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Controlling the Expression of Rhizobial Genes During Nodule Development with Elements and An Inducer of the lac Operon

April 2011 , Volume 24 , Number  4
Pages  478 - 486

Jodie Box and K. Dale Noel

Department of Biological Sciences, Marquette University, PO Box 1881, Milwaukee, WI 53201 U.S.A.

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Accepted 22 November 2010.

A simple strategy was tested for imposing artificial regulation of rhizobial genes during nodule development. Isopropyl-β-D-1-thiogalactoside (IPTG) was added to liquid root media to sustain expression of rhizobial genes controlled by Escherichia coli lac promoter/operators and repressor gene lacI. Conversely, a rinsing protocol was devised to remove IPTG sufficiently that genes could be repressed after having been induced. gusA under this control exhibited clearly delineated expression and repression in both the determinate Rhizobium etliPhaseolus vulgaris and the indeterminate Sinorhizobium melilotiMedicago sativa symbioses. Apparently, IPTG was taken up in sufficiently undegraded concentrations that gene expression was derepressed even in interior portions of the nodule. Moreover, the rinsing protocol led to obvious repression of gusA. Importantly, no deleterious effects of IPTG on nodule development, infection, or nitrogen fixation were observed. An R. etli CE3 gene required for lipopolysaccharide O antigen and infection on bean was put under this control by means of a two-plasmid construct. When this construct was added to a strain with a null mutation in this gene, infection, nodule development, and nitrogenase activity all depended on the length of time before IPTG was rinsed from the roots after inoculation.

© 2011 The American Phytopathological Society