Identification of an Na+-dependent malonate transporter of Malonomonas rubra and its dependence on two separate genes

Abstract

Two membrane proteins encoded by the malonate fermentation gene cluster of Malonomonas rubra, MadL and MadM, have been synthesized in Escherichia coli. MadL and MadM were shown to function together as a malonate transport system, whereas each protein alone was unable to catalyze malonate transport. Malonate transport by MadLM is Na+ dependent, and imposition of a DeltapNa+ markedly enhanced the rate of malonate uptake. The kinetics of malonate uptake into E. coli BL21(DE3) cells synthesizing MadLM at different pH values indicated that Hmalonate- is the transported malonate species. The stimulation of malonate uptake by Na+ ions showed Michaelis-Menten kinetics, and a Km for Na+ of 1.2 mM was determined. These results suggest that MadLM is an electroneutral Na+/Hmalonate- symporter and that it is dependent on two separate genes.

FIG. 1

Malonate uptake into E. coli cells expressing madL and/or madM. E. coli BL21(DE3) cells transformed with pET-LM (expressing madL and madM in cis) (•) or pACYC-M and pET-L (trans) (○) or pACYC-L and pET-M (trans) (▾) were compared. In controls, E. coli BL21(DE3) was transformed with pET-24a(+) (vector control) (▴), pET-L (expression of madL) (⧫), pET-M (madM) (◊), pACYC-L (madL) (▾), or pACYC-M (madM) (▿). Cells in 50 mM potassium phosphate buffer, pH 7.0, containing 10 mM NaCl (99 μl) were incubated for 1 min at room temperature, prior to the addition of 1 μl of [2-¹⁴C]malonate (1.8 mM; specific activity, 113 cpm/pmol). After the times indicated, samples were treated as described in Materials and Methods.

FIG. 2

Release of intracellular [2-¹⁴C]malonate from E. coli BL21(DE3)/pET-LM by addition of 20 mM external malonate. E. coli BL21(DE3)/pET-LM cells suspended in 50 mM potassium phosphate, pH 7.0, containing 10 mM NaCl (99 μl) were preincubated for 1 min at room temperature. Subsequently, the transport assay was initiated by addition of 1 μl of [2-¹⁴C]malonate (1.8 mM; specific activity, 113 cpm/pmol). After 5 min, 100 μl of 40 mM potassium malonate (pH 7.0) was added (○). In the control (•), 100 μl of 50 mM potassium phosphate buffer (pH 7.0) was added. After the times indicated, samples were treated as described in Materials and Methods.

FIG. 3

Na⁺ dependency of malonate transport in E. coli BL21(DE3)/pET-LM. Transport was initiated by addition of 2 μl of 2.9 mM [2-¹⁴C]malonate (34,730 cpm/nmol) to 98 μl of cells suspended in 50 mM potassium phosphate buffer, pH 7.5, without NaCl addition (<25 μM Na⁺) (▾) or with 50 mM NaCl (○). ΔpNa⁺-driven malonate uptake was initiated by addition of 3 μl of 1.67 M NaCl, containing 1.93 mM [2-¹⁴C]malonate (34,730 cpm/nmol), to 97 μl of Na⁺-free cells (•). After the times indicated, samples were treated as described in Materials and Methods.

FIG. 4

Model for the Na⁺ cycle during malonate fermentation in M. rubra. The malonate transporter (MadLM) and the intrinsic membrane part of the malonate decarboxylase (MadB) are depicted as boxes in the membrane. The biotin-containing subunit MadF, drawn at the cytoplasmic side of the membrane, functions as a CO₂ shuttle between MadB and the soluble parts of the malonate decarboxylase (MadACDE). The direction of the H⁺ and Na⁺ fluxes during malonate fermentation is indicated with arrows. Acetate is postulated to leave the cell by passive diffusion or via a specific carrier. Further details are explained in the text.