Abstract
SpoIIIE directionally pumps DNA across membranes during Bacillus subtilis sporulation and vegetative growth. The sequence-reading domain (γ domain) is required for directional DNA transport, and its deletion severely impairs sporulation. We selected suppressors of the spoIIIEΔγ sporulation defect. Unexpectedly, many suppressors were intragenic missense mutants, and some restore sporulation to near-wild-type levels. The mutant proteins are likely not more abundant, faster at translocating DNA, or sequence-sensitive, and rescue does not involve the SpoIIIE homolog SftA. Some mutants behave differently when co-expressed with spoIIIEΔγ, consistent with the idea that some, but not all, variants may form mixed oligomers. In full-length spoIIIE, these mutations do not affect sporulation, and yet the corresponding residues are rarely found in other SpoIIIE/FtsK family members. The suppressors do not rescue chromosome translocation defects during vegetative growth, indicating that the role of the γ domain cannot be fully replaced by these mutations. We present two models consistent with our findings: that the suppressors commit to transport in one arbitrarily-determined direction or delay spore development. It is surprising that missense mutations somehow rescue loss of an entire domain with a complex function, and this raises new questions about the mechanism by which SpoIIIE pumps DNA and the roles SpoIIIE plays in vivo.
Highlights
Impairing a protein and looking for ways in which its function can be restored can lead to new discoveries about how the protein works
We set out to learn more about the role of the SpoIIIE γ domain in sporulating cells by exploring how cells would cope with its absence
We selected suppressors of the sporulation defect in spoIIIEΔγ strains by successive rounds of sporulation and outgrowth and found that spontaneously arising intragenic mutations could rescue sporulation to near-wild-type levels
Summary
Impairing a protein and looking for ways in which its function can be restored can lead to new discoveries about how the protein works. In the absence of that dedicated DNA-reading domain, missense mutations elsewhere in the protein somehow compensate for a defect which normally results from sequence insensitivity These simple mutations surprisingly rescue the loss of a complex domain function, and uncover new questions about the role SpoIIIE plays in sporulation, a process for which it is essential. The SpoIIIE γ domain detects 8-nucleotide motifs called SRSs (SpoIIIE-Recognition Sequences) to determine directionality of DNA transport [33] These types of sequences are highly conserved, and their orientation on the B. subtilis chromosome is extremely biased; 82% are found on the leading strand [33, 36, 37]. We propose two models for how the suppressors might function that are consistent with these observations and that highlight how the mutants could be used to further explore and understand the role of SpoIIIE in sporulation
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