Dataset Information

Phage ORF family recombinases: conservation of activities and involvement of the central channel in DNA binding.

ABSTRACT: Genetic and biochemical evidence suggests that ? Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Red? recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its ? relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the ? Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination.

SUBMITTER: Curtis FA

PROVIDER: S-EPMC4118853 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Phage ORF family recombinases: conservation of activities and involvement of the central channel in DNA binding.

Curtis Fiona A FA Malay Ali D AD Trotter Alexander J AJ Wilson Lindsay A LA Barradell-Black Michael M H MM Bowers Laura Y LY Reed Patricia P Hillyar Christopher R T CR Yeo Robert P RP Sanderson John M JM Heddle Jonathan G JG Sharples Gary J GJ

PloS one 20140801 8

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redβ recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic propha ...[more]

PMID: 25083707

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Project description:Several strains in the Pseudomonas genus are categorized as plant growth-promoting rhizobacteria (PGPR). Although several of these strains are strong candidates for applications as biofertilizers or biopesticides, genome editing approaches are generally limited and require further development. Editing genomes in PGPR could enable more robust agricultural applications, persistence, and biosafety measures. In this study, we investigate the use of five phage-encoded recombinases to develop a recombineering workflow in three PGPR strains: Pseudomonas protegens Pf-5, Pseudomonas protegens CHA0, and Pseudomonas putida KT2440. Using point mutations in the rpoB gene, we reach maximum recombineering efficiencies of 1.5 × 10-4, 3 × 10-4, and 5 × 10-5, respectively, in these strains using λ-Red Beta recombinase from Escherichia coli. We further examine recombineering efficiencies across these strains as a function of selected mutation, editing template concentration, and phosphorothiolate bond protection. This work