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1.
Methods Mol Biol ; 1968: 63-78, 2019.
Article in English | MEDLINE | ID: mdl-30929206

ABSTRACT

The ability of Streptococcus pneumoniae (the pneumococcus) to transform is particularly convenient for genome engineering. Several protocols relying on sequential positive and negative selection strategies have been described to create directed markerless modifications, including deletions, insertions, or point mutations. Transformation with DNA fragments carrying long flanking homology sequences is also used to generate mutations without selection but it requires high transformability. Here, we present an optimized version of this method. As an example, we construct a strain harboring a translational fusion ftsZ-mTurquoise at the ftsZ locus. We provide instructions to produce a linear DNA fragment containing the chimeric construction and give details of the conditions to obtain optimal pneumococcal transformation efficiencies.


Subject(s)
Chromosomes, Bacterial/genetics , DNA, Bacterial/genetics , Streptococcus pneumoniae/genetics , Mutagenesis, Insertional , Mutation/genetics , Recombination, Genetic/genetics
2.
Proc Natl Acad Sci U S A ; 110(11): E1035-44, 2013 Mar 12.
Article in English | MEDLINE | ID: mdl-23440217

ABSTRACT

Natural bacterial transformation is a genetically programmed process allowing genotype alterations that involves the internalization of DNA and its chromosomal integration catalyzed by the universal recombinase RecA, assisted by its transformation-dedicated loader, DNA processing protein A (DprA). In Streptococcus pneumoniae, the ability to internalize DNA, known as competence, is transient, developing suddenly and stopping as quickly. Competence is induced by the comC-encoded peptide, competence stimulating peptide (CSP), via a classic two-component regulatory system ComDE. Upon CSP binding, ComD phosphorylates the ComE response-regulator, which then activates transcription of comCDE and the competence-specific σ(X), leading to a sudden rise in CSP levels and rendering all cells in a culture competent. However, how competence stops has remained unknown. We report that DprA, under σ(X) control, interacts with ComE∼P to block ComE-driven transcription, chiefly impacting σ(X) production. Mutations of dprA specifically disrupting interaction with ComE were isolated and shown to map mainly to the N-terminal domain of DprA. Wild-type DprA but not ComE interaction mutants affected in vitro binding of ComE to its promoter targets. Once introduced at the dprA chromosomal locus, mutations disrupting DprA interaction with ComE altered competence shut-off. The absence of DprA was found to negatively impact growth following competence induction, highlighting the importance of DprA for pneumococcal physiology. DprA has thus two key roles: ensuring production of transformants via interaction with RecA and competence shut-off via interaction with ComE, avoiding physiologically detrimental consequences of prolonged competence. Finally, phylogenetic analyses revealed that the acquisition of a new function by DprA impacted its evolution in streptococci relying on ComE to regulate comX expression.


Subject(s)
Bacterial Proteins/metabolism , DNA Transformation Competence/physiology , Membrane Proteins/metabolism , Rec A Recombinases/metabolism , Streptococcus pneumoniae/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial/physiology , Membrane Proteins/genetics , Mutation , Protein Structure, Tertiary , Rec A Recombinases/genetics , Streptococcus pneumoniae/genetics , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transcription, Genetic/physiology
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