Auxins upregulate expression of the indole-3-pyruvate decarboxylase gene in Azospirillum brasilense

Abstract

Transcription of the Azospirillum brasilense ipdC gene, encoding an indole-3-pyruvate decarboxylase involved in the biosynthesis of indole-3-acetic acid (IAA), is induced by IAA as determined by ipdC-gusA expression studies and Northern analysis. Besides IAA, exogenously added synthetic auxins such as 1-naphthaleneacetic acid, 2,4-dichlorophenoxypropionic acid, and p-chlorophenoxyacetic acid were also found to upregulate ipdC expression. No upregulation was observed with tryptophan, acetic acid, or propionic acid or with the IAA conjugates IAA ethyl ester and IAA-L-phenylalanine, indicating structural specificity is required for ipdC induction. This is the first report describing the induction of a bacterial gene by auxin.

FIG. 1

Growth and expression of ipdC in A. brasilense Sp245 and in the IpdC⁻ mutant FAJ009. Cultures of Sp245(pFAJ64) and FAJ009(pFAJ64) were grown in LB* broth. Periodically, samples were taken to determine the expression of the ipdC-gusA fusion, pFAJ64. Quantitative analysis of β-glucuronidase activity was carried out in microtiter plates with p-nitrophenyl-β-d-glucuronide as substrate (14). Growth was monitored by measuring the OD₆₀₀. The data presented are from one representative experiment, which was confirmed twice in additional independent experiments. Triangles, Sp245(pFAJ64); squares, FAJ009(pFAJ64); open symbols, cell density (OD₆₀₀); solid symbols, β-glucuronidase activity in Miller units (17).

FIG. 2

Induction of the ipdC-gusA fusion by spent culture supernatants. An exponential-phase culture of Sp245(pFAJ64) (OD₆₀₀ = 0.6) grown in LB* broth was centrifuged and resuspended in fresh LB* broth. Two-milliliter samples of this culture were then transferred to sterile test tubes, supplemented with different amounts of spent culture supernatant (ranging from 0 to 2 ml), and diluted up to a total volume of 4 ml with fresh LB* broth. After 2.5 h of growth at 30°C, the cultures were assayed for β-glucuronidase activity as detailed in the legend of Fig. 1. The spent culture supernatants were obtained from late-stationary-phase cultures (OD₆₀₀ = 2.0) of Sp245 (solid bars) or FAJ009 (stippled bars) grown in LB* broth. Open bar, control culture to which no supernatant was added. All data are the means from three replicates. Error bars denote the standard deviations.

FIG. 3

Effect of exogenously added IAA on ipdC expression in Sp245 and the IpdC⁻ mutant, FAJ009. To obtain cultures at various cell densities of Sp245(pFAJ64) (A) and FAJ009(pFAJ64) (B) different flasks containing 100 ml of LB* medium were inoculated with different volumes of preculture and grown overnight at 30°C. From each culture, six samples of 3 ml were transferred to sterile test tubes. Three test tubes of each series were supplemented with IAA to a final concentration of 1 mM (solid bars); to the remaining three test tubes no IAA was added (open bars). After 3.5 h of additional growth at 30°C, β-glucuronidase activity was assayed as detailed in the legend of Fig. 1. Data are the means from the three replicates. Error bars denote the standard deviations.

FIG. 4

Northern blot analysis of RNA extracted from cells of A. brasilense wild-type and IpdC⁻ mutant strains. (A) Blot of total RNA (10 μg per lane) isolated from A. brasilense IpdC⁻ mutant (lane 1) and wild-type strains (lanes 2 to 5). RNA was visualized by epi-illumination with UV light. Cells were harvested at an OD₆₀₀ of 0.9 (lanes 1 and 3), 0.5 (lane 2), or 0.4 (lanes 4 and 5). To measure the effect of IAA on the transcription of the ipdC gene, the wild-type culture at OD₆₀₀ of 0.4 was divided into two and further incubated for 3 h at 30°C in the absence (lane 4) or presence of exogenously added IAA at 1 mM (lane 5). The positions of the 16S and 23S ribosomal RNAs are indicated at the right. (B) RNA blot analysis of the same samples as in panel A after hybridization with a digoxygenin-labeled ipdC-derived riboprobe. The size of the ipdC transcript (in kilobases) is indicated at the right and was determined relative to RNA standards that were electrophoresed in the same gel.