Natural transformation protein ComFA exhibits single-stranded DNA translocase activity

Abstract

SummaryNatural transformation is one of the major mechanisms of horizontal gene transfer in bacterial populations and has been demonstrated in numerous species of bacteria. Despite the prevalence of natural transformation, much of the molecular mechanism remains unexplored. One major outstanding question is how the cell powers DNA import, which is rapid and highly processive. ComFA is one of a handful of proteins required for natural transformation in gram-positive bacteria. Its structural resemblance to the DEADbox helicase family has led to a long-held hypothesis that ComFA acts as a motor to help drive DNA import into the cytosol. Here, we explored the helicase and translocase activity of ComFA to address this hypothesis. We followed the DNA-dependent ATPase activity of ComFA and, combined with mathematical modeling, demonstrated that ComFA likely translocates on single-stranded DNA from 5’ to 3’. However, this translocase activity does not lead to DNA unwinding in the conditions we tested. Further, we analyzed the ATPase cycle of ComFA and found that ATP hydrolysis stimulates the release of DNA, providing a potential mechanism for translocation. These findings help define the molecular contribution of ComFA to natural transformation and support the conclusion that ComFA plays a key role in powering DNA uptake.ImportanceCompetence, or the ability of bacteria to take up and incorporate foreign DNA in a process called natural transformation is common in the bacterial kingdom, but understanding of the mechanism is still limited. Increasing evidence in several bacteria confirms that long, contiguous stretches of DNA are imported into cells, and yet how bacteria power processive transformation remains unclear. Our finding that ComFA, a DExD-box helicase required for competence in gram-positive bacteria, translocates on single-stranded DNA from 5’ to 3’, supports the long held hypothesis that ComFA may be the motor powering DNA transport during natural transformation. Moreover, ComFA may be a previously unidentified type of DExD-box helicase—one with the capability of extended translocation on single-stranded DNA.