Type III Pilus of Corynebacteria: Pilus Length Is Determined by the Level of Its Major Pilin Subunit

Abstract

Multiple pilus gene clusters have been identified in several gram-positive bacterial genomes sequenced to date, including the Actinomycetales, clostridia, streptococci, and corynebacteria. The genome of Corynebacterium diphtheriae contains three pilus gene clusters, two of which have been previously characterized. Here, we report the characterization of the third pilus encoded by the spaHIG cluster. By using electron microscopy and biochemical analysis, we demonstrate that SpaH forms the pilus shaft, while SpaI decorates the structure and SpaG is largely located at the pilus tip. The assembly of the SpaHIG pilus requires a specific sortase located within the spaHIG pilus gene cluster. Deletion of genes specific for the synthesis and polymerization of the other two pilus types does not affect the SpaHIG pilus. Moreover, SpaH but not SpaI or SpaG is essential for the formation of the filament. When expressed under the control of an inducible promoter, the amount of the SpaH pilin regulates pilus length; no pili are assembled from an SpaH precursor that has an alanine in place of the conserved lysine of the SpaH pilin motif. Thus, the spaHIG pilus gene cluster encodes a pilus structure that is independently assembled and antigenically distinct from other pili of C. diphtheriae. We incorporate these findings in a model of sortase-mediated pilus assembly that may be applicable to many gram-positive pathogens.Over 35 years ago, using electron microscopy of corynebacterial species, Yanagawa and colleagues observed proteinaceous filaments, namely, pili or fimbriae, on the surface of many corynebacterial species (30, 31). Since then, pili have been identified in several gram-positive bacteria (2, 4, 10-13, 19, 21, 29). However, the mechanisms of pilus assembly have remained obscure. It has been proposed that gram-positive bacterial pili are covalently linked to the cell wall peptidoglycan and require sortase (14,20,25,32), a transpeptidase that is found in all gram-positive organisms (5, 17, 23). The available genome sequences have revealed multiple pilus gene clusters with a putative sortase(s) in each of several gram-positive bacterial species, including the Actinomycetales, clostridia, corynebacteria, and streptococci (9, 26). In Corynebacterium diphtheriae, with genes encoding pilin subunits and putative sortases organized into three clusters, it has been shown that the organism assembles two different pilus structures encoded by spaABC and spaDEF gene clusters, designated SpaA-type and SpaD-type pili, respectively (9, 26). It is likely that the third cluster, spaHIG, also encodes a distinct pilus organelle.Previous work has shown that the SpaA-type pilus of corynebacteria is composed of three pilin subunits with SpaA polymers forming the pilus shaft, SpaB observed at regular intervals, and SpaC positioned at the tip (26). Similarly, the SpaD-type pilus is composed of SpaD, SpaE, and SpaF, which are counterparts of SpaA, SpaB, and SpaC, respectively (9). Although C. diphtheriae has six sortase genes, srtA, srtB, srt...

The Corynebacterium diphtheriae shaft pilin SpaA is built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds

“…The general principles of assembly were first established through studies on the SpaA pili expressed by Corynebacterium diphtheriae. These pili, encoded by the gene cluster spaA-spaB-srtA-spaC, comprise a polymeric shaft formed by SpaA, SpaC located at the tip, and SpaB found at the base and occasionally along the shaft (6,(8)(9)(10). In our current model, successive major pilin SpaA subunits are joined by the action of the pilus-specific sortase SrtA.…”

Genomics ofActinobacteria: Tracing the Evolutionary History of an Ancient Phylum

“…Each fimbrial gene cluster is composed of at least one pilin-specific sortase gene (srtA to -E) and three sortase-mediated pilus assembly genes (spaA to -I). Morphological and genetic studies have clearly demonstrated that each pilus of C. diphtheriae is composed of three pilin subunits and that the pilus assembly process requires pilin-specific sortases (60,141,417,435,436). Thus, the genomic islands contribute substantially to the structure of the cell surface.…”

“…Five contain phage-related genes and may be prophage remnants. Several pathogenicity-related functions are encoded in the genomic islands, including a siderophore biosynthesis and export system and a potential lantibiotic biosynthesis system, as well as three sortase-related fimbrial systems (60,141,417,436). Each fimbrial gene cluster is composed of at least one pilin-specific sortase gene (srtA to -E) and three sortase-mediated pilus assembly genes (spaA to -I).…”

Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis

“…A nonexhaustive number of pilus gene clusters also described in the literature and/or present in sequence databases were retrieved manually, i.e. L. rhamnosus (4), Corynebacterium glutamicum (26) (41,42) and C. diphtheriae (43)(44)(45). Their C-terminal domains were subsequently analyzed with the MEME suite (25).…”