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1.
Nat Commun ; 15(1): 5625, 2024 Jul 10.
Article in English | MEDLINE | ID: mdl-38987237

ABSTRACT

Competence for natural transformation is a central driver of genetic diversity in bacteria. In the human pathogen Streptococcus pneumoniae, competence exhibits a populational character mediated by the stress-induced ComABCDE quorum-sensing (QS) system. Here, we explore how this cell-to-cell communication mechanism proceeds and the functional properties acquired by competent cells grown under lethal stress. We show that populational competence development depends on self-induced cells stochastically emerging in response to stresses, including antibiotics. Competence then propagates through the population from a low threshold density of self-induced cells, defining a biphasic Self-Induction and Propagation (SI&P) QS mechanism. We also reveal that a competent population displays either increased sensitivity or improved tolerance to lethal doses of antibiotics, dependent in the latter case on the competence-induced ComM division inhibitor. Remarkably, these surviving competent cells also display an altered transformation potential. Thus, the unveiled SI&P QS mechanism shapes pneumococcal competence as a health sensor of the clonal population, promoting a bet-hedging strategy that both responds to and drives cells towards heterogeneity.


Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Quorum Sensing , Streptococcus pneumoniae , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/physiology , Anti-Bacterial Agents/pharmacology , Quorum Sensing/drug effects , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Humans , Gene Expression Regulation, Bacterial/drug effects , Transformation, Bacterial
2.
Nucleic Acids Res ; 51(6): 2800-2817, 2023 04 11.
Article in English | MEDLINE | ID: mdl-36806960

ABSTRACT

RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report by direct microscopic analysis of SpRecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of SpRecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.


Subject(s)
Rec A Recombinases , Streptococcus pneumoniae , Adenosine Triphosphate/metabolism , DNA/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Rec A Recombinases/metabolism , Rec A Recombinases/ultrastructure , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/metabolism , Cryoelectron Microscopy
3.
Proc Natl Acad Sci U S A ; 120(8): e2213867120, 2023 02 21.
Article in English | MEDLINE | ID: mdl-36795748

ABSTRACT

Homologous recombination (HR) is a crucial mechanism of DNA strand exchange that promotes genetic repair and diversity in all kingdoms of life. Bacterial HR is driven by the universal recombinase RecA, assisted in the early steps by dedicated mediators that promote its polymerization on single-stranded DNA (ssDNA). In bacteria, natural transformation is a prominent HR-driven mechanism of horizontal gene transfer specifically dependent on the conserved DprA recombination mediator. Transformation involves internalization of exogenous DNA as ssDNA, followed by its integration into the chromosome by RecA-directed HR. How DprA-mediated RecA filamentation on transforming ssDNA is spatiotemporally coordinated with other cellular processes remains unknown. Here, we tracked the localization of fluorescent fusions to DprA and RecA in Streptococcus pneumoniae and revealed that both accumulate in an interdependent manner with internalized ssDNA at replication forks. In addition, dynamic RecA filaments were observed emanating from replication forks, even with heterologous transforming DNA, which probably represent chromosomal homology search. In conclusion, this unveiled interaction between HR transformation and replication machineries highlights an unprecedented role for replisomes as landing pads for chromosomal access of tDNA, which would define a pivotal early HR step for its chromosomal integration.


Subject(s)
Rec A Recombinases , Streptococcus pneumoniae , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/metabolism , Rec A Recombinases/genetics , Rec A Recombinases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chromosomes/metabolism , DNA/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism
4.
Elife ; 92020 11 02.
Article in English | MEDLINE | ID: mdl-33135635

ABSTRACT

Competence is a widespread bacterial differentiation program driving antibiotic resistance and virulence in many pathogens. Here, we studied the spatiotemporal localization dynamics of the key regulators that master the two intertwined and transient transcription waves defining competence in Streptococcus pneumoniae. The first wave relies on the stress-inducible phosphorelay between ComD and ComE proteins, and the second on the alternative sigma factor σX, which directs the expression of the DprA protein that turns off competence through interaction with phosphorylated ComE. We found that ComD, σX and DprA stably co-localize at one pole in competent cells, with σX physically conveying DprA next to ComD. Through this polar DprA targeting function, σX mediates the timely shut-off of the pneumococcal competence cycle, preserving cell fitness. Altogether, this study unveils an unprecedented role for a transcription σ factor in spatially coordinating the negative feedback loop of its own genetic circuit.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Sigma Factor/metabolism , Streptococcus pneumoniae/metabolism , Bacterial Proteins/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Fluorescence , Sigma Factor/genetics , Streptococcus pneumoniae/cytology , Streptococcus pneumoniae/genetics , Transformation, Genetic
5.
Nat Commun ; 8: 15638, 2017 05 31.
Article in English | MEDLINE | ID: mdl-28561029

ABSTRACT

Homologous recombination (HR) is a central process of genome biology driven by a conserved recombinase, which catalyses the pairing of single-stranded DNA (ssDNA) with double-stranded DNA to generate a D-loop intermediate. Bacterial RadA is a conserved HR effector acting with RecA recombinase to promote ssDNA integration. The mechanism of this RadA-mediated assistance to RecA is unknown. Here, we report functional and structural analyses of RadA from the human pathogen Streptococcus pneumoniae. RadA is found to facilitate RecA-driven ssDNA recombination over long genomic distances during natural transformation. RadA is revealed as a hexameric DnaB-type helicase, which interacts with RecA to promote orientated unwinding of branched DNA molecules mimicking D-loop boundaries. These findings support a model of DNA branch migration in HR, relying on RecA-mediated loading of RadA hexamers on each strand of the recipient dsDNA in the D-loop, from which they migrate divergently to facilitate incorporation of invading ssDNA.


