title: 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.
authors.affiliation: MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK.
authors.forename: Ishwori
authors.initials: I
authors.lastname: Gurung
authors.affiliation: MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK.
authors.forename: Jamie-Lee
authors.initials: JL
authors.lastname: Berry
authors.affiliation: MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK.
authors.forename: Alexander M J
authors.initials: AMJ
authors.lastname: Hall
authors.affiliation: MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Pelicic
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D060846:Cell Engineering; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000235:genetics* / Q000502:physiology; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D005819:Genetic Markers; D006639:Histidine; D020125:Mutation, Missense; D009842:Oligopeptides; D015202:Protein Engineering; D013291:Streptococcus / Q000235:genetics* / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D005819:Genetic Markers; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D036781:Fimbriae Proteins; D006639:Histidine; D010450:Endopeptidases; C067127:prepilin peptidase protein, Bacteria