Functional and practical insights into three lactococcal antiphage systems DOI
Andriana Grafakou, Cas Mosterd,

Paul P. de Waal

et al.

Applied and Environmental Microbiology, Journal Year: 2024, Volume and Issue: 90(9)

Published: Aug. 13, 2024

ABSTRACT The persistent challenge of phages in dairy fermentations requires the development starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems found to confer high levels resistance against various Skunavirus members. In present study, their effectiveness infection was confirmed milk-based medium, thus validating potential ensure reliable fermentations. We furthermore demonstrated that Rhea Kamadhenu do not directly hinder genome replication, transcription, or associated translation. Conversely, Aristaios interfere transcription. Two are encoded on pMRC01-like conjugative plasmids, Kamadhenu-encoding plasmid successfully transferred by conjugation strains, each which acquired substantially Such advances our knowledge resistome possibility mobilizing these protective functions bolster protection sensitive strains provide practical solutions ongoing problem industrial food IMPORTANCE current we characterized evaluated mechanistic diversity recently described, systems. many members most prevalent problematic genus, rendering them particular interest industry, where characteristics. Our highlights understanding encoding plasmids can rational effective enduring issue infections fermentation facilities.

Language: Английский

Insight into crRNA Processing in Streptococcus mutans P42S and Application of SmutCas9 in Genome Editing DOI Open Access
Cas Mosterd, Sylvain Moineau

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(5), P. 2005 - 2005

Published: Feb. 25, 2025

CRISPR-Cas is an adaptive immune system found in bacteria and archaea that provides resistance against invading nucleic acids. Elements of this natural have been harnessed to develop several genome editing tools, including CRISPR-Cas9. This technology relies on the ability nuclease Cas9 cut DNA at specific locations directed by a guide RNA. In addition, activity requires presence short nucleotide motif (5'-NGG-3' for from Streptococcus pyogenes) called PAM, flanking targeted region. As reliance PAM typically strict, diverse variants recognising different motifs studied target broader range genomic sites. study, we assessed potential mutans strain P42S (SmutCas9) gene editing. SmutCas9 recognises rarely 5'-NAA-3' 5'-NGAA-3' PAMs. To test its efficacy, two genes virulent lactococcal phage p2 were edited, thereby demonstrating purposes, particularly AT-rich genomes. Sequencing total RNA also revealed components system, allowing further molecular characterisation type II-A S. mutans.

Language: Английский

Citations

0
Deciphering the RNA-based regulation mechanism of the phage-encoded AbiF system inClostridioides difficile DOI Creative Commons

Marion Saunier,

Adeline Humbert,

Victor Kreis

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: April 15, 2025

Abstract Clostridioides difficile is the major cause of nosocomial infections associated with antibiotic therapy. The severity C. increased worldwide emergence hypervirulent strains, including 027 ribotype epidemic strains. Many aspects ’s adaptation strategies during pathogenesis remain poorly understood. This pathogen thrives in gut communities that are rich microbes and phages. To regulate horizontal transfer genetic material its infection cycle, relies on diverse mechanisms. More specifically, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas Toxin-Antitoxin (TA) systems contribute to prophage maintenance, prevention phage infection, stress response. Abortive (Abi) can provide additional lines anti-phage defense. RNAs have emerged as key components these antitoxin within type I III TA. We report here identification a new AbiF-like system strain R20291. It an Abi_2/AbiD/F protein family largely distributed Bacillota Pseudomonadota structural links ancestral Cas13 proteins at origin RNA-targeting CRISPR-Cas13 systems. demonstrated toxic activity AbiF Cd Escherichia coli negative regulation abiF expression by non-coding RNA RCd22. RCd22 contains two conserved motifs active both cis trans neutralize toxin direct RNA-protein interaction, similar A mass spectrometry interactomics analysis fractions from MS2-Affinity Purification coupled sequencing (MAPS) revealed among most enriched partners . Structural modeling complex mutagenesis positions for this interaction activity. In summary, findings valuable insights into mechanisms between bacteria phages, which pertinent advancement therapy, genome editing, epidemiological surveillance, formulation novel therapeutic approaches.

Language: Английский

Citations

0
A Plasmid-Encoded Surface Polysaccharide Partly Blocks Ceduovirus Infection in Lactococci DOI Open Access
Claudia Rendueles, Javier Nicolás Garay‐Novillo, Martin Holm Rau

et al.

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(6), P. 2508 - 2508

Published: March 11, 2025

Bacteriophages (or phages) remain the leading cause of failure in dairy fermentations. Thereby, phage-resistant Lactococcus lactis and cremoris starters are continuous demand. In this work, our goal was to identify phage defense mechanisms against ceduoviruses encoded by two wild isolates origin named L. IPLA517 IPLA1064. These strains were previously subjected experimental evolution select derivatives that resistant bacteriocin Lcn972. It observed Lcn972R became sensitive infection; however, underlying mechanism not defined. The long-read sequencing technologies applied work reveal all shared loss a 41 kb endogenous plasmid (p41) harbors putative exopolysaccharide (EPS) gene cluster with significant homology one described garvieae. Using CRISPR-Cas9-based approach, p41 selectively cured from Phage infection assays three demonstrated curing restored sensitivity at levels comparable Lcn972R-IPLA1064 derivatives. adsorption Δp41 cells also increased, consistent hypothesis EPS production hindering access receptor protein Pip. Our results reinforce role EPSs protecting infection, phenomenon is rarely reported for ceduoviruses. Moreover, exemplify likely horizontal transfer can occur between garvieae environment.

Language: Английский

Citations

0
Functional and practical insights into three lactococcal antiphage systems DOI
Andriana Grafakou, Cas Mosterd,

Paul P. de Waal

et al.

Applied and Environmental Microbiology, Journal Year: 2024, Volume and Issue: 90(9)

Published: Aug. 13, 2024

ABSTRACT The persistent challenge of phages in dairy fermentations requires the development starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems found to confer high levels resistance against various Skunavirus members. In present study, their effectiveness infection was confirmed milk-based medium, thus validating potential ensure reliable fermentations. We furthermore demonstrated that Rhea Kamadhenu do not directly hinder genome replication, transcription, or associated translation. Conversely, Aristaios interfere transcription. Two are encoded on pMRC01-like conjugative plasmids, Kamadhenu-encoding plasmid successfully transferred by conjugation strains, each which acquired substantially Such advances our knowledge resistome possibility mobilizing these protective functions bolster protection sensitive strains provide practical solutions ongoing problem industrial food IMPORTANCE current we characterized evaluated mechanistic diversity recently described, systems. many members most prevalent problematic genus, rendering them particular interest industry, where characteristics. Our highlights understanding encoding plasmids can rational effective enduring issue infections fermentation facilities.

Language: Английский

Citations

1