The Bacillus subtilis dCMP deaminase ComEB acts as a dynamic polar localization factor for ComGA within the competence machinery

Abstract

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.

ComEB protein is dispensable for the transformation but must be translated for the optimal synthesis of comEC

“…In several experiments with this construct, a low level of transformation was measured, about 40-fold lower than that of the wild-type strain, but still well above the transformation frequency of a ΔcomEC mutant, which is completely deficient (Table 1). This result confirms the finding of Burghard-Schrod et al (2020), which led to the conclusion that the ComEB protein is needed for transformation, admitting two possible interpretations. Either ComEB protein is needed for transformation or the deletion has removed sequences that are essential for the expression of comEC.…”

“…This arrangement suggests that a ribosomal-binding site (RBS) for comEC may be embedded in the coding sequence of comEB and even that the two genes may be translationally coupled, so that the translation of the comEB coding sequence is required for the translation of comEC as suggested previously (Inamine & Dubnau, 1995). The transformation deficient ΔcomEB::tet construct used by Burghard-Schrod et al (2020) removed the entire comEB reading frame including any sequences embedded in comEB that might contain an RBS for comEC translation.…”

“…We also show that the comEB and comEC open reading frames are translationally coupled, explaining why deletion of comEB causes a loss of transformability. Finally, we speculate as to the reason for coupling and for why ComEB, a dCMP deaminase (Burghard-Schrod et al, 2020, Oehlenschlaeger et al, 2015, is expressed by cells that are competent for transformation.…”

“…Although this conclusion is consistent with the absence of comEB in some transformable bacteria that encode ComEA and ComEC, it remained possible that the major portion of ComEA still encoded by comEB Δ84 was sufficient for normal transformability. A recent report that ComEB plays an important role in transformation (Burghard-Schrod et al, 2020) suggests that this may indeed be the case and caused us to re-examine the role of this protein.…”

“…ComEA is a DNA‐binding protein that participates in the uptake of transforming DNA to the periplasm (Gangel et al., 2014; Matthey & Blokesch, 2016; Provvedi & Dubnau, 1999; Schwarzenlander et al., 2009), while ComEC is a large, multipass membrane protein that most likely forms a channel for DNA transport to the cytoplasm (Draskovic & Dubnau, 2005). In Bacillus subtilis and in many other firmicutes, the comE operon also encodes ComEB, a dCMP deaminase (Burghard‐Schrod et al., 2020; Oehlenschlaeger et al., 2015), while in other bacteria only comEA and comEC are present. It has been suggested that ComEB is dispensable for the transformation of B. subtilis , because a short in‐frame deletion ( comEB Δ84 ), removing 28 out of 189 codons from the 5ʹ end of the gene, had no effect on the transformability (Inamine & Dubnau, 1995).…”