Analysis ofBrevundimonas subvibrioidesdevelopmental signaling systems reveals unexpected differences between phenotypes and c-di-GMP levels

The DivJ-DivK-PleC signaling system of Caulobacter crescentus is a signaling network that regulates polar development and the cell cycle. This system is conserved in related bacteria, including the sister genus Brevundimonas . Previous studies had shown unexpected phenotypic differences between the C. crescentus divK mutant and the analogous mutant of Brevundimonas subvibrioides , but further characterization was not performed. Here, phenotypic assays analyzing motility, adhesion, and pilus production (the latter characterized by a newly discovered bacteriophage) revealed that divJ and pleC mutants have mostly similar phenotypes as their C. crescentus homologs, but divK mutants maintain largely opposite phenotypes than expected. Suppressor mutations of the B. subvibrioides divK motility defect were involved in cyclic-di-GMP (c-di-GMP) signaling, including the diguanylate cyclase dgcB , and cleD which is hypothesized to affect flagellar function in a c-di-GMP dependent fashion. However, the screen did not identify the diguanylate cyclase pleD. Disruption of pleD in B. subvibrioides caused hypermotility in wild-type, but not in the divK background. Analysis of c-di-GMP levels in these strains revealed incongruities between c-di-GMP levels and displayed phenotypes with a notable result that suppressor mutations altered phenotypes but had little impact on c-di-GMP levels in the divK background. Conversely, when c-di-GMP levels were artificially manipulated, alterations of c-di-GMP levels in the divK strain had minimal impact on phenotypes. These results suggest that DivK performs a critical function in the integration of c-di-GMP signaling into the B. subvibrioides cell cycle. Importance Cyclic-di-GMP signaling is one of the most broadly conserved signaling systems in bacteria, but there is little understanding of how this system directly affects the physiology of the organism. In C. crescentus , c-di-GMP has been integrated into the developmental cell cycle, but there is increasing evidence that environmental factors can impact this system as well. The research presented here suggests that developmental signaling could impact physiological Processes in c-di-GMP dependent and independent ways. This hints that the integration of these signaling networks could be more complex than previously hypothesized, which could have a bearing on the larger field of c-di-GMP signaling. In addition, this work further examines how much models developed in one organism can be extrapolated to related organisms.