Expression of flcA, a gene regulating differentiation and plant interaction in 'Azospirillum'

Abstract

'Azospirillum brasilense' strains benefit field crops by excreting plant growth factors, promoting root proliferation and enhancing water uptake. FlcA protein is a transcriptional response regulator of the LuxR-UhpA family, which controls flocculation of 'A. brasilense' cells in solution and plant root surface colonisation by 'A. brasilense'. The expression of the flcA gene, studied here using a lacZ fusion controlled by the flcA promoter, was found to be partially constitutive in rich media. However, it could be further induced 3-4 fold by high ratios of C to N in the surrounding medium when transferred to minimal medium supplemented with fructose. Such conditions also induced flocculation by wild-type strains of 'A. brasilense' in liquid media. Relatively high expressions of flcA were detected in root-associated cells, suggesting a positive effect of plant exudates on flcA expression. Although flocculation conditions increased the expression of flcA in wild-type 'A. brasilense' strain Sp7 during the first 4-5 h of growth, the expression was reduced to low levels with the initiation of flocculation. Moreover, flcA expression in wild-type strain producing FlcA, was found to be significantly lower (3.5-5.5 fold in rich medium and 3-4 fold in flocculation-inducing conditions) than its expression in flcA⁻ Tn5 induced mutant strains Sp72001/2/4, which lack the FlcA protein in their cells. Similar to the wild-type strain Sp7, the mutants Sp72001/2/4 also showed 3-4 fold induction of flcA expression in minimal media containing high fructose to N ratio. However, in contrast to the wild-type strain, the mutants did not flocculate in any stage of their growth and the expression of flcA continued to rise after 6 h of growth. Therefore, it seems highly likely that flcA expression is negatively autoregulated by its own product, FlcA. Such regulation may be either direct, via transcriptional regulation of the flcA gene or indirect, by controlling the activity of other genes and proteins that may be involved in the differentiation process of 'A. brasilense' from vegetative to cyst-like cells. A model for self-regulation of flcA is presented.