Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor

Abstract

Bacteria employ extracellular appendages called type IV pili (T4P) to interact with their environment. T4P are essential for diverse microbial behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility1. While T4P have been studied extensively, our understanding of these nanomachines largely comes from work on a few model species. Here, we develop Acinetobacter baylyi as a new model organism to study T4P and uncover several unreported mechanisms of T4P regulation. First, using recently-developed T4P-labeling methods2,3, we demonstrate that A. baylyi T4P are synthesized on one side of the cell body along the long axis of the cell, and we uncover that this pattern is dependent on components of a conserved chemosensory pathway. Second, we overturn the current dogma that T4P extension occurs through the action of a single, highly conserved ATP-hydrolyzing motor (ATPase) called PilB by showing that T4P synthesis in A. baylyi is dependent on two partially redundant and phylogenetically distinct motors, PilB and PilB2. Third, we uncover a small protein inhibitor of T4P synthesis that specifically inhibits PilB but not PilB2 activity. Together, these results demonstrate novel mechanisms of T4P regulation, which have broad implications for the unexplored diversity of T4P biology in microbial species.

Pilus Production in Acinetobacter baumannii Is Growth Phase Dependent and Essential for Natural Transformation

“…2C , the bacteria produced pili that, for some cells, extended far beyond the outer membrane. Notably, such extended pili were not restricted to one side of the cell body as recently described for A. baylyi in a preprinted study ( 53 ). The bodies of the cells were also stained by the dye in a significant fraction of the population, which suggested incorporation of the pilin subunit into the inner membrane.…”

“…As shown in Figure 2C, the bacteria produced pili that, for some cells, extended far beyond the outer membrane. Notably, such extended pili were not restricted to one side of the cell body as recently described for A. baylyi in a preprinted study (52).…”

“…4A ), as homologs for these regulatory proteins exist in A. baumannii ( Table 3 ). Notably, Leong et al recently showed that, while important for twitching motility, these signal transduction-related systems (e.g., PilS, PilR, and PilG) were fully dispensable for natural transformation in A. baylyi (strains ADP1 and BD413) ( 48 ), which was recently confirmed for the Pil-Chp system ( 53 ). Given the differences we found in natural transformability and TFP production between A. baumannii and A. baylyi , we were interested in these systems in A. baumannii .…”

Type IV pili share a conserved mechanism of motor-independent retraction that is an inherent property of the pilus filament

“…S4 ). A. baylyi competence pili can be studied using the same cysteine knock-in ( comP T123C ) and maleimide dye labeling approach described above (39), and we can functionally assess competence pilus dynamic activity through natural transformation assays. Unlike V. cholerae , A. baylyi Δ pilTU mutants do not exhibit any residual natural transformation ( Fig.…”

Retraction ATPase motors from three orthologous type IVa pilus systems support promiscuous retraction of the Vibrio cholerae competence pilus

“…Dynamic extension of all T4aP relies on the activity of a dedicated motor ATPase, which is commonly called PilB (2). Some systems also have an additional accessory extension ATPase motor called TfpB (3). Dynamic retraction of T4aP canonically relies on a dedicated retraction ATPase called PilT.…”