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

Abstract

Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility. Dynamic extension and retraction of T4P is essential for their function, and T4P extension is thought to occur through the action of a single, highly conserved motor, PilB. Here, we develop Acinetobacter baylyi as a model to study T4P by employing a recently developed pilus labeling method. By contrast to previous studies of other bacterial species, we find that T4P synthesis in A. baylyi is dependent not only on PilB but also on an additional, phylogenetically distinct motor, TfpB. Furthermore, we identify a protein (CpiA) that inhibits T4P extension by specifically binding and inhibiting PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to disrupt T4P synthesis.

Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA

“…While the frequency and timing of pilus extension and retraction are crucial to their function, many questions remain about how pilus dynamics are regulated. Some progress has been made on this front, including the characterization of regulators that impact dynamic activity ( 39 – 42 ) and the impact of spatial organization of the extension and retraction motors ( 43 – 45 ). Our study highlights another mode of regulation for pilus dynamic activity, wherein environmental regulatory cues rapidly induce pilus retraction through the modulation of a secondary messenger (c-di-GMP) that is required for pilus biogenesis.…”

Subcellular localization of type IV pili regulates bacterial multicellular development

“…To identify factors involved in the unusual linear long-axis T4P localization of A. baylyi , we examined T4P in a collection of mutants in genes associated with T4P function in other species 7 . Many of these mutants displayed no T4P, inhibiting analysis of their effects on T4P localization, but one subset of mutants disrupted long-axis localization and instead resulted in dispersed T4P.…”

“…2b ). To more directly test whether T4P synthesis is regulated by Pil-Chp components, we performed natural transformation assays as prior work established that T4P are essential for DNA uptake in A. baylyi 7 , 14 . Pil-Chp mutants exhibited equal levels of natural transformation compared to the parent (Fig.…”

“…To date, T4P have only been reported to localize either to the cell poles or dispersed around the cell body, and no role for these differences in multicellular organization has been identified. We recently developed the gram-negative coccobacillus-shaped bacterium Acinetobacter baylyi as a model for T4P study by introducing a cysteine residue into the major pilin ComP for subsequent labeling with fluorescent, thiol-reactive maleimide dyes 7 . A. baylyi is a soil-dwelling species that is closely related to emerging pathogens like Acinetobacter baumannii , and both use their T4P to take up DNA from the environment to acquire antibiotic resistance (AbR) genes 7 , 8 .…”

“…We recently developed the gram-negative coccobacillus-shaped bacterium Acinetobacter baylyi as a model for T4P study by introducing a cysteine residue into the major pilin ComP for subsequent labeling with fluorescent, thiol-reactive maleimide dyes 7 . A. baylyi is a soil-dwelling species that is closely related to emerging pathogens like Acinetobacter baumannii , and both use their T4P to take up DNA from the environment to acquire antibiotic resistance (AbR) genes 7 , 8 . Upon fluorescently labeling T4P in this species, we noticed that T4P are produced along a line that is parallel to the long axis of the cell body (Fig.…”