ComEA homologs from different naturally competent bacteria compensate for the absence of ComEA in <i>V. cholerae</i>.

<p>First row: Schematic representation of the ComEA protein of <i>V. cholerae</i> from amino acid 1 to the end. Domains such as the helix-hairpin-helix motifs (HhH) and the signal sequence (signal) are also depicted. The signal sequence cleavage site (scissor) was predicted through the SignalP 4.1 server <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Petersen1" target="_blank">[73]</a> for ComEA<i>^V.c.</i> and for its homologs. Designed constructs replacing wild-type ComEA on the chromosome of the respective <i>V. cholerae</i> strains are indicated below the WT ComEA scheme. First, the two ComEA mutants lacking either of both HhH motifs are indicated. In gray: the ComEA protein of <i>B. subtilis</i> (<i>B.s.</i>; not to scale compared to the other constructs and as indicated by the two diagonal lines). The transmembrane domain of ComEA<i>^B.s.</i> was removed to avoid toxicity/insolubility problems <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Provvedi1" target="_blank">[21]</a>. ComEA(C-term) of <i>B. subtilis</i> refers to the C-terminal part of the protein, which still allowed DNA binding <i>in vitro </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Provvedi1" target="_blank">[21]</a>. Both ComEA<i>^B.s.</i> and ComEA(C-term)<i>^B.s.</i> were tested without and with mCherry fused to the C-terminus (− mCherry/ + mCherry). In white: ComEA homologs (ComE1) of the Gram-negative bacteria <i>Neisseria gonorrhoeae</i> (<i>N.g.</i>), <i>Haemophilus influenzae</i> (<i>H.i.</i>), and <i>Pasteurella multocida</i> (<i>P.m.</i>). The original signal sequence of each of those proteins was removed. All constructs were expressed from the native <i>comEA</i> promoter and encoded the <i>V. cholerae</i> ComEA-specific signal sequence (residues 1–25) to allow proper translocation across the inner membrane. Natural transformation was tested for all strains and the transformation frequencies (TF) are indicated on the right. The average of at least three independent biological replicates is shown (± SD).