Identification and Characterization of Negative Regulators of Rgg1518 Quorum Sensing in Streptococcus pneumoniae.

Streptococcus pneumoniae is an agent of otitis media, septicemia, and meningitis and remains the leading cause of community-acquired pneumonia regardless of vaccine use. Of the various strategies that S. pneumoniae takes to enhance its potential to colonize the human host, quorum sensing (QS) is an intercellular communication process that provides coordination of gene expression at a community level.

Markerless Genome Editing in Competent Streptococci.

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S.

Competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake.

The competence pili of transformable Gram-positive species are phylogenetically related to the diverse and widespread class of extracellular filamentous organelles known as type IV pili. In Gram-negative bacteria, type IV pili act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether competence pili in Gram-positive species exhibit similar dynamic activity, and their mechanism of action for DNA uptake remains unclear.

ComWΔ6 Stimulates Transcription of Pneumococcal Competence Genes .

The alternative streptococcal σ-factor and master competence regulator, σ, stimulates transcription from competence promoters, As the only known alternative σ-factor in streptococci, σ expression is tightly controlled in each species and has a specific physiological role. Pneumococcal transformation also requires the DNA binding activity of ComW, a known σ activator and stabilizer. Mutations to the housekeeping σ factor, σ, partially alleviate the ComW requirement, suggesting that ComW is a key player in the σ factor swap during the pneumococcal competence response.

Multiplex gene transfer by genetic transformation between isolated S. pneumoniae cells confined in microfluidic droplets.

Gene exchange via genetic transformation makes major contributions to antibiotic resistance of the human pathogen, Streptococcus pneumoniae (pneumococcus). The transfers begin when a pneumococcal cell, in a transient specialized physiological state called competence, attacks and lyses another cell, takes up fragments of the liberated DNA, and integrates divergent genes into its genome. Recently, it has been demonstrated that the pneumococcal cells can be enclosed in femtoliter-scale droplets for study of the transformation mechanism, offering the ability to characterize individual cell-cell interactions and overcome the limitations of current methods involving bulk mixed cultures.

HtrA-mediated selective degradation of DNA uptake apparatus accelerates termination of pneumococcal transformation.

Natural transformation mediates horizontal gene transfer, and thereby promotes exchange of antibiotic resistance and virulence traits among bacteria. Streptococcus pneumoniae, the first known transformable bacterium, rapidly activates and then terminates the transformation state, but it is unclear how the bacterium accomplishes this rapid turn-around at the protein level. This work determined the transcriptomic and proteomic dynamics during the window of pneumococcal transformation.

The pneumococcal σ activator, ComW, is a DNA-binding protein critical for natural transformation.

Natural genetic transformation via horizontal gene transfer enables rapid adaptation to dynamic environments and contributes to both antibiotic resistance and vaccine evasion among bacterial populations. In (pneumococcus), transformation occurs when cells enter competence, a transient state in which cells express the competence master regulator, SigX (σ), an alternative σ factor (σ), and a competence co-regulator, ComW. Together, ComW and σ facilitate expression of the genes required for DNA uptake and genetic recombination.

Competence in and Close Commensal Relatives: Mechanisms and Implications.

The mitis group of streptococci comprises species that are common colonizers of the naso-oral-pharyngeal tract of humans. and are close relatives and share ~60-80% of orthologous genes, but still present striking differences in pathogenic potential toward the human host. has long been recognized as a reservoir of antibiotic resistance genes for , as well as a source for capsule polysaccharide variation, leading to resistance and vaccine escape.

Competence beyond Genes: Filling in the Details of the Pneumococcal Competence Transcriptome by a Systems Approach.

DNA uptake by natural competence is a central process underlying the genetic plasticity, biology, and virulence of the human respiratory opportunistic pathogen A study reported in this issue (J. Slager, R. Aprianto, and J.

Hidden Gems in the Transcriptome Maps of Competent Streptococci.

Natural transformation is regarded as an important mechanism in bacteria that allows for adaptation to different environmental stressors by ensuring genome plasticity. Since the discovery of this phenomenon in , remarkable progress has been made in the understanding of the molecular mechanisms and pathways coordinating this process. Recently, the advent of high-throughput sequencing allows the posing of questions that address the system at a larger scale but also allow for the creation of high-resolution maps of transcription.

Femtoliter droplet confinement of Streptococcus pneumoniae: bacterial genetic transformation by cell-cell interaction in droplets.

Streptococcus pneumoniae (pneumococcus), a deadly bacterial human pathogen, uses genetic transformation to gain antibiotic resistance. Genetic transformation begins when a pneumococcal strain in a transient specialized physiological state called competence, attacks and lyses another strain, releasing DNA, taking up fragments of the liberated DNA, and integrating divergent genes into its genome. While many steps of the process are known and generally understood, the precise mechanism of this natural genetic transformation is not fully understood and the current standard strategies to study it have limitations in specifically controlling and observing the process in detail.

Evolution via recombination: Cell-to-cell contact facilitates larger recombination events in Streptococcus pneumoniae.

Homologous recombination in the genetic transformation model organism Streptococcus pneumoniae is thought to be important in the adaptation and evolution of this pathogen. While competent pneumococci are able to scavenge DNA added to laboratory cultures, large-scale transfers of multiple kb are rare under these conditions. We used whole genome sequencing (WGS) to map transfers in recombinants arising from contact of competent cells with non-competent 'target' cells, using strains with known genomes, distinguished by a total of ~16,000 SNPs.

Flavin Reductase Contributes to Pneumococcal Virulence by Protecting from Oxidative Stress and Mediating Adhesion and Elicits Protection Against Pneumococcal Challenge.

