Antibiotic-induced replication stress triggers bacterial competence by increasing gene dosage near the origin

Streptococcus pneumoniae (pneumococcus) kills nearly 1 million children annually, and the emergence of antibiotic-resistant strains poses a serious threat to human health. Because pneumococci can take up DNA from their environment by a process called competence, genes associated with antibiotic resistance can rapidly spread. Remarkably, competence is activated in response to several antibiotics. Here, we demonstrate that antibiotics targeting DNA replication cause an increase in the copy number of genes proximal to the origin of replication (oriC). As the genes required for competence initiation are located near oriC, competence is thereby activated. Transcriptome analyses show that antibiotics targeting DNA replication also upregulate origin-proximal gene expression in other bacteria. This mechanism is a direct, intrinsic consequence of replication fork stalling. Our data suggest that evolution has conserved the oriC-proximal location of important genes in bacteria to allow for a robust response to replication stress without the need for complex gene-regulatory pathways. PAPERCLIP

The ParB-parS Chromosome Segregation System Modulates Competence Development in Streptococcus pneumoniae

ParB proteins bind centromere-like DNA sequences called parS sites and are involved in plasmid and chromosome segregation in bacteria. We previously showed that the opportunistic human pathogen Streptococcus pneumoniae contains four parS sequences located close to the origin of replication which are bound by ParB. Using chromatin immunoprecipitation (ChIP), we found here that ParB spreads out from one of these parS sites, parS(-1.6 degrees), for more than 5 kb and occupies the nearby comCDE operon, which drives competence development. Competence allows S. pneumoniae to take up DNA from its environment, thereby mediating horizontal gene transfer, and is also employed as a general stress response. Mutating parS(-1.6 degrees) or deleting parB resulted in transcriptional up-regulation of comCDE and ssbB (a gene belonging to the competence regulon), demonstrating that ParB acts as a repressor of competence. However, genome-wide transcription analysis showed that ParB is not a global transcriptional regulator. Different factors, such as the composition of the growth medium and antibiotic-induced stress, can trigger the sensitive switch driving competence. This work shows that the ParB-parS chromosome segregation machinery also influences this developmental process. IMPORTANCE Streptococcus pneumoniae (pneumococcus) is an important human pathogen responsible for more than a million deaths each year. Like all other organisms, S. pneumoniae must be able to segregate its chromosomes properly. Not only is understanding the molecular mechanisms underlying chromosome segregation in S. pneumoniae therefore of fundamental importance, but also, this knowledge might offer new leads for ways to target this pathogen. Here, we identified a link between the pneumococcal chromosome segregation system and the competence-developmental system. Competence allows S. pneumoniae to take up and integrate exogenous DNA in its chromosome. This process plays a crucial role in successful adaptation to-and escape from-host defenses, antibiotic treatments, and vaccination strategies. We show that the chromosome segregation protein ParB acts as a repressor of competence. To the best of our knowledge, this is the first example of a ParB protein controlling bacterial competence

Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae

Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in 'transcription traffic jams' on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes

Role of the Single-Stranded DNA–Binding Protein SsbB in Pneumococcal Transformation: Maintenance of a Reservoir for Genetic Plasticity

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

Silently transformable: the many ways bacteria conceal their built-in capacity of genetic exchange

International audienceBacteria can undergo genetic transformation by actively integrating genetic information from phylogenetically related or unrelated organisms. The original function of natural transformation remains a subject of debate, but it is well established as a major player in genome evolution. Naturally transformable bacteria use a highly conserved DNA uptake system to internalize DNA and integrate it in their chromosome by homologous recombination. Expression of the DNA uptake system, often referred to as competence, is tightly controlled and induced by signals that are often elusive. Initially thought to be restricted to a few bacterial species, natural transformation increasingly seems widespread in bacteria. Yet, the triggering signals and regulatory mechanisms involved appear diverse and are understood only in a limited set of species. As a result, natural transformation in most bacterial species remains poorly documented and the potential impact of this mechanism on global genetic mobilization is likely underappreciated. Indeed, even when a conserved activator can be identified to artificially induce the expression of the DNA uptake system, the considered species may still remain non-transformable. Recent works indicate that the DNA uptake system is directly subjected to silencing. At least in Legionella pneumophila and possibly in other species, a small non-coding RNA prevents expression of the DNA uptake system. Silencing constitutes one more way bacteria control expression of their engine of genetic exchange. It may also be the underlying reason of the undetectable natural transformation of many bacterial species grown under laboratory conditions even though they possess a DNA uptake system

Déterminants de stabilité et de maturation de l'ADN internalisé lors de la transformation génétique naturelle chez Streptococcus pneumoniae

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

RNA Chaperones Step Out of Hfq's Shadow

International audienceThe stability and function of regulatory small RNAs (sRNAs) often require a specialized RNA-binding protein called an RNA chaperone. Recent findings show that proteins containing a ProQ/FinO domain constitute a new class of RNA chaperones that could play key roles in post-transcriptional gene regulation throughout bacterial species

RadC, a Misleading Name?▿ †

The pfam04002 annotation describes RadC as a bacterial DNA repair protein. Although the radC gene is expressed specifically during competence for genetic transformation in Streptococcus pneumoniae, we report that radC mutants exhibit normal uptake and processing of transforming DNA. They also display normal sensitivity to DNA-damaging agents, providing no support for the rad epithet