Diversification of the type IV filament superfamily into machines for adhesion, protein secretion, DNA uptake, and motility

Abstract

Processes of molecular innovation require tinkering and shifting in the function of existing genes. How this occurs in terms of molecular evolution at long evolutionary scales remains poorly understood. Here, we analyse the natural history of a vast group of membrane-associated molecular systems in Bacteria and Archaea-the type IV filament (TFF) superfamily-that diversified in systems involved in flagellar or twitching motility, adhesion, protein secretion, and DNA uptake. The phylogeny of the thousands of detected systems suggests they may have been present in the last universal common ancestor. From there, two lineages-a bacterial and an archaeal-diversified by multiple gene duplications, gene fissions and deletions, and accretion of novel components. Surprisingly, we find that the 'tight adherence' (Tad) systems originated from the interkingdom transfer from Archaea to Bacteria of a system resembling the 'EppA-dependent' (Epd) pilus and were associated with the acquisition of a secretin. The phylogeny and content of ancestral systems suggest that initial bacterial pili were engaged in cell motility and/or DNA uptake. In contrast, specialised protein secretion systems arose several times independently and much later in natural history. The functional diversification of the TFF superfamily was accompanied by genetic rearrangements with implications for genetic regulation and horizontal gene transfer: systems encoded in fewer loci were more frequently exchanged between taxa. This may have contributed to their rapid evolution and spread across Bacteria and Archaea. Hence, the evolutionary history of the superfamily reveals an impressive catalogue of molecular evolution mechanisms that resulted in remarkable functional innovation and specialisation from a relatively small set of components.

Fig 1. Schematic representation of the different systems and associated genes.

Homologous components are represented in the same colour. The table below the drawing indicates the colour code and the name of the different components in each type of system. For the Archaeal-T4P, the representation of the systems is based on the representation of the archaellum, and the genes mentioned in the legend are the names of the genes used in the literature (not the arCOG database’s names). Some systems have multiple homologues of the ATPase, and these are shown as multiple clusters in the figure (with same shape and colour). Archael-T4P, type IV-related pili in Archaea; arCOG, archaeal Cluster of Orthologous Genes; Com, competence pilus; IM, integral membrane; MSH, mannose-sensitive hemagglutinin pilus; SDA, secretin-dynamic–associated; Tad, tight adherence; TFF, type IV filament; T2SS, type II protein secretion system; T4aP, type IVa pilus; T4bP, type IVb pilus.

Fig 2. Results of the HMM–HMM alignments (HHSearch) between all the components of the TFF superfamily.

The colour of the nodes represents the known or predicted function of the protein. The size of the outlines is proportional to the frequency of the profiles in the detected systems (thicker outlines indicate higher frequencies). Com, competence pilus; HMM, hidden Markov model; IM, integral membrane; MSH, mannose-sensitive hemagglutinin pilus; Tad, tight adherence; TFF, type IV filament.

Fig 3. Rooted phylogeny of the TFF superfamily.

The tree was built with the concatenate of the IM platform (using TadC) and the AAA+ ATPase (using PilB). The branches are in green if the ultrafast bootstrap is >95%. The supports of the significant nodes are indicated in text. The different coloured strips indicate the classification of the systems with the MacSyFinder annotation (with the initial model and with the final one) and the annotation of the systems in the literature. The systems known to be implicated in natural transformation are indicated in dark purple. Known subtypes of Archaeal-T4P are indicate by text in red. The tree was built using IQ-Tree, 10,000 replicates of UFBoot, with a partition model. Halo pilus indicates two pili characterised in Halobacteria. Aap, adhesive archaeal pilus; Archaeal-T4P, type IV-related pili in Archaea; ComM, competence pilus of monoderms; Epd, EppA dependent; IM, integral membrane; MSH, mannose-sensitive hemagglutinin pilus; Tad, tight adherence; TFF, type IV filament; T2SS, type II protein secretion system; T4aP, type IVa pilus; T4bP, type IVb pilus; UFBoot, Ultrafast Bootstrap Approximation; Ups, UV-inducible pilus of Sulfolobus.

Fig 4. Taxonomic distribution of the systems in Bacteria and Archaea obtained using the final models.

Cells indicate the number of genomes with at least one detected system. The cell’s colour gradient represents the proportion of genomes with at least one system in the clade. The bar plot shows the total number of detected systems. The bars are separated in two categories: Alpha-, Beta-, and Gamma-proteobacteria versus the other clades. The cladogram symbolises approximated relationships between the bacterial and archaeal taxa analysed in this study. Archaeal-T4P, type IV-related pili in Archaea; Com, competence pilus; ComM, Com in monoderms; MSH, mannose-sensitive hemagglutinin pilus; Tad, tight adherence; T2SS, type II protein secretion system; T4aP, type IVa pilus; T4bP, type IVb pilus.

Fig 5. Genetic organisation of the detected systems.

For each detected system (those indicated in Fig 4), the edge width represents the number of times the two genes are contiguous divided by the number of times the rarest gene is present in the system. The colour of the edge represents the number of times the two genes are contiguous in the system divided by the number of systems. Com, competence pilus; ComM, Com in monoderms; Epd, EppA dependent; IM, integral membrane; MSH, mannose-sensitive hemagglutinin pilus; Tad, tight adherence; T2SS, type II protein secretion system; T4aP, type IVa pilus; T4bP, type IVb pilus.

Fig 6. Association between organisation and horizontal transfer of the different systems.

For each system, we compared the subtree of the systems with the 16S tree of the same species using ALE v0.4 to obtain the proportion of transfers. The panel above the graphic indicates the proportion of systems in a single locus and the proportion of systems on chromosomes (the others being found on plasmids). ComM, competence pilus of monoderms; Tad, tight adherence; T2SS, type II protein secretion system; T4aP, type IVa pilus.

Fig 7. Evolutionary scenario of the TFF superfamily.

The tree was based on the information of the trees of the concatenate and simplified to highlight the key clades and events. The colour of the triangles indicates the type of the systems. Each vertical bar on the branch indicates a numbered evolutionary event, whose details are specified under the corresponding number in the list ‘Key events’. The hypotheses for the composition of the last common ancestor of the TFF superfamily are indicated at the root, and the distant homologues of these systems are indicated in the list ‘Homologous of ancestral components’, in which homology was observed by sequence (‘aa’) or structural (‘struct’) similarity. Halo pilus indicates two pili characterised in Halobacteria. Aap, adhesive archaeal pilus; Epd, EppA dependent; IM, integral membrane; MSH, mannose-sensitive hemagglutinin pilus; Tad, tight adherence; TFF, type IV filament; T2SS, type II protein secretion system; T3SS, type III protein secretion system; T4aP, type IVa pilus; T4bP, type IVb pilus; Ups, UV-inducible pilus of Sulfolobus.