Biology subjects

Berge, M.

Publications and source records attributed to Berge, M..

6 recordsLinked to original sources

bioRxiv2024

Competence induction of homologous recombination genes protects pneumococcal cells from genotoxic stress.

Homologous recombination (HR) is a universally conserved mechanism of DNA strand exchange between homologous sequences, driven in bacteria by the universal recombinase RecA. HR is key for the maintenance of bacterial genomes via replication fork restart and DNA repair, as well as for their plasticity via the widespread mechanism of natural transformation. Transformation involves the capture and internalisation of exogenous DNA in the form of single strands (ssDNA), followed by chromosomal integration via HR. In the human pathogen Streptococcus pneumoniae, transformation occurs during a transient, stress-induced physiological state called competence. RecA and its partner DNA branch migration translocase RadA both cooperate in HR during transformation and in some recombinational DNA repair pathways of genome maintenance. Both recA and radA genes are basally expressed and transcriptionally induced during competence. In this study, we explored the importance of competence induction of recA and radA expression in transformation and genome maintenance processes. We confirmed that competence induction of recA was important for optimal transformation, but found this was not the case for radA. In contrast, the competence induction of both genes was required for optimal tolerance faced with transient exposure to the lethal genotoxic agent methyl methanesulfonate (MMS). However, this was not the case for another DNA-damaging agent, norfloxacin. These results show that competence induction of HR effectors is important for the increased tolerance to genotoxic stress provided to competent pneumococcal cells. This reinforces the finding that pneumococcal competence is a stress-sensing mechanism, transiently increasing the expression of some genes not to optimise transformation but to improve survival faced with specific lethal stresses. ImportanceHomologous recombination (HR) is a mechanism of DNA strand exchange important for both the maintenance and plasticity of bacterial genomes. Bacterial HR is driven by the recombinase RecA along with many accessory partner proteins, which define multiple dedicated pathways crucial to genome biology. Thus, a main mechanism of genome plasticity in bacteria is natural genetic transformation, which involves uptake and chromosomal integration of exogenous DNA via HR. In the human pathogen Streptococcus pneumoniae, transformation occurs during a transient, stress-induced physiological state called competence. RecA and the helicase RadA are key for both genome maintenance and transformation, and both are over-produced during competence. Here, we explore the importance of this over-production for transformation and genome maintenance, quantified by tolerance to genotoxic stress. While over-production of RecA was important for both processes, over-production of RadA was required only for genotoxic stress tolerance. This highlights the importance of competence as a stress-responsive mechanism, with induction of HR genes important for genotoxic stress tolerance.

microbiology↗

bioRxiv2023

Fever-like temperature bursts promote competence development via an HtrA-dependent pathway in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is well known for its ability to develop competence for natural DNA transformation. Competence development is regulated by an autocatalytic loop driven by variations in the basal level of transcription of the comCDE and comAB operons. These genes are part of the early gene regulon that controls expression of the late competence genes known to encode the apparatus of transformation. Several stressful conditions are known to promote competence development, although the induction pathways are remain poorly understood so far. To date there have been no reports of the response of the pneumococcal competence regulon to changes in the physical conditions of the environment, oxidative potential and temperature. Here we demonstrate that transient temperature elevation induces an immediate increase in the basal expression level of the comCDE operon and early genes that, in turn, stimulates its full induction, including that of the late competence regulon. This thermal regulation depends on the HtrA chaperone/protease and its proteolytic activity. We find that certain other competence induction stimuli are not conveyed by the HtrA-dependent pathway. This finding strongly supports that competence can be induced by any of several independent pathways and thus reinforces the view that competence is a general stress response system in the pneumococcus. Authors summaryStreptococcus pneumoniae is a commensal bacterium and an opportunistic pathogen of humans. Certain environmental stimuli, such as a variety of antibiotics targeting distinct cellular functions, trigger the induction of the distinct physiological state of competence, in which cells can, among other things, import and integrate external DNA. Competence is thus considered a general stress response in this highly adaptable species. To understand the role of competence in pneumococcal interaction with its host and in pathogenicity, we attempted to decipher the pathways that enable appropriate reactions to environmental stress, and have focused here on induction of competence by elevation of temperature to levels similar to that of a host in fever. We found that elevated temperature raises the basal expression level of the competence control operon, and thus lowers the threshold of transition to full competence induction. By genetic characterisation of the thermal induction pathway of competence, we demonstrated that the chaperone/protease HtrA is essential to relaying of the thermal signal but is not involved in transmitting other stimuli such as those arising from the presence of certain antibiotics. Our work supports the view that competence can be induced through various pathways in response to distinct aggressions, but also by fever-like bursts of temperature that the pneumococcus could face in its natural habitat.

microbiology↗

bioRxiv2020

Cryptosporulation in Kurthia spp. forces a rethinking of asporogenesis in Firmicutes

