Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology

Abstract

Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)—exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable.

Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacteriumStreptococcus sanguinis

“…The study of Tff in phylogenetically distant species has the potential to move the field forward, which has recently sparked studies in Archaea (2) and in distant phyla of Bacteria (21). One of the most promising new Tfp models that has emerged is the Gram-positive opportunistic pathogen S. sanguinis (24,25). A recent systematic genetic analysis of Tfp biology in this species -the first to be realised in a non Proteobacterium -showed that S. sanguinis uses a simpler machinery (with fewer components) to assemble canonical Tfpa that generate high tensile forces and power twitching motility (24).…”

“…First, in contrast to all previously available pilin structures, the C-terminus in PilE1 is unstructured and highly flexible. This explains why it is a permissive insertion site and why it even can be deleted and replaced by a 6His tag (25), without interfering with the ability of PilE1/PilE2 to be polymerised into filaments. Therefore, the common rule that the C-terminus of major pilins must be stabilised to preserve pilin integrity and its ability to be polymerised (3), is not always true.…”

“…Next, we sought to answer the question whether S. sanguinis Tfp are heteropolymers of PilE1 and PilE2 or whether two homo-polymers co-exist. Recently, using a markerless gene editing strategy (25), we showed that pilE1 could be engineered in situ to encode a protein with a C-terminal 6His tag, without affecting piliation or Tfp functionality. We therefore used this property to design an affinity copurification procedure to answer the above question (Fig.…”

“…1B and Fig. S2), we focused our efforts on PilE1 and used our gene editing strategy (25) to construct markerless mutant strains expressing PilE1 variants in which the Gly-1 and Glu5 residues would be mutated. Next, we tested whether these mutants proteins were processed by PilD and assembled into filaments.…”

“…Until recently, Tfp have not been extensively studied in Gram-positive species although it has been recognised that this represents a promising new research avenue as these bacteria possess a simpler surface architecture (21). Although Gram-positive Tfp were first described in Clostridiales (22,23), Streptococcus sanguinis has emerged as a model because it is genetically tractable (24,25). A comprehensive genetic analysis of S. sanguinis Tfpa (24) has revealed that they: (i) are assembled by a similar machinery as in Gram-negative species but with fewer components, (ii) are retracted by a PilT-dependent mechanism, generating tensile forces very similar to those measured in Gram-negative species, and (iii) power intense twitching motility.…”