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 .