Al-Haideri Halah, White Michael A, Kelly David J
Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.
Environ Microbiol. 2016 Feb;18(2):721-35. doi: 10.1111/1462-2920.13092. Epub 2015 Dec 21.
Campylobacter jejuni, the leading cause of human bacterial gastroenteritis, requires low environmental oxygen and high carbon dioxide for optimum growth, but the molecular basis for the carbon dioxide requirement is unclear. One factor may be inefficient conversion of gaseous CO2 to bicarbonate, the required substrate of various carboxylases. Two putative carbonic anhydrases (CAs) are encoded in the genome of C. jejuni strain NCTC 11168 (Cj0229 and Cj0237). Here, we show that the deletion of the cj0237 (canB) gene alone prevents growth in complex media at low (1% v/v) CO2 and significantly reduces the growth rate at high (5% v/v) CO2. In minimal media incubated under high CO2, the canB mutant grew on L-aspartate but not on the key C3 compounds L-serine, pyruvate and L-lactate, showing that CanB is crucial in bicarbonate provision for pyruvate carboxylase-mediated oxaloacetate synthesis. Nevertheless, purified CanB (a dimeric, anion and acetazolamide sensitive, zinc-containing type II beta-class enzyme) hydrates CO2 actively only above pH 8 and with a high Km (∼ 34 mM). At typical cytoplasmic pH values and low CO2, these kinetic properties might limit intracellular bicarbonate availability. Taken together, our data suggest CanB is a major contributor to the capnophilic growth phenotype of C. jejuni.
空肠弯曲菌是人类细菌性肠胃炎的主要病因，其最佳生长需要低环境氧和高二氧化碳，但对二氧化碳需求的分子基础尚不清楚。一个因素可能是气态二氧化碳向碳酸氢盐（各种羧化酶所需的底物）的转化效率低下。空肠弯曲菌菌株NCTC 11168的基因组中编码了两种假定的碳酸酐酶（CAs）（Cj0229和Cj0237）。在此，我们表明单独缺失cj0237（canB）基因会阻止在低（1% v/v）二氧化碳的复杂培养基中生长，并显著降低在高（5% v/v）二氧化碳时的生长速率。在高二氧化碳条件下孵育的基本培养基中，canB突变体在L-天冬氨酸上生长，但在关键的C3化合物L-丝氨酸、丙酮酸和L-乳酸上不生长，这表明CanB在为丙酮酸羧化酶介导的草酰乙酸合成提供碳酸氢盐方面至关重要。然而，纯化的CanB（一种二聚体、对阴离子和乙酰唑胺敏感的含锌II型β类酶）仅在pH 8以上且具有高Km（约34 mM）时才积极水合二氧化碳。在典型的细胞质pH值和低二氧化碳条件下，这些动力学特性可能会限制细胞内碳酸氢盐的可用性。综上所述，我们的数据表明CanB是空肠弯曲菌嗜二氧化碳生长表型的主要贡献者。
