MsmX as model for functional studies of Multitask ATPases from pathogenic bacteria

Abstract

The ABC-type transporters constitute one of the largest and most diverse transporter superfamilies characterized by a highly conserved ATP-binding cassette, and are widespread among all domains of life. Recent studies, performed in our laboratory demonstrated that MsmX ATPase from Bacillus subtilis interacts with several distinct ABC sugar importers thus, unlike other NBDs MsmX was shown to be multitask serving as energy-generating component to several sugar importers. Sharing of an ATPase among carbohydrate ABC transporters in both Gram-positive and Gram-negative bacteria seems to be a common strategy for adaption and survival and may represent novel therapeutic approaches for targeting since ABC importers are exclusive to prokaryotes. To characterize multipurpose ATPases and to assess their intra- and interspecies interchangeability, we fine-tuned a genetic system in B. subtilis for controlled ectopic gene expression. The functionality of distinct multitask ATPases alleles was determined by their ability to complement the role of MsmX in a B. subtilis msmX-null mutant. Moreover, this genetic system allowed the determination of intracellular accumulation of the tested ATPases by Western-Blot analysis. The results show that an ATPase from B. thuringiensis was able to fulfill the role of MsmX in its absence, while another ATPase from B. subtilis YurJ was only able to partially play MsmX role. In addition to intra- and interspecies interchangeability of Bacillus ATPases, we found that ATPases from Streptococcus pneumoniae and Staphylococcus aureus were also able to complement to a certain degree the B. subtilis MsmX function in vivo. In contrast ATPases from the Gram-negative bacterium Escherichia coli were not functional in B. subtilis. Furthermore, all the tested ATPases accumulate in the cells. Our study shows that B. subtilis can be use as model for the study of bacterial multitask ATPases. Furthermore, it provides a genetic tool for the characterization of this phenomenon in bacterial carbohydrate transport and particularly in bacterial pathogens