Distribution and diversity of the Rap-Phr family in theBacillus genus
The Rap-Phr family of regulatory proteins is highly diversified and widespread in the Bacillus genus. Rap homologs have been found in the genomes of B. subtilis , Bacillus amyloliquefaciens,Bacillus pumilus, Bacillus anthracis, Bacillus cereus, Bacillus halodurans , Bacillus stearothermophilus, and Bacillus clausii . Also, one of these Rap-Phr cassettes has been studied heterologously in another Bacillus species (RapQ-PhrQ), and was found to be both functional and comparable to the native Rap phosphatases of the host . These reports support the idea that the Rap proteins and their Phr peptides play a common regulatory role in the entire Bacillus genus.
Interestingly, Bacillus species that have at least one Rap-Phr cassette normally possess multiple of these regulatory elements encoded in their genomes. The first study to identify RapA as a Spo0F phosphatase already established RapA and RapB as members of a protein-aspartate phosphatase family with multiple members within the same organism: B. subtilis strain JH642 . Subsequent studies have identified further members of this phosphatase family in diverseB. subtilis strains (see Table 1). In 2016, Even-Tov et al . compared over 400 Bacillus genomes, searching for Rap homologs based on their conserved N-terminal 3-helix bundle and C-terminal TPR domain structure. They found over 2500 raphomologs among the studied genomes, and that B. subtilis strains have, on average, 11 rap genes per strain, while B. cereusstrains usually possess around 6 of these phosphatases .
How has the Rap-Phr family achieved such a widespread presence and diversity among Bacilli ? There are two main factors that can be considered when answering this question. First, bacteria commonly pass on genes among sibling cells or cells from closely related species. This ability, known as horizontal gene transfer (HGT), is an efficient mechanism for individual organisms to acquire genes, regardless of functionality . HGT of Rap-Phr cassettes is heightened due to the fact that many rap-phr genes are encoded within mobile genetic elements . In addition, the genes related to natural competence for the uptake of DNA from the environment are widely conserved inBacilli . Even-Tov et al . recently estimated that up to 75% of Rap-Phr cassettes may be mobile, based on a GC-content comparison with their host strain . Furthermore, some Rap-Phr cassettes are able to regulate the mobility of the genetic element that contains them, be them plasmids , or transposons . These features could then favor a rapid expansion of Rap-Phr cassettes through HGT amongBacilli . Similarly, experimental selection for spores of B. subtilis increases the copy number of a cryptic prophage, phi3T, that carries Rap and Phr proteins . Interestingly, certain prophages, like SPβ, that are similar to phi3T do not carry such rap gene, but encode a biosynthetic gene cluster for a bacteriocin that presumably benefit the fitness of the host bacterium . The Rap protein coded within the phi3T prophage has been also hypothesized to contribute to phage fitness . Further, genome analysis combined with targeted experimental validation revealed that diversification of the autoinducer Phr peptides might be driven by promiscuous duplication events followed by adjustment of the Phr peptide in accordance with the respective evolutionary change of its cognate Rap phosphatase .
A second factor that can help explain the diversity of the Rap-Phr family is functional diversification through social selection. Experimental analyses and modeling suggest that acquisition of additional Rap-Phr system is facilitated by a facultative social cheating mechanism in B. subtilis . At low frequency, a strain harbouring an extra Rap-Phr system acts a cheater (i.e. exploiting the public good produced by the wild type), while at high frequency it returns to cooperation without fitness loss. Such social selection processes in combination with HGT ensure the diversification and maintainance of multiple copies of Rap-Phr systems in Bacilli .