Pneumococcal flavin reductase (FlaR) is known to be cell-wall associated and possess age dependent antigenicity in children. This study aimed at characterizing FlaR and elucidating its involvement in pneumococcal physiology and virulence. Bioinformatic analysis of FlaR sequence identified three-conserved cysteine residues, suggesting a transition metal-binding capacity.

A positive feedback loop mediated by Sigma X enhances expression of the streptococcal regulator ComR.

Natural transformation is used by bacteria to take up DNA from their surroundings and incorporate it into their genomes. Streptococci do so during a transient period of competence, triggered by pheromones that they produce, secrete and sense under conditions influenced by the environment. In Streptococcus mutans, Streptococcus suis, and species of the bovis, salivarius and pyogenic groups of streptococci, the pheromone XIP is sensed by the intra-cellular regulator ComR, that in turn activates the transcription of comS, encoding the XIP precursor, and of sigX, encoding the only known alternative sigma factor in streptococci.

Markerless Genome Editing in Competent Streptococci.

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S.

Natural Transformation of Oral Streptococci by Use of Synthetic Pheromones.

The discovery that Streptococcus pneumoniae uses a competence-stimulating peptide (CSP) to induce competence for natural transformation, and that other species of the mitis and the anginosus streptococcal groups use a similar system, has expanded the tools to explore gene function and regulatory pathways in streptococci. Two other classes of pheromones have been discovered since then, comprising the bacteriocin-inducing peptide class found in Streptococcus mutans (also named CSP, although different from the former) and the SigX-inducing peptides (XIP), in the mutans, salivarius, bovis, and pyogenes groups of streptococci. The three classes of peptide pheromones can be ordered from peptide synthesis services at affordable prices, and used in transformation assays to obtain competent cultures consistently at levels usually higher than those achieved during spontaneous competence.

Pheromone Recognition and Selectivity by ComR Proteins among Streptococcus Species.

Natural transformation, or competence, is an ability inherent to bacteria for the uptake of extracellular DNA. This process is central to bacterial evolution and allows for the rapid acquirement of new traits, such as antibiotic resistance in pathogenic microorganisms. For the Gram-positive bacteria genus Streptococcus, genes required for competence are under the regulation of quorum sensing (QS) mediated by peptide pheromones.

Overcoming the Barrier of Low Efficiency during Genetic Transformation of Streptococcus mitis.

Objective: Streptococcus mitis is a predominant oral colonizer, but difficulties in genetic manipulation of this species have hampered our understanding of the mechanisms it uses for colonization of oral surfaces. The aim of this study was to reveal optimal conditions for natural genetic transformation in S. mitis and illustrate its application in direct genome editing.

Competence for Genetic Transformation in Streptococcus pneumoniae: Mutations in σA Bypass the ComW Requirement for Late Gene Expression.

Unlabelled: Streptococcus pneumoniae is able to integrate exogenous DNA into its genome by natural genetic transformation. Transient accumulation of high levels of the only S. pneumoniae alternative σ factor is insufficient for development of full competence without expression of a second competence-specific protein, ComW.

Addiction of Hypertransformable Pneumococcal Isolates to Natural Transformation for In Vivo Fitness and Virulence.

Natural genetic transformation of Streptococcus pneumoniae, an important human pathogen, mediates horizontal gene transfer for the development of drug resistance, modulation of carriage and virulence traits, and evasion of host immunity. Transformation frequency differs greatly among pneumococcal clinical isolates, but the molecular basis and biological importance of this interstrain variability remain unclear. In this study, we characterized the transformation frequency and other associated phenotypes of 208 S.

Competence for genetic transformation in Streptococcus pneumoniae: mutations in σA bypass the comW requirement.

Competence for genetic transformation in the genus Streptococcus depends on an alternative sigma factor, σ(X), for coordinated synthesis of 23 proteins, which together establish the X state by permitting lysis of incompetent streptococci, uptake of DNA fragments, and integration of strands of that DNA into the resident genome. Initiation of transient accumulation of high levels of σ(X) is coordinated between cells by transcription factors linked to peptide pheromone signals. In Streptococcus pneumoniae, elevated σ(X) is insufficient for development of full competence without coexpression of a second competence-specific protein, ComW.

Control of competence for DNA transformation in streptococcus suis by genetically transferable pherotypes.

Here we show that S. suis, a major bacterial pathogen of pigs and emerging pathogen in humans responds to a peptide pheromone by developing competence for DNA transformation. This species does not fall within any of the phylogenetic clusters of streptococci previously shown to regulate competence via peptide pheromones suggesting that more species of streptococci may be naturally competent.

Exit from competence for genetic transformation in Streptococcus pneumoniae is regulated at multiple levels.

Development of natural competence in S. pneumoniae entails coordinated expression of two sets of genes. Early gene expression depends on ComE, a response regulator activated by the pheromone CSP (Competence-Stimulating-Peptide).

NADH oxidase functions as an adhesin in Streptococcus pneumoniae and elicits a protective immune response in mice.

The initial event in disease caused by S. pneumoniae is adhesion of the bacterium to respiratory epithelial cells, mediated by surface expressed molecules including cell-wall proteins. NADH oxidase (NOX), which reduces free oxygen to water in the cytoplasm, was identified in a non-lectin enriched pneumococcal cell-wall fraction.

Competence for natural genetic transformation in the Streptococcus bovis group streptococci S. infantarius and S. macedonicus.

Natural genetic transformation is common among many species of the genus Streptococcus, but it has never, or rarely, been reported for the Streptococcus pyogenes and S. bovis groups of species, even though many streptococcal competence genes and the competence regulators SigX, ComR, and ComS are well conserved in both groups. To explore the incidence of competence in the S.