Functional analysis of an unusual type IV pilus in the Gram-positive Streptococcus sanguinis

Abstract

Type IV pili (Tfp), which have been studied extensively in a few Gram-negative species, are the paradigm of a group of widespread and functionally versatile nano-machines. Here, we performed the most detailed molecular characterisation of Tfp in a Gram-positive bacterium. We demonstrate that the naturally competent Streptococcus sanguinis produces retractable Tfp, which like their Gram-negative counterparts can generate hundreds of piconewton of tensile force and promote intense surface-associated motility. Tfp power 'train-like' directional motion parallel to the long axis of chains of cells, leading to spreading zones around bacteria grown on plates. However, S. sanguinis Tfp are not involved in DNA uptake, which is mediated by a related but distinct nano-machine, and are unusual because they are composed of two pilins in comparable amounts, rather than one as normally seen. Whole genome sequencing identified a locus encoding all the genes involved in Tfp biology in S. sanguinis. A systematic mutational analysis revealed that Tfp biogenesis in S. sanguinis relies on a more basic machinery (only 10 components) than in Gram-negative species and that a small subset of four proteins dispensable for pilus biogenesis are essential for motility. Intriguingly, one of the piliated mutants that does not exhibit spreading retains microscopic motility but moves sideways, which suggests that the corresponding protein controls motion directionality. Besides establishing S. sanguinis as a useful new model for studying Tfp biology, these findings have important implications for our understanding of these widespread filamentous nano-machines.

Figure 1

S . sanguinis exhibits PilT‐powered motility and exerts huge pulling forces, hallmarks of retractile Tfp. A. Macroscopic motility assay. Spreading zones, or lack thereof, around series of human isolates and a Δpil T mutant of 2908. B. Microscopic motility assay. Representative 30 s trajectories of movement of small chains of 2908 and Δpil T cells. Scale bar represents 5 μm. Corresponding movies are available as supplementary information (Movies S1 and S2). The histogram represents the distribution curve of velocities (in 100 nm s⁻¹ intervals) measured for 2908 in three independent experiments. C. Measure of pulling forces exerted by 2908 using PoMPs force sensors. Movies showing the displacement of the micro‐pillar tips for WT and Δpil T are available as supplementary information (Movies S3 and S4). The histogram represents the distribution curve of pulling forces (in 25 pN intervals) measured in five independent experiments.

Figure 2

Genomic organisation of the pil locus in S . sanguinis 2908. All the genes are drawn to scale, with the scale bar representing 1 kb. Genes essential for Tfp biogenesis are boxed by a thick line. Genes encoding proteins predicted to play a role in Tfp biology based on the presence of signature sequence motifs have been colour coded and were named, when possible, after their homologues in N . meningitidis. Genes encoding proteins with no signature motifs but shown in this study to play a role in Tfp biology are in grey, whereas genes with (so far) no role in Tfp biology are in white. Values (in %) under each gene indicate the level of aa identity of the corresponding proteins in 21 other sequenced S . sanguinis genomes.

Figure 3

PilE1 and PilE2 are type IVa prepilins produced during growth in vitro and processed by the prepilin peptidase PilD. A. Sequence alignment of PilE1 and PilE2 prepilins encoded in 2908 genome. Residues were shaded in dark blue (identical), light blue (conserved) or unshaded (different). The class III signal peptide is highlighted, with the predicted processing site by the prepilin peptidase PilD indicated by a vertical arrow. Peptides (two per protein) used to generate antibodes are also indicated. B. Immunoblot analysis of PilE1 and PilE2 expression and processing by PilD. Whole‐cell protein extracts were probed using anti‐PilE1 or anti‐PilE2 antibodies. Protein extracts were quantified and equalised, and equivalent amounts of total proteins were loaded in each lane. Molecular weights are indicated in kDa.

Figure 4

S . sanguinis 2908 produces Tfp. Piliation in the WT strain and Δpil D mutant was assessed by SEM. The scale bar represents 500 nm.

Figure 5

Purified S . sanguinis 2908 Tfp are composed of two pilins in comparable amounts. A. SDS‐PAGE/Coomassie analysis of purified S . sanguinis  Tfp. Samples were prepared from cultures adjusted to the same OD ₆₀₀, separated by SDS‐PAGE and stained with Coomassie blue. Identical volumes were loaded in each lane. A molecular weight marker (MW) was run in the first lane. Molecular weights are indicated in kDa. B. Immunoblot analysis of pilus preparations using anti‐PilE1 or anti‐PilE2 antibodies. Molecular weights are indicated in kDa. C. Analysis of WT pilus preparations by TEM after negative staining. Scale bars represent 100 nm.

Figure 6

Ten proteins are essential for Tfp biogenesis in S . sanguinis. Pilus preparations made from mutants in each gene in the pil locus were separated by SDS‐PAGE and stained with Coomassie blue. The WT strain was included as a control. Samples were prepared from cultures adjusted to the same OD ₆₀₀.

Figure 7

Four proteins dispensable for piliation modulate twitching motility. A. Macroscopic motility assay. Mutants in each gene in the pil locus were analysed for their ability to produce spreading zones on plates. WT strain was included as a control. B. Microscopic motility assay for Δpil I, Δpil J and Δpil K piliated mutants. Representative 30 s trajectories of movement of small chains of cells are shown. Corresponding movie for Δpil K is available as supplementary information (Movie S5).The histogram represents the distribution curve of velocities (in 100 nm s⁻¹ intervals) measured for Δpil K in three independent experiments.

Figure 8

S . sanguinis Tfp are not involved in natural competence. Competence for DNA transformation was quantified using a PCR fragment conferring resistance to streptomycin. In Δcom GB, we deleted a component of the (pseudo)pilus Tff nano‐machine involved in DNA uptake in other naturally competent species. Results are expressed as transformation frequencies, i.e. number of Str^R CFU relative to total number of CFU, and are the mean ± standard deviation of at least four independent experiments. The dotted line indicates the lower limit of detection.