Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis . In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis . We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins, i.e. a methylated N terminus; (iii) are found in the same heteropolymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin-fold with a highly unusual flexible C terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff than bona fide Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that S. sanguinis Tfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.

The twin-arginine protein translocation (Tat) system has a unique ability to translocate folded and co-factor-containing proteins across lipid bilayers. The Tat pathway is present in bacteria, archaea and in the thylakoid membranes of chloroplasts and, depending on the organism and environmental conditions, it can be deemed important for cell survival, virulence or bioproduction. This review provides an overview of the current understanding of the Tat system with specific focus on Gram-positive bacteria. The 'universal minimal Tat system' is composed of a TatA and a TatC protein. However, this pathway is more commonly composed of two TatA-like proteins and one TatC protein. Often the TatA-like proteins have diverged to have two different functions and, in this case, the second TatA-like protein is usually referred to as TatB. The correct folding and/or incorporation of co-factors are requirements for translocation, and the known quality control mechanisms are examined in this review. A number of examples of crosstalk between the Tat system and other protein transport systems, such as the Sec-YidC translocon and signal peptidases or sheddases are also discussed. Further, an overview of specific Gram-positive bacterial Tat systems found in monoderm and diderm species is detailed. Altogether, this review highlights the unique features of Gram-positive bacterial Tat systems and pinpoints key questions that remain to be addressed in future research. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Gram-positive bacteria are known to export many proteins to the cell wall and growth medium, and accordingly, many studies have addressed the respective protein export mechanisms. In contrast, very little is known about the subsequent fate of these proteins. The present studies were therefore aimed at determining the fate of native exported proteins in the model organism Bacillus subtilis. Specifically, we employed a gel electrophoresis-based liquid chromatography-mass spectrometry approach to distinguish the roles of the membrane-associated quality control proteases HtrA and HtrB from those of eight other proteases that are present in the cell wall and/or growth medium of B. subtilis. Notably, HtrA and HtrB were previously shown to counteract potentially detrimental "protein export stresses" upon overproduction of membrane or secreted proteins. Our results show that many secreted proteins, lipoproteins, and membrane proteins of B. subtilis are potential substrates of extracytoplasmic proteases. Moreover, potentially important roles of HtrA and HtrB in the folding of native secreted proteins into a protease-resistant conformation, the liberation of lipoproteins from the membrane-cell wall interface, and the degradation of membrane proteins are uncovered. Altogether, our observations show that HtrA and HtrB are crucial for maintaining the integrity of the B. subtilis cell even under nonstress conditions.