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

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 pr okaryotic filaments. To enhance genetic manipulation of S. 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) del eting single or mutiple genes, (ii) altering specific bases in genes of int erest, 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 that processes pilins, and (iii) that pilin subunits with a C terminally ap pended hexa- histidine (6His) tag are still assembled into functional Tfp. T he methodology for genetic manipulation we describe here should be broadly applicable