Visualizing dynamic competence pili and DNA capture throughout the long axis ofBacillus subtilis

Abstract

AbstractThe first step in the process of bacterial natural transformation is DNA capture. Although long-hypothesized based on genetics and functional experiments, the pilus structure responsible for initial DNA-binding had not yet been visualized forBacillus subtilis. Here, we visualize functional competence pili inBacillus subtilisusing fluorophore-conjugated maleimide labeling in conjunction with epifluorescence microscopy. In strains that produce pilin monomers within ten-fold of wild type levels, the median length of detectable pili is 300nm. These pili are retractile and associate with DNA. Analysis of pilus distribution at the cell surface reveals that they are predominantly located along the long axis of the cell. The distribution is consistent with localization of proteins associated with subsequent transformation steps, DNA-binding and DNA translocation in the cytosol. These data suggest a distributed model forB. subtilistransformation machinery, in which initial steps of DNA capture occur throughout the long axis of the cell and subsequent steps may also occur away from the cell poles.ImportanceThis work provides novel visual evidence for DNA translocation across the cell wall duringBacillus subtilisnatural competence, an essential step in the natural transformation process. Our data demonstrate the existence of natural competence associated, retractile pili that can bind exogenous DNA. Furthermore, we show that pilus biogenesis occurs throughout the cell long axis. These data strongly support DNA translocation occurring all along the lateral cell wall during natural competence, wherein pili are produced, bind to free DNA in the extracellular space, and finally retract to pull the bound DNA through the gap in the cell wall created during pilus biogenesis.