Transcriptional activation of ydeA, which encodes a member of the major facilitator superfamily, interferes with arabinose accumulation and induction of the Escherichia coli arabinose PBAD promoter

Abstract

Induction of genes expressed from the arabinose PBAD promoter is very rapid and maximal at low arabinose concentrations. We describe here two mutations that interfere with the expression of genes cloned under arabinose control. Both mutations map to the ydeA promoter and stimulate ydeA transcription; overexpression of YdeA from a multicopy plasmid confers the same phenotype. One mutation is a large deletion that creates a more efficient -35 region (ATCACA changed to TTCACA), whereas the other affects the initiation site (TTTT changed to TGTT). The ydeA gene is expressed at extremely low levels in exponentially growing wild-type cells and is not induced by arabinose. Disruption of ydeA has no detectable effect on cell growth. Thus, ydeA appears to be nonessential under usual laboratory growth conditions. The ydeA gene encodes a membrane protein with 12 putative transmembrane segments. YdeA belongs to the largest family of bacterial secondary active transporters, the major facilitator superfamily, which includes antibiotic resistance exporters, Lac permease, and the nonessential AraJ protein. Intracellular accumulation of arabinose is strongly decreased in mutant strains overexpressing YdeA, suggesting that YdeA facilitates arabinose export. Consistent with this interpretation, very high arabinose concentrations can compensate for the negative effect of ydeA transcriptional activation. Our studies (i) indicate that YdeA, when transcriptionally activated, contributes to the control of the arabinose regulon and (ii) demonstrate a new way to modulate the kinetics of induction of cloned genes.

FIG. 1

Effects of suppressor mutations on kinetics of PAI2-AP export upon induction with arabinose. The two suppressor mutations of strains SB1 and SB34 were transduced into the parental strain SB0, generating suppressor strains SB01 and SB034, respectively. All strains express the chimeric PAI2-AP protein. Cells grown to an A₆₀₀ of 0.1 in NZ medium at 37°C were induced with 0.2% arabinose at time zero. AP activity was assayed at the indicated times. Each curve represents the average of three independent cultures; error bars indicate standard deviations.

FIG. 2

Effects of suppressor mutations on kinetics of PAI2-AP transcription upon induction with arabinose. Cells grown to an A₆₀₀ of 0.1 in NZ medium at 37°C were induced with 0.2% arabinose at time zero. Total RNA was extracted from samples collected at the indicated times. Two micrograms of RNA was hybridized to a ³²P-labeled PAI2 cRNA probe (290 nt). After digestion with pancreatic RNase, the hybrids were denatured and electrophoresed in a 6% urea-polyacrylamide gel. The protected cRNA fragment is 193 nt long (arrow); traces of undigested probe are visible in the upper portion of the gel. Lanes 1 to 5, parental strain SB0; lanes 6 to 10, suppressor strain SB1; lanes 11 to 15, suppressor strain SB34.

FIG. 3

Nucleotide sequences of mutations in the ydeA promoter. The nucleotide sequence of the wild-type (wt) ydeA promoter region is shown at the top; numbering corresponds to that of database entry ECAE000250. The putative −35 and −10 promoter regions are boxed, and the potential start sites are underlined. The mutation in strain SB34 introduces a G residue in the transcription initiation region. The large deletion in strain SB1 changes the −35 region, which now shows a five-of-six-position match with the TTGACA consensus sequence; the insert of pSB11 starts at the underlined Sau3AI site. The sequence upstream of the deletion is in italics and boxed, as is the wild-type sequence at the beginning of terC, shown at the bottom; numbering corresponds to that of database entry ECAE000249. The ACAAAT repeats (thick lines) upstream of terC (bottom) and ydeA (top) may have been involved in the spontaneous deletion of intervening sequences.

FIG. 4

ydeA mRNA levels in wild-type and mutant strains. For each strain, two independent cultures were grown at 37°C to an A₆₀₀ of 0.4 in NZ medium, and total RNA was isolated. (A) Two micrograms of RNA was hybridized to a ³²P-labeled ydeA cRNA probe (450 nt). After digestion with pancreatic RNase, the hybrids were denatured and electrophoresed in a 5% polyacrylamide gel. The protected fragment is 360 nt long (arrow); traces of undigested probe are visible in the upper portion of the gel. The wild-type (wt) strain was SB0. (B) Two cultures of DB530 (ydeA⁺) and DB529 (SB1 derivative) grown in LB medium were induced for 20 min with 2% arabinose (+) or not induced (−). Ten micrograms of RNA was hybridized to detect the very low levels of ydeA mRNA in wild-type cells. The gel was exposed for 4 days (wild type) and 20 h (SB1).

FIG. 5

Effect of the ydeA promoter mutations on arabinose uptake. (A) Two independent cultures of DB529 (SB1 derivative) and wild-type (wt) strain DB530 (ydeA⁺) were assayed in duplicate for arabinose uptake during a 1-min pulse at the indicated arabinose concentrations. Intracellular arabinose levels (in picomoles/0.2 ml of cells) were normalized to the optical density of the cultures. Error bars indicate ranges. (B) The kinetics of arabinose uptake was measured with two independent cultures of DB529 (SB1 derivative), DB531 (SB34 derivative), and DB530 (ydeA⁺ [wt]), the average of the two cultures is presented. The arabinose concentration was 62.5 μM. (C) Arabinose uptake by the AraFGH system was assayed in strain AD126 (unc) carrying pKKATEB and either pSB13 or pACYC184 (pAC). Cells were assayed in triplicate for arabinose uptake during a 2-min pulse at 14 μM arabinose. Glucose-fed cells were pretreated for 30 s with 16 μM CCCP (+CCCP) or with the same volume of solvent (no). Intracellular arabinose levels (in picomoles/0.2 ml of cells) were normalized to the optical density of the cultures. Error bars indicate standard deviations.

FIG. 6

Effect of YdeA on the induction of PAI2-AP by increasing arabinose concentrations. Cells were grown at 37°C in NZ medium to an A₆₀₀ of 0.4, induced for 20 min with the indicated concentrations of arabinose, and assayed for AP activity. Each curve represents the average of three independent cultures. Error bars represent standard deviations. Both the wild-type (wt; SB0) and suppressor (SB1) strains carry pBAD72K and express the PAI2-AP chimeric protein.