Markerless Genome Editing in Competent Streptococci

Junges, Roger; Khan, Rabia; Tovpeko, Yanina; Åmdal, Heidi Aarø; Petersen, Fernanda Cristina; Morrison, Donald A.

doi:10.1007/978-1-4939-6685-1_14

Cited by 17 publications

(13 citation statements)

References 15 publications

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“…The transformation efficiency also depends on the growth phase of the cells. The efficiency is highest in mid-exponential phase, similar to other streptococci transformed with XIP [19,20].…”

Section: Resultssupporting

confidence: 59%

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A ComRS competence pathway in the oral pathogen Streptococcus sobrinus

Wyllie

Jensen

2020

Preprint

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Streptococcus sobrinus is one of two species of bacteria that cause dental caries (tooth decay) in humans. Our knowledge of S. sobrinus is limited despite the organism's important role in oral health. It is widely believed that S. sobrinus lacks the natural competence pathways that are used by other streptococci to regulate growth, virulence, and quorum sensing. The lack of natural competence has also prevented genetic manipulation of S. sobrinus, limiting our knowledge of its pathogenicity.We discovered a functional ComRS competence system in S. sobrinus. The ComRS pathway in S. sobrinus has a unique structure, including two copies of the transcriptional regulator ComR and a peptide pheromone (XIP) that lacks aromatic amino acids. We show that synthetic XIP allows transformation of S. sobrinus with plasmid or linear DNA, and we leverage this newfound genetic tractability to confirm that only one of the ComR homologs is required for induced competence.Although S. sobrinus is typically placed among the mutans group streptococci, the S. sobrinus ComRS system is structurally and functionally similar to the competence pathways in the salivarius group. Like S. salivarius, the ComRS gene cluster in S. sobrinus includes a peptide cleavage/export gene, and the ComRS system appears coupled to a bacteriocin response system. These findings raise questions about the true phylogenetic placement of S. sobrinus.Finally, we identified two strains of S. sobrinus appear to be "cheaters" by either not responding to or not producing XIP. While the mechanisms of cheating could be independent, we show how a recombination event in the non-responsive strain would restore function of the ComRS pathway but delete the gene encoding XIP. Thus the S. sobrinus ComRS pathway provides a lens into the evolution of ecological cheaters.

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Section: Resultssupporting

confidence: 59%

“…S. sobrinus is widely believed to be genetically intractable. The genome of the well-studied S. mutans can be modified by hĳacking the organism's natural competence pathways [18][19][20]. Adding synthetic analogues of CSP or XIP causes S. mutans to readily uptake exogenous DNA.…”

Section: Introductionmentioning

confidence: 99%

A ComRS competence pathway in the oral pathogen Streptococcus sobrinus

Wyllie

Jensen

2020

Preprint

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“…given the fact that maximum concentrations were in most cases observed only close to the stationary phase, which is a period during which competence under laboratory conditions is already shut-off. For some of the strains that produced low levels of XIP, transformation was only detected by using a large PCR amplicon as DNA donor, which is known to result in increased recovery of transformants (35,47).…”

