Radiolabeled ATP and sucrose uptake assays.

[¹⁴C]sucrose and [α-³²P]ATP were purchased from Perkin-Elmer (Waltham, MA). Isotopic sucrose and ATP uptake assays were performed as described previously by Vahling et al. (28) with some modifications. Five-day-old L. crescens wild-type BT-1, sugar transporter interruption mutant Sut-1, and pMJ056 (lacZ::nttA)-transformed cells (25 ml, A₆₀₀ = 0.5) were collected via centrifugation and washed in 50 mM phosphate-buffered saline (PBS; pH 7.0). The bacteria were resuspended in PBS to A₆₀₀ = 1.0, followed by incubation in the presence of [¹⁴C]sucrose (1 μCi 10 μl⁻¹; specific activity, 435 mCi mmol⁻¹) or [α-³²P]ATP (10 μCi μl⁻¹; specific activity, 3,000 Ci mmol⁻¹) at room temperature. At the indicated time points, 500-μl culture aliquots were withdrawn, washed twice in ice-cold 50 mM PBS (pH 7.0), and resuspended in 2 ml of a solution of PBS and scintillation fluid (1:1, vol/vol) before being measured on a LS6500 multipurpose scintillation counter (Beckman Coulter, Brea, CA).