Characterization of SotA and SotB, two Erwinia chrysanthemi proteins which modify isopropyl-beta-D-thiogalactopyranoside and lactose induction of the Escherichia coli lac promoter

Abstract

The expression, in Escherichia coli, of variants of the Erwinia chrysanthemi secretion genes outB and outS under the Ptac promoter is toxic to the cells. During attempts to clone E. chrysanthemi genes able to suppress this toxicity, I identified two genes, sotA and sotB, whose products are able to reduce the isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of the E. coli lac promoter. SotA and SotB belong to two different families of the major facilitator superfamily. SotA is a member of the sugar efflux transporter family, while SotB belongs to the multidrug efflux family. The results presented here suggest that SotA and SotB are sugar efflux pumps. SotA reduces the intracellular concentration of IPTG, lactose, and arabinose. SotB reduces the concentration of IPTG, lactose, and melibiose. Expression of sotA and sotB is not regulated by their substrates, but sotA is activated by the cyclic AMP receptor protein (CRP), while sotB is repressed by CRP. Lactose is weakly toxic for E. chrysanthemi. This toxicity is increased in a sotB mutant which cannot efficiently efflux lactose. This first evidence for a physiological role of sugar efflux proteins suggests that their function could be to reduce the intracellular concentration of toxic sugars or sugar metabolites.

FIG. 1

Effect of the presence of sotA and sotB on the expression of genes cloned into plasmid pACT3. E. coli NM522 cells containing plasmids pACTS1 (pelBsp-outS) (A), pABSC3 (pelBsp-outB) (B), pABTC2 (outB) (C), and pACTPB (pelB) (D) were transformed with plasmids pUC18, pUCS1 (sotA), and pUCS6 (sotB). Cells were grown for 3 h in the absence or presence of 10⁻⁴ M IPTG. Aliquots were loaded onto SDS-PAGE gel and immunoblotted with anti-OutS (A), anti-OutB (B and C), and anti-PelB (D) antibodies.

FIG. 2

Effect of the presence of sotA and sotB on the IPTG induction of pelB cloned into pACT3. E. coli NM522 cells containing pACTPB (pelB) and pUC18 (A), pUCS1 (sotA) (B), or pUCS6 (sotB) (C) were grown in LB medium with 0 (lane 1), 10⁻⁶ (lane 2), 10⁻⁵ (lane 3), 10⁻⁴ (lane 4), or 10⁻³ M IPTG (lane 5). Aliquots were loaded onto an SDS-PAGE gel (10% polyacrylamide) and immunoblotted with anti-PelB antibody.

FIG. 3

Effect of the presence of sotA and sotB on the induction of the lac and ara promoter in E. coli by various compounds. Strains P4X (induction by IPTG, lactose, or melibiose) containing plasmid pBR322 (□), pBRS1 (sotA) (◊), or pBRS6 (sotB) (○), or NM522/pBADuid (induction by arabinose) carrying plasmid pSU9 (□), pS1A (sotA) (◊), or pS6E (sotB) (○) were grown in LB medium to an optical density at 600 nm of 0.5. Inducer was added at the concentration indicated, and the β-galactosidase (β-gal) or β-glucuronidase (β-glu) activity of the culture was measured over time. (A) IPTG (4 × 10⁻⁵ M). (B) IPTG (3 × 10⁻⁶ M). (C) Lactose (20 mg/liter). (D) Lactose (5 mg/liter). (E) Melibiose (40 mg/liter). (F) Arabinose (4 mg/liter).

FIG. 4

Growth of strain A350 and of sotA and sotB mutants in the presence of lactose. M63 medium containing 0.2% glycerol and various concentrations of lactose was inoculated at a 1:100 dilution with a culture of strain A350, A3156, or A3501. After 16 h of growth, the optical density at 600 nm (OD₆₀₀) was measured.