Social regulation of B. subtilis phenotypic adaptability through Rap phosphatases
Rap phosphatases fine-tune the sociomicrobiology of B. subtilisby modulating the activity of the master regulators Spo0A, ComA and DegU and therefore their regulons (Fig. 2). This fine-tuning occurs at different levels: first, the Rap phosphatases themselves that may appear at first sight to have directly redundant regulatory roles, are expressed under different conditions. This leads to regulatory differences among Rap proteins with the same target. As an example, both RapA and RapB dephosphorylate Spo0F∼P, however, rapA expression is promoted by QS-dependent ComA, while rapB seems to be promoted only by the house-keeping sigma factor σA . This difference means that RapB will be produced earlier and more consistently than RapA, leading to differences in the Spo0A/Spo0A∼P ratio in the cell population. A second level of fine-tuning is given by the Phr peptides, which may comprise the most diverse family of QS autoinducers known to date . QS systems are used to coordinate social behavior that is most effective if a large number of cells participate, such as expression of virulence, or production of a biofilm matrix. The different Rap-Phr pairs have distinct influence on biofilm development of B. subtilis and colonization of the roots of the model plantArabidopsis thaliana . In biofilm settings, the process-export-import regulatory pathway of Phr peptides providesB. subtilis cells with the opportunity to detect and integrate further environmental signals into their complex gene regulatory network. Cells in a biofilm live in micro-niches that lead to population heterogeneity , thus, the biofilm subpopulations will secrete different types and amounts of mature Phr peptides to the extracellular milieu. Furthermore, the flexibility of Phr peptides to serve as cell-cell communication signals has been demonstrated by the ability of non-producing cells to detect the Phr signals produced by other cell . Therefore, Phr peptides can form a biofilm-spanning communication network, where each biofilm subpopulation can participate in the developmental process of their neighbors.
B. subtilis is an environmentally ubiquitous bacterium, with numerous strains obtained from soil, animals, plants and aquatic habitats . Interestingly, although B. subtilis strains commonly show conservation among their main population heterogeneity regulators (Spo0A, ComA, DegU) , they show high variation among their Rap-Phr cassette content . Rap phosphatases determine the phenotypic memory ofB. subtilis spores, the timing of spore formation and germination speed: the earlier the spores are formed, the faster these spores germinate driven by higher level of alanine dehydrogenase (i.e. high-quality spores), while delayed establishment of spores lead to higher number of spores in the population with reduced revival ability (i.e. high quantity spores) . Overexpression of kinA gene decelerates sporulation and therefore increase the spores yield, however, this gives rise to a lower fraction of spores growing out . The differences in the number and the diversity of the Rap-Phr family play an important role for the environmental adaptability of specific strains of B. subtilis by allowing them to fine-tune their metabolism to different ecological niches. For example, strains isolated from the digestive tract of animals can show differences in the timing of sporulation initiation influenced by Rap-Phr cassette variation. This variation effectively serves as an adaptation that allows them to sporulate at optimum rates according to the ecological niche in which they live . Ultimately, these differences in the amount of Rap phosphatases and the timing of sporulation eventually influence the quality-yield spore tradeoff in natural isolates .
Motivated by these observations related to the divergence inrap -phr modules and their influence on timing of sporulation, Gallegos-Monterrosa and colleagues constructed all possible combinations of single and double rap -phr deletions inB. subtilis NCIB 3610 and tested the fitness of these strains in a selection experiment . A mixture of the single and double mutants along with the wild-type strain was cultivated either as biofilms or as plaktonic cultures for 2- or 5-days, and spores were selected for re-inoculation again. After 9 cycles of spore selection, the abundance of strains in each culture condition was examined, which revealed that a shorter incubation time selects for higher diversity of strains, while longer cultivation selects for specific combination of rap-phrdeletions . Additionally, fitness assays using the mutants that were selected under these conditions, e.g. derivative carrying a rapBor rapH deletion in addition to other rap -phrmutation, demonstrated the competitiveness of these strains compared to the wild-type strain. However, as these evolution experiments included spore selection, the cryptic phi3T prophage and its encoded Rap protein could have potentially influenced the selection dynamics . Nevertheless, this experimental approach highlighted the complexity of how the different Rap-Phr systems contribute to fitness of B. subtilis .