Section: Discussionmentioning

confidence: 99%

Conserved pheromone production, response and degradation byStreptococcus mutans

Filho

Khan

Åmdal

et al. 2019

Preprint

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Streptococcus mutans, a bacterium with high cariogenic potential, coordinates competence for natural transformation and bacteriocin production via the XIP and CSP pheromones. CSP is effective in inducing bacteriocin responses, but not competence in chemically defined media (CDM). This is in contrast to XIP, which is a strong inducer of competence in CDM, but can also stimulate bacteriocin genes as a late response. Inter-connections between the pathways activated by the two pheromones have been characterized in certain detail in S. mutans UA159, but it is mostly unknown whether such findings are representative for the species. In this study, we used bioassays based on luciferase reporters for the bacteriocin gene cipB and the alternative sigma factor sigX to investigate various S. mutans isolates for production and response to CSP and XIP pheromones in CDM. Similar to S. mutans UA159, endogenous CSP was undetectable in the culture supernatants of all tested strains. During optimization of the bioassay using the cipB reporter, we discovered that the acivity of exogenous CSP used as a standard was reduced over time during S. mutans growth. Using a FRET-CSP reporter peptide, we found that S. mutans UA159 was indeed able to degrade CSP, and that such proteolytic activity was not significantly different in isogenic mutants with deletion of the protease gene htrA, or the competence genes sigX, oppD, and comR. CSP proteolysis was also detected in all the wild type strains, indicating that such activity is conserved in S. mutans. For the XIP pheromone, endogenous production was observed in the supernatants of all 34 tested strains at peak concentrations in culture supernatants that varied between 200 nM and 26000 nM. Transformation in the presence of exogenous XIP was detected in all, but one, of the isolates. The efficiency of transformation varied, however, among the different strains, and for those with the highest transformation rates, endogenous XIP peak concentrations in the supernatants were above 2000 nM XIP. We conclude that XIP production and inducing effect on transformation, as well as proteolytic activity leading to the inactivation of CSP are conserved functions among different S. mutans isolates. Understanding the functionality and conservation of pheromone systems in S. mutans may lead to novel strategies to prevent or treat unbalances in oral microbiomes that may favour diseases.

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“…For mutant selection and recovery, antibiotics were used at the following concentrations: kanamycin, 500 g/ml; erythromycin, 10 g/ml; and spectinomycin, 500 g/ml. Preculture stocks were prepared as described previously (57). All peptides utilized in the assays were obtained from GenScript (GenScript, Piscataway, NJ) as custom synthetic peptides and stored as sterile 10 mM solutions at Ϫ20°C (Table 3).…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Signaling System in Streptococcus mitis That Mediates Interspecies Communication with Streptococcus pneumoniae

Junges

Sturød

Salvadori

et al. 2019

Appl Environ Microbiol

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Streptococcus mitis is found in the oral cavity and nasopharynx and forms a significant portion of the human microbiome. In this study, in silico analyses indicated the presence of an Rgg regulator and short hydrophobic peptide (Rgg/SHP) cell-to-cell communication system in S. mitis. Although Rgg presented greater similarity to a repressor in Streptococcus pyogenes, autoinducing assays and genetic mutation analysis revealed that in S. mitis Rgg acts as an activator. Transcriptome analysis showed that in addition to shp, the system regulates two other downstream genes, comprising a segment of a putative lantibiotic gene cluster that is in a conjugative element locus in different members of the mitis group. Close comparison to a similar lantibiotic gene cluster in Streptococcus pneumoniae indicated that S. mitis lacked the full set of genes. Despite the potential of SHP to trigger a futile cycle of autoinduction, growth was not significantly affected for the rgg mutant under normal or antibiotic stress conditions. The S. mitis SHP was, however, fully functional in promoting cross-species communication and increasing S. pneumoniae surface polysaccharide production, which in this species is regulated by Rgg/SHP. The activity of SHPs produced by both species was detected in cocultures using a S. mitis reporter strain. In competitive assays, a slight advantage was observed for the rgg mutants. We conclude that the Rgg/SHP system in S. mitis regulates the expression of its own shp and activates an Rgg/SHP system in S. pneumoniae that regulates surface polysaccharide synthesis. Fundamentally, cross-communication of such systems may have a role during multispecies interactions. IMPORTANCE Bacteria secrete signal molecules into the environment which are sensed by other cells when the density reaches a certain threshold. In this study, we describe a communication system in Streptococcus mitis, a commensal species from the oral cavity, which we also found in several species and strains of streptococci from the mitis group. Further, we show that this system can promote cross-communication with S. pneumoniae, a closely related major human pathogen. Importantly, we show that this cross-communication can take place during coculture. While the genes regulated in S. mitis are likely part of a futile cycle of activation, the target genes in S. pneumoniae are potentially involved in virulence. The understanding of such complex communication networks can provide important insights into the dynamics of bacterial communities.

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Markerless Genome Editing in Competent Streptococci

Cited by 17 publications

References 15 publications

A ComRS competence pathway in the oral pathogen Streptococcus sobrinus

A ComRS competence pathway in the oral pathogen Streptococcus sobrinus

Conserved pheromone production, response and degradation byStreptococcus mutans

Characterization of a Signaling System in Streptococcus mitis That Mediates Interspecies Communication with Streptococcus pneumoniae

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