Nonfermentative thermoalkaliphilic growth is restricted to alkaline environments

Abstract

Caldalkalibacillus thermarum strain TA2.A1 grew in pH-controlled batch culture containing a fermentable growth substrate (i.e., sucrose) from pH 7.5 to 10.0 with no significant change in the specific growth rate, suggesting that this bacterium was a facultative alkaliphile. However, when strain TA2.A1 was grown on a nonfermentable carbon source, such as succinate or malate, no growth was observed until the external pH was >9.0, suggesting that this bacterium was an obligate alkaliphile. Succinate transport and sucrose transport by strain TA2.A1 showed pH profiles similar to that of growth on these carbon sources, and the molar growth yield on sucrose was higher at pH 9.5 than at pH 7.5, despite the increased energy demands on the cell for intracellular pH regulation. Succinate transport, succinate-dependent oxygen consumption, and succinate dehydrogenase and F(1)F(o)-ATPase specific activities were all significantly lower in cultures of strain TA2.A1 grown at pH 7.5 than in those cultured at pH 9.5. No significant ATP synthesis via the F(1)F(o)-ATP synthase was detected until the external pH was >8.5. On the basis of these results, we propose that nonfermentative thermoalkaliphilic growth is specialized to function at high pH values, but not at pH values near neutral pH.

FIG. 1.

Effects of external pH and carbon source on the growth of C. thermarum strain TA2.A1 in pH-controlled batch culture using either alkaline basal medium (no added carbon source) or alkaline basal medium supplemented with 50 mM succinate, malate, or sucrose. (A) Final optical density (600 nm) of cells grown for 48 h (stationary phase) at 65°C. (B) Weight (dry weight) of cells grown on various carbon sources. The values reported are the means of two to four biological replicates (triplicate technical replicates), and the standard errors of the means (error bars) are shown.

FIG. 2.

Uptake of [¹⁴C]succinate into whole cells of C. thermarum strain TA2.A1. (A) Accumulation of [¹⁴C]succinate (10 μM) by whole cells (black squares) or cells treated with 1% toluene (vol/vol) (black circles) are expressed as counts per minute (determined by liquid scintillation counting) over time. Data are normalized to a cell suspension in the assay of OD₆₀₀ of 1.0. (B) Effects of CCCP (100 μM) and monensin (Mon) (10 μM) on [¹⁴C]succinate uptake. (C and D) Kinetics of [¹⁴C]succinate transport by strain TA2.A1. The initial uptake values of [¹⁴C]succinate, expressed as nanomoles of succinate per minute per milligram of protein, were measured over 180 s at succinate concentrations between 1 μM and 75 μM (C) and at sodium concentrations of 0 to 50 mM NaCl and 50 μM succinate (D). Data are shown as Michaelis-Menten plots. The insets show Lineweaver-Burk plots of the same data. V, succinate transport in nmol succinate min⁻¹ mg protein⁻¹; S, succinate concentration in μM. Experiments were carried out in triplicate, and representative results are shown.

FIG. 3.

Uptake of [¹⁴C]succinate (10 μM) and [¹⁴C]sucrose (10 μM) into whole cells of C. thermarum strain TA2.A1 resuspended in a 50 mM MES-MOPS-Tris-HCl buffer system containing 50 mM NaCl. (A) Effects of external pH on [¹⁴C]succinate (black squares) and [¹⁴C]sucrose (black circles) uptake by cells grown at pH 9.5 on 50 mM succinate. (B) Effects of external pH on [¹⁴C]succinate (black squares) and [¹⁴C]sucrose (black circles) uptake by cells grown at pH 9.5 on 50 mM sucrose. (C) Effects of external pH on [¹⁴C]succinate (black squares) and [¹⁴C]sucrose (black circles) uptake by cells grown at pH 7.5 on 50 mM sucrose and 50 mM succinate. (D) Effects of competing substrates (100-fold excess) on [¹⁴C]succinate uptake. One hundred percent succinate uptake is equivalent to 0.40 nmol succinate·min⁻¹ mg protein⁻¹, and competitor substrates were added 15 min prior to the addition of [¹⁴C]succinate (final concentration of 50 μM). The competing substrates were succinate (Succ), malate (Mal), fumarate (Fum), aspartate (Asp), α-ketoglutarate (Kg), glutamate (Glu), and pyruvate (Pyr). The initial uptake values of [¹⁴C]succinate and [¹⁴C]sucrose are expressed as nmol substrate·min⁻¹ mg protein⁻¹. The values reported are the means of two to four biological replicates (triplicate technical replicates), and the standard errors of the means (error bars) are shown.

FIG. 4.

(A) Effect of external pH on oxygen consumption by whole cells of strain TA2.A1 grown on 50 mM succinate (pH 9.5). Oxygen consumption was determined at 65°C in 50 mM MES-MOPS-Tris-HCl buffer and initiated by the addition of 20 mM succinate. (B and C) Succinate dehydrogenase (SDH) oxidoreductase (B) and ATP hydrolysis (C) specific activities in inverted membrane vesicles of strain TA2.A1 grown at either pH 7.5 (sucrose or sucrose and succinate) or pH 9.5 (sucrose or succinate). Sucr, sucrose; succ, succinate. (D) Effect of external pH on ATP synthesis in ADP- plus P_i-loaded RSO membrane vesicles of strain TA2.A1. The time course of the ATP synthesis assay at pH 7.0 to 10.5 with 0.5 mg RSO membrane vesicles using the ATP synthesis assay described in Materials and Methods. Black squares, no DCCD addition; black circles, a 5-min preincubation with 150 μM DCCD. The values reported are the means of two to four biological replicates (triplicate technical replicates), and the standard errors of the means (error bars) are shown.