Structure and Properties of a Natural Competence-Associated Pilin Suggest a Unique Pilus Tip-Associated DNA Receptor

Abstract

Natural competence is the term used to describe the uptake of “naked” extracellular DNA by bacteria; it plays a significant role in horizontal genetic exchange. It is associated with type IV pili, and specialized competence pili mediate DNA uptake. Here, we show that the crystal structure of a competence-associated protein from Thermus thermophilus, ComZ, consists of a type II secretion pseudopilin-like domain, with a large β-solenoid domain inserted into the β-sheet of the pilin-like fold. ComZ binds with high affinity to another competence-associated pilin, PilA2, which lies adjacent to the comZ gene in the genome. The crystal structure of PilA2 revealed a similar type II secretion pseudopilin-like fold, with a small subdomain; docking simulations predicted that PilA2 binds between the pseudopilin-like and β-solenoid domains of ComZ. Electrophoretic shift analysis and DNase protection studies were used to show that ComZ alone and the ComZ/PilA2 complex are able to bind DNA. Protection against reductive dimethylation was used in combination with mass spectrometry and site-directed mutagenesis to identify two lysine residues in ComZ which are involved in DNA binding. They are located between the two domains in ComZ, on the opposite side from the predicted PilA2 binding site. These results suggest a model in which PilA2 assists ComZ in forming the competence pilus tip and DNA binds to the side of the fiber. The results demonstrate how a type IV pilin can be adapted to a specific function by domain insertion and provide the first structural insights into a tip-located competence pilin. IMPORTANCE Thermus thermophilus is a thermophilic bacterium which is capable of natural transformation, the uptake of external DNA with high efficiency. DNA uptake is thought to be mediated by a competence-associated pilus, which binds the DNA substrate and mediates its transfer across the outer membrane and periplasm. Here, we describe the structural and functional analysis of two pilins which are known to be essential for DNA uptake, ComZ and PilA2. ComZ adopts an unusual structure, incorporating a large β-solenoid domain into the pilin structural framework. We argue on structural grounds that this structure cannot readily be accommodated into the competence pilus fiber unless it is at the tip. We also show that ComZ binds DNA and identify two lysine residues which appear to be important for DNA binding. These results suggest a model in which ComZ and PilA2 form a tip-associated DNA receptor which mediates DNA uptake.

Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium

“…9). However, recent studies have shown that the tip of the filament is important for DNA binding in Vibrio cholerae 49 , and it has been suggested that the Thermus filament cap may be comprised of the minor pilin PilA2 and ComZ, a DNA-binding protein 50 . In addition, functional experiments in Thermus have shown that pili are not absolutely required for natural transformation.…”

Structure and function of minor pilins of type IV pili

“…With the emergence of antibiotic resistance, T4P are considered as a target for innovative antibacterial therapeutics [61]. T4aP of EHEC, among others, have been shown to be involved in biofilm formation, twitching motility and adherence [60]. The core minor pilins that likely form the tip of the pilus might play multiple roles, being at the same time responsible for the initiation of pilus formation, capping the pilus, and mediate specific biological functions.…”

“…Some non-core minor pilins have large structural insertions. The recently determined structure of the minor T4a pilin ComZ from T. thermophilus [60] shows an unusual structure with two domains, a pilin-like domain and a large additional domain inserted into its β-sheet. Hence, although not involved in pilus assembly, this minor pilin is likely located at the tip of the pilus, since the structural organization and the large size of this additional domain cannot allow upward incorporation of major pilins.…”

The Role of the Cyanobacterial Type IV Pilus Machinery in Finding and Maintaining a Favourable Environment

“…The uptake of exogenous DNA, known as natural competence, allows cells to adapt to environmental conditions through the exchange of situationally useful genes. Competence is known to depend on functional T4P in several bacteria [ 81 , 127 ], with DNA binding by T4P likely occurring at the pilus tip [ 128 , 129 ].…”