Sporulation is a complex morphophysiological process resulting in a cellular structure that is more resistant than the vegetative form. In Firmicutes, this structure is produced within the mother cell, and is called an endospore. Endospore formation is thought to have evolved in the common ancestor of Firmicutes. However, sporulation has apparently been lost in some extant lineages that are defined as asporogenic. We isolated strain 11kri321, a representative of the genus Kurthia, from an oligotrophic geothermal reservoir. While Kurthia spp. is considered to comprise only asporogenic species, strain 11kri321 produced spores. Genomic reconstruction of the sporulation pathway shows elements typical of sporulation in Bacilli, including the signaling for sporulation onset. However, key genes were missing, including those involved in engulfment and dipicolinic acid synthesis. Based on the results for strain 11kri321, sporulation was investigated in other Kurthia strains. Genes involved in signaling, cell division and spore coat formation were detected in three available Kurthia genomes. Moreover, endosporulation was clearly visualized in at least two of the four strains tested. These results show that Kurthia is an endospore-forming Firmicute lineage. However, the genetic background of sporulation in this genus deviates from the known sporulation pathway in Firmicutes and even within Bacilli, suggesting that a revision of the minimal set of genes used for genomic detection of sporulation is required. Based on our findings we propose the term cryptosporulant to refer to putative asporogenic Firmicutes for which a detailed genomic and physiological characterization of sporulating is missing. ImportanceEndospore-forming Firmicutes include many environmental and medical relevant bacterial clades. In these microorganisms, the ability to produce endospores is essential for survival in the environment and even for pathogenesis. The minimum core of genes required to produce a viable and resistant spore, the distinction between endospore-forming and asporogenic groups, as well as the evolution of sporulation have been a subject of investigation and debate for decades. Here, we demonstrate endosporulation in the genus Kurthia, considered as asporogenic. Morphological, physiological and genomic analyses were undertaken to demonstrate that sporulation is not lost within this lineage. Based on our results we propose a re-examination of the minimal genetic requirements of sporulation and the use of the term cryptosporulant to describe lineages of Firmicutes that have not previously been observed to sporulate, but for which a detailed analysis is still missing.

microbiology↗

bioRxiv2020

The alternative sigma factor σX mediates competence shut-off at the cell pole in Streptococcus pneumoniae.

Bacterial competence for genetic transformation is a well-known species-specific differentiation program driving genome plasticity, antibiotic resistance and virulence in many pathogens. How competence regulation is spatiotemporally integrated in the cell is ill-defined. Here, we unraveled the localization dynamics of the key regulators that master the two intertwined transcription waves controlling competence in Streptococcus pneumoniae. The first wave relies on a stress-inducible phosphorelay system, made up of the ComD and ComE proteins, and the second is directed by an alternative sigma factor, {sigma}X, which includes in its regulon the DprA protein that turns off competence through interaction with phosphorylated ComE. Remarkably, we found that ComD, {sigma}X and DprA stably co-localize at a single cell pole over the competence period. In contrast, ComE assembles into dynamic patches in the cell periphery, colocalizing temporarily with DprA and ComD at the pole. Furthermore, we provide evidence that {sigma}X directly conveys DprA polar anchoring. Through this protein targeting function, {sigma}X is shown to be actively involved in the timely shut-off of the competence cycle, hence preserving cell fitness. Altogether, this study unveils an unprecedented role for a bacterial transcription {sigma} factor in spatially coordinating the negative feedback loop of its own genetic circuit.

microbiology↗

bioRxiv2019

Bacterial cell cycle control by citrate synthase independent of enzymatic activity

Coordination of cell cycle progression with central metabolism is fundamental to all cell types and likely underlies differentiation into dispersal cells in bacteria. How central metabolism is monitored to regulate cell cycle functions is poorly understood. A forward genetic selection for cell cycle regulators in the polarized alpha-proteobacterium Caulobacter crescentus unearthed the uncharacterized CitA citrate synthase, a TCA (tricarboxylic acid) cycle enzyme, as unprecedented checkpoint regulator of the G1[->]S transition. We show that loss of the CitA protein provokes a (p)ppGpp alarmone-dependent G1-phase arrest without apparent metabolic or energy insufficiency. While S-phase entry is still conferred when CitA is rendered catalytically inactive, the paralogous CitB citrate synthase has no overt role other than sustaining TCA cycle activity when CitA is absent. With eukaryotic citrate synthase paralogs known to fulfill regulatory functions, our work extends the moonlighting paradigm to citrate synthase coordinating central (TCA) metabolism with development and perhaps antibiotic tolerance in bacteria.

cell biology↗

bioRxiv2018

Dynamic modeling of Streptococcus pneumoniae competence provides regulatory mechanistic insights

In the human pathogen Streptococcus pneumoniae, the gene regulatory circuit leading to the transient state of competence for natural transformation is based on production of an auto-inducer that activates a positive feedback loop. About one hundred genes are activated in two successive waves linked by a central alternative sigma factor ComX. This mechanism appears to be fundamental to the biological fitness of S. pneumoniae. We have developed a knowledge-based model of the competence cycle that describes average cell behavior. It reveals that the expression rates of the two competence operon, comAB and comCDE, involved in the positive feedback loop must be coordinated to elicit spontaneous competence. Simulations revealed the requirement for an unknown late com gene product that shuts of competence by impairing ComX activity. Further simulations led to the predictions that the membrane protein ComD bound to CSP reacts directly to pH change of the medium and that blindness to CSP during the post-competence phase is controlled by late DprA protein. Both predictions were confirmed experimentally.

systems biology↗