FOLLOWING THE AGING PROCESS OF BACILLUS SUBTILIS COLONIES

When grown on a solid surface bacterial cells form complex multicellular colonies, which can survive for long periods of time. However, little is known about the adaptation and the physiology of the colony aging cells. Furthermore, it is unrevealed whether and how the colony population is evolved and modified throughout time. The goal of our research is to characterize the aging process of colonies derived from the sporulating soil bacterium Bacillus subtilis by characterizing physiological and genetic features of the aging cells. Investigation of aging colonies (up to 15 days) revealed that at least 30% of the aging population composed of non sporulating cells, suggesting that this significant portion is able to adapt and acquire mutations. To enrich this adaptive population, we have followed colonies deriving from a non-sporulating strain in the course of days. Utilizing a strain harboring a GFP fusion to the major cell division protein FtsZ, we defined basic features of aging colonies. Furthermore, by re-plating cells of parental aging colonies, we identified progeny colonies with altered morphologies (morphomutants) that became abundant within the surviving population. Whole genome sequence analysis of such morphomutants revealed the majority to contain mutations in genes encoding components necessary for bacterial differentiation, with the key target being the pathway controlling the developmental master regulator Spo0A. Importantly, similar mutations were prevalent in mutants derived from colonies of a sporulating strain. Characterization of these morphomutants revealed that they exhibit a shorter lag phase in comparison to the parental strain upon growth resumption. We propose that the obtained mutants are transition state deficient, unable to differentiate to any of the bacterium developmental states: sporulation, biofilm formation, competence or stationary phase. This deficiency leaves the bacterium captured in an undifferentiated state, capable of rapidly resuming growth when resources become available.