Multicellular development in Bacillus subtilis

Wild type B. subtilis strain A164 (ATCC 6051a) exhibits robust social behaviors, which include swarming, biofilm formation, and the production of aerial structures known as fruiting bodies. These behaviors require cooperative intercellular interactions that are controlled by a sophisticated network of regulatory pathways. This innate complexity led to the hypothesis that B. subtilis evolved overlapping control mechanisms to coordinate distinct social behaviors. To investigate this hypothesis, I attempted to discover new factors that functioned in specific multicellular behaviors, and study how these factors influenced other forms of multicellular development. My first aim was to identify extracellular factors necessary for swarming in B. subtilis A164. By examining a set of strains, containing ten sequential deletions, I established that extracellular proteolytic activity has a role in initiating swarming. In addition, I found that the requirement for extracellular proteases was alleviated by the biosurfactant, surfactin. Finally, I discovered that in addition to its swarming phenotype, a strain deficient for the production of extracellular proteases and surfactin, also manifested deficiencies in biofilm formation. My second aim was to determine if the Ser/Thr kinase, PrkC, which has been implicated in biofilm formation, functions in other forms of multicellular development. While loss of PrkC function had no apparent affect on other multicellular behaviors, I discovered the altered expression of yloQ, a gene downstream of prkC, influenced fruiting body formation, swarm migration and biofilm development. The gene yloQ encodes a small GTPase that has an essential, but undefined role in cell growth. To identify other genes involved in the genetic program(s) affected by a change in yloQ expression, I used an insertional mutagenesis approach. Screening for mutations that suppressed the effect of altered yloQ expression, I characterized three genes involved in fruiting body formation. These are nusG (encoding a transcription antiterminator factor), med (encoding a cell surface protein and positive regulator of comK expression), and pyrB (encoding aspartate carbamoyltransferase). My discovery that distinct social behaviors are influenced by protease activity as well as altered yloQ expression, lends support to the hypothesis that in B. subtilis multicellular developmental processes are at least partly controlled by a related means.