Saturation mutagenesis of an Escherichia coli rRNA promoter and initial characterization of promoter variants.

Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p according to the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoter fragments containing single base substitutions, insertions, deletions, and multiple mutations, covering the whole length of the promoter including the upstream activation sequence (UAS). The activities of 112 mutant promoters were assayed as operon fusions to lacZ in lambda lysogens. The activities of most mutants with changes in the core promoter recognition region (i.e., substitutions, insertions, or deletions in the region of the promoter spanning the -10 and -35 E. coli consensus hexamers) correlated with changes toward or away from the consensus in the hexamer sequences or in the spacing between them. However, changes at some positions in the core promoter region not normally associated with transcriptional activity in other systems also had significant effects on rrnB P1. Since rRNA promoter activity varies with cellular growth rate, changes in activity can be the result of changes in promoter strength or of alterations in the regulation of the promoter. The accompanying paper (R. R. Dickson, T. Gaal, H. A. deBoer, P. L. deHaseth, and R. L. Gourse, J. Bacteriol. 171:4862-4870, 1989) distinguishes between these two alternatives. Several mutations in the UAS resulted in two- to fivefold reductions in activity. However, two mutants with changes just upstream of the -35 hexamer in constructs containing the UAS had activities 20- to 100-fold lower than the wild-type level. This collection of mutant rRNA promoters should serve as an important resource in the characterization of the mechanisms responsible for upstream activation and growth rate-dependent regulation of rRNA transcription.

Induction of Bradyrhizobium japonicum common nod genes by isoflavones isolated from Glycine max.

The early events in legume nodulation by Rhizobium spp. involve a conserved gene cluster known as the common nod region. A broad-host-range plasmid (pEA2-21) containing a Bradyrhizobium japonicum nodDABC-lacZ translational fusion was constructed and used to monitor nod gene expression in response to soybean root extract. Two inducing compounds were isolated and identified. Analysis using ultraviolet absorption spectra, proton nuclear magnetic resonance, and mass spectrometry showed that the two inducers were 4',7-dihydroxyisoflavone (daidzein) and 4',5,7-trihydroxyisoflavone (genistein). Induction was also seen with some, but not all, of the flavonoid compounds that induce nod genes in fast-growing Rhizobium strains that nodulate clover, alfalfa, or peas. When pEA2-21 was introduced into Rhizobium trifolii, it was inducible by flavones but not by daidzein and genistein. In Rhizobium fredii, pEA2-21 was induced by isoflavones and flavones. Thus, the specificity of induction appears to be influenced by the host-strain genome.