Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology
File(s)
Published version
Author(s)
Gurung, I
Berry, J
Hall, A
Pelicic, V
Type
Journal Article
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 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.
, 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.
Date Issued
2016-11-28
Date Acceptance
2016-11-10
Citation
Nucleic Acids Research, 2016, 45 (45)
ISSN
1362-4962
Publisher
Oxford University Press (OUP)
Journal / Book Title
Nucleic Acids Research
Volume
45
Issue
45
Copyright Statement
© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Sponsor
Medical Research Council (MRC)
Grant Number
MR/J006874/1B
Subjects
Science & Technology
Life Sciences & Biomedicine
Biochemistry & Molecular Biology
GRAM-POSITIVE BACTERIA
PREPILIN PEPTIDASES
TWITCHING MOTILITY
BIOGENESIS
IDENTIFICATION
COMPETENCE
PROTEINS
PILIN
ENDOCARDITIS
MUTAGENESIS
Developmental Biology
05 Environmental Sciences
06 Biological Sciences
08 Information And Computing Sciences
Publication Status
Published
Article Number
e40