RESUMO
Bacillus subtilis can import DNA from the environment by an uptake machinery that localizes to a single cell pole. We investigated the roles of ComEB and of the ATPase ComGA during the state of competence. We show that ComEB plays an important role during competence, possibly because it is necessary for the recruitment of GomGA to the cell pole. ComEB localizes to the cell poles even upon expression during exponential phase, indicating that it can serve as polar marker. ComEB is also a deoxycytidylate monophosphate (dCMP) deaminase, for the function of which a conserved cysteine residue is important. However, cysteine-mutant ComEB is still capable of natural transformation, while a comEB deletion strain is highly impaired in competence, indicating that ComEB confers two independent functions. Single-molecule tracking (SMT) reveals that both proteins exchange at the cell poles between bound and unbound in a time scale of a few milliseconds, but turnover of ComGA increases during DNA uptake, whereas the mobility of ComEB is not affected. Our data reveal a highly dynamic role of ComGA during DNA uptake and an unusual role for ComEB as a mediator of polar localization, localizing by diffusion-capture on an extremely rapid time scale and functioning as a moonlighting enzyme.
Assuntos
Adenosina Trifosfatases/fisiologia , Bacillus subtilis/fisiologia , Proteínas de Bactérias/fisiologia , DCMP Desaminase/fisiologia , Transformação Bacteriana , Adenosina Trifosfatases/genética , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Polaridade Celular , DCMP Desaminase/genética , DNA Bacteriano , Proteínas de Ligação a DNA , Proteínas de Fluorescência Verde , Mutação , Ligação Proteica , Proteínas Recombinantes de Fusão , Imagem Individual de MoléculaRESUMO
Gemcitabine is a nucleoside analog which exhibits metabolic characteristics that distinguish it from related compounds and may explain its activity in solid tumors. The active nucleotide forms are effectively accumulated to high concentrations in cells. This is due to both efficient phosphorylation and relatively slow elimination. The diphosphate is a potent inhibitor of ribonucleotide reductase, an action that reduces deoxynucleotide pools. Decreased cellular concentrations of deoxycytidine triphosphate permit more rapid phosphorylation of gemcitabine and decreases the metabolic clearance of gemcitabine nucleotides by deoxycytidine monophosphate deaminase. Most importantly, the ratio of the cellular concentrations of gemcitabine triphosphate to deoxycytidine triphosphate increases, favoring analog incorporation into DNA, which is strongly associated with loss of viability.