Physiological Roles of Carbonic Anhydrases and an MpsAB-like Bicarbonate Transporter in Bacillus subtilis.

UNLABELLED: Carbonic anhydrase (E.C. 4.2.1.1) is an enzyme that catalyzes the reversible hydration of CO 2 to carbonic acid (H 2 CO 3 ), which dissociates to bicarbonate (HCO 3 - ) at intracellular pH. Copious amounts of CO 2 are produced by catabolism, although much of this is lost by diffusion from the cell. In Bacillus subtilis , anabolic processes rely largely on bicarbonate as a substrate rather than CO 2 . While CO 2 reacts spontaneously with water to yield bicarbonate, the rate of this reaction is too slow to keep up with cell requirements. B. subtilis encodes three putative β-class carbonic anhydrases, here renamed canA ( yvdA) , canB ( ytiB ), and canC ( ybcF) . The canC gene is encoded in an operon with ndhF - mpsB ( ybcC) , which encodes a candidate MpsAB-type bicarbonate transporter. Here we demonstrate that a strain lacking canA, canB, canC, and mpsB (Δ4) has a severe growth defect at atmospheric CO 2 . This defect can be overcome by growing cells with supplemental CO 2 or by plating at high cell density. We isolated suppressors of Δ4 and identified mutations in resD that suppress the requirement for supplemental CO 2 to support growth. ResD functions as a global regulator of genes important for both aerobic and anaerobic respiration. We demonstrate that a resD null mutation results in metabolic changes that lead to an increased generation of CO 2 . We infer that this results in an increase in spontaneous bicarbonate formation that is sufficient to support cell growth. We conclude that the requirement for bicarbonate concentrating mechanisms may be bypassed under conditions that increase endogenous CO 2 generation.

IMPORTANCE: Carbonic anhydrase (CA) catalyzes the reversible hydration of CO 2 to bicarbonate. This enzyme is found in all domains of life and has evolved multiple times. In heterotrophic organisms, CA is important for concentrating bicarbonate for anabolic reactions such as fatty acid, amino acid, and menaquinone synthesis. Bicarbonate can also be directly imported by dedicated transporters. Free-living bacteria are often unable to grow in the absence of a bicarbonate concentrating mechanism, and CAs and bicarbonate transporters are also important in pathogenesis. Thus, understanding how cells obtain bicarbonate may lead to the development of novel antibiotics and highlight new therapeutic targets.