabolic status of the cells or by expression of dctD in the absence of dctB.
Mavridou
A
A
John Innes Centre, Norwich, UK.
Barny
M A
MA
Poole
P
P
Plaskitt
K
K
Davies
A E
AE
Johnston
A W
AW
Downie
J A
JA
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000483:Alleles; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D003227:Conjugation, Genetic; D017931:DNA Primers; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids / Q000378:metabolism*; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics*; D015183:Restriction Mapping; D016961:Rhizobium leguminosarum / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D017931:DNA Primers; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids; C060773:NodD protein, Bacteria; D011956:Receptors, Cell Surface; C073787:DCTB protein, Sinorhizobium; D017351:N-Acetylglucosaminyltransferases; C060771:NodC protein, Rhizobiales; D011494:Protein Kinases; D000469:Alkaline Phosphatase