Haloferax Volcanii Strategies to Regulate Type IV Pilus Dependent Adhesion and Microcolony Formation

Microorganisms can utilize type IV pili to initiate and maintain biofilms microbial communities that provide protection against stressful conditions. Because environmental conditions change suddenly, microorganisms have evolved multiple mechanisms to rapidly transition from a planktonic to sessile cell state. Despite the presence of archaea alongside bacteria throughout the environment, including the human microbiome, little is known about how these organisms form and maintain biofilms. Here we use genetic, microscopic and biochemical techniques to investigate multiple strategies the model archaeon Haloferax volcanii employs to permit effective adhesion and microcolony formation, early steps in biofilm formation and maturation, as well as eventual dispersal from biofilms. First, we identified six pilins, PilA1-6, each with a highly conserved hydrophobic stretch (H-domain). Each of these pilins can adhere to surfaces when expressed individually in trans but with diverse phenotypes. PilA1 and PilA2, which in wild-type planktonic cells appear to be the most abundant pilins, adhere less well than wild-type, while PilA3 and PilA4 adhere better. Conversely, PilA5 and PilA6 form microcolonies significantly earlier than wild-type. We furthermore showed that N-glycosylation, dependent on the oligosaccharyltransferase, AglB, regulates the functions of these pilins. A mutant lacking all six pilins, ΔpilA[1-6], has a severe motility defect that can be complemented by expression of any individual PilA pilin in trans. Surprisingly, a hybrid protein containing only the H-domain of the PilA pilins could restore motility in the ΔpilA[1-6] strain, contributing to a model in which pilins sequester a motility inhibitor within the membrane of planktonic cells. Motility was also shown to be regulated by the flagellin FlgA2. Strains lacking flgA2 are hypermotile with longer, more abundant flagella, implicating FlgA2 as an additional factor in inhibiting dispersal from biofilms. From these results, we have demonstrated numerous mechanisms to regulate biofilm formation and dispersal in Hfx. volcanii. These novel mechanisms, some of which are likely conserved across the bacterial and archaeal domains, will advance our understanding of critically understudied members of the microbiome. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Mecky Pohlschroder