Subject(s)
Bacterial Proteins/metabolism , DNA, Single-Stranded/chemistry , DNA-Binding Proteins/metabolism , DnaB Helicases/metabolism , Rec A Recombinases/metabolism , Crystallography, X-Ray , DNA Helicases/metabolism , Homologous Recombination , Mutagenesis, Site-Directed , Protein Domains , Protein Structure, Quaternary , Recombination, Genetic , Streptococcus pneumoniae/enzymology , Two-Hybrid System Techniques
6.
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
7.
Mol Microbiol ; 87(2): 394-411, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23216914

ABSTRACT

Since 1996, induction of competence for genetic transformation of Streptococcus pneumoniae is known to be controlled by the ComD/ComE two-component regulatory system. The mechanism of induction is generally described as involving ComD autophosphorylation, transphosphorylation of ComE and transcriptional activation by ComE~P of the early competence (com) genes, including comX which encodes the competence-specific σ(X) . However, none of these features has been experimentally established. Here we document the autokinase activity of ComD proteins in vitro, and provide an estimate of the stoichiometry of ComD and ComE in vivo. We report that a phosphorylmimetic mutant, ComE(D58E), constructed because of the failure to detect transphosphorylation of purified ComE in vitro, displays full spontaneous competence in ΔcomD cells, an that in vitro ComE(D58E) exhibits significantly improved binding affinity for P(comCDE). We also provide evidence for a differential transcriptional activation and repression of P(comCDE) and P(comX). Altogether, these data support the model of ComE~P-dependent activation of transcription. Finally, we establish that ComE antagonizes expression of the early com genes and propose that the rapid deceleration of transcription from P(comCDE) observed even in cells lacking σ(X) is due to the progressive accumulation of ComE, which outcompetes ComE~P.


Subject(s)
Bacterial Proteins/metabolism , DNA Transformation Competence , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Streptococcus pneumoniae/physiology , Models, Biological , Protein Binding , Protein Interaction Mapping , Streptococcus pneumoniae/genetics , Transcription, Genetic
8.
Proc Natl Acad Sci U S A ; 109(37): E2466-75, 2012 Sep 11.
Article in English | MEDLINE | ID: mdl-22904190

ABSTRACT

Transformation promotes genome plasticity in bacteria via RecA-driven homologous recombination. In the gram-positive human pathogen Streptococcus pneumoniae, the transformasome a multiprotein complex, internalizes, protects, and processes transforming DNA to generate chromosomal recombinants. Double-stranded DNA is internalized as single strands, onto which the transformation-dedicated DNA processing protein A (DprA) ensures the loading of RecA to form presynaptic filaments. We report that the structure of DprA consists of the association of a sterile alpha motif domain and a Rossmann fold and that DprA forms tail-to-tail dimers. The isolation of DprA self-interaction mutants revealed that dimerization is crucial for the formation of nucleocomplexes in vitro and for genetic transformation. Residues important for DprA-RecA interaction also were identified and mutated, establishing this interaction as equally important for transformation. Positioning of key interaction residues on the DprA structure revealed an overlap of DprA-DprA and DprA-RecA interaction surfaces. We propose a model in which RecA interaction promotes rearrangement or disruption of the DprA dimer, enabling the subsequent nucleation of RecA and its polymerization onto ssDNA.


Subject(s)
Bacterial Proteins/metabolism , Membrane Proteins/metabolism , Models, Molecular , Protein Conformation , Rec A Recombinases/metabolism , Streptococcus pneumoniae/metabolism , Transformation, Bacterial/physiology , Bacterial Proteins/chemistry , Blotting, Western , Crystallization , DNA/metabolism , DNA Primers/genetics , Dimerization , Membrane Proteins/chemistry , Mutagenesis, Site-Directed , Transformation, Bacterial/genetics , Two-Hybrid System Techniques
9.
PLoS Genet ; 7(6): e1002156, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21738490

ABSTRACT

Bacteria encode a single-stranded DNA (ssDNA) binding protein (SSB) crucial for genome maintenance. In Bacillus subtilis and Streptococcus pneumoniae, an alternative SSB, SsbB, is expressed uniquely during competence for genetic transformation, but its precise role has been disappointingly obscure. Here, we report our investigations involving comparison of a null mutant (ssbB(-)) and a C-ter truncation (ssbBΔ7) of SsbB of S. pneumoniae, the latter constructed because SSBs' acidic tail has emerged as a key site for interactions with partner proteins. We provide evidence that SsbB directly protects internalized ssDNA. We show that SsbB is highly abundant, potentially allowing the binding of ~1.15 Mb ssDNA (half a genome equivalent); that it participates in the processing of ssDNA into recombinants; and that, at high DNA concentration, it is of crucial importance for chromosomal transformation whilst antagonizing plasmid transformation. While the latter observation explains a long-standing observation that plasmid transformation is very inefficient in S. pneumoniae (compared to chromosomal transformation), the former supports our previous suggestion that SsbB creates a reservoir of ssDNA, allowing successive recombination cycles. SsbBΔ7 fulfils the reservoir function, suggesting that SsbB C-ter is not necessary for processing protein(s) to access stored ssDNA. We propose that the evolutionary raison d'être of SsbB and its abundance is maintenance of this reservoir, which contributes to the genetic plasticity of S. pneumoniae by increasing the likelihood of multiple transformation events in the same cell.


Subject(s)
Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/metabolism , Transformation, Bacterial/genetics , Bacterial Proteins/genetics , Chromosomes, Bacterial/genetics , Chromosomes, Bacterial/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/genetics , Models, Biological , Mutation/genetics , Plasmids/genetics , Plasmids/metabolism
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