A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis

Abstract

Ca ²⁺ plays an important role in the physiology of bacteria. Intracellular Ca ²⁺ concentrations are tightly maintained in the nanomolar range. Molecular mechanisms of Ca ²⁺ uptake in bacteria remain elusive. Here we show that CtpE is responsible for Ca ²⁺ uptake in Mycobacterium smegmatis . It represents a previously uncharacterized P-type ATPase family in bacteria. Disruption of ctpE in M. smegmatis resulted in a mutant with impaired growth under Ca ²⁺ -deficient conditions. The growth defect of the mutant could be rescued by Ca ²⁺ or by ectopic expression of ctpE from M. smegmatis or the orthologous gene ( Rv0908 ) from Mycobacterium tuberculosis H37Rv. Radioactive transport assays revealed that CtpE is a Ca ²⁺ -specific transporter. Ca ²⁺ deficiency increased expression of ctpE , resulting in increased ⁴⁵ Ca ²⁺ accumulation in cells. ctpE is a gene that is part of an operon, which is negatively regulated by Ca ²⁺ . The ctpE mutant also showed hypersensitivity to polymyxin B, increased biofilm formation, and higher cell aggregation, indicating cell envelope defects. Our work establishes, for the first time, the presence of Ca ²⁺ uptake pumps of the energy-dependent P-type ATPase superfamily in bacteria and also implicates that intracellular Ca ²⁺ is essential for growth and cell envelope integrity in M. smegmatis . IMPORTANCE Ca ²⁺ is essential for gene regulation, enzymatic activity, and maintenance of structural integrity of cell walls in bacteria. Bacteria maintain intracellular calcium concentrations in a narrow range, creating a gradient with low cytoplasmic calcium concentration and high extracellular calcium concentration. Due to this steep gradient, active pumps belonging to family 2 of P-type ATPases and antiporters are used for Ca ²⁺ efflux, whereas Ca ²⁺ uptake is usually carried out by channels. Molecular mechanisms of Ca ²⁺ uptake in bacteria are still elusive and are mainly limited to a nonproteinaceous channel in Escherichia coli and a pH-dependent channel protein from Bacillus subtilis . Energy-dependent active transporters are not reported for Ca ²⁺ uptake from any organism. Here we show that CtpE belonging to a family of previously uncharacterized bacterial P-type ATPases is involved in specific uptake of Ca ²⁺ into Mycobacterium smegmatis . We also demonstrate that intracellular Ca ²⁺ obtained through CtpE is essential for growth and maintenance of cell surface properties under Ca ²⁺ -deficient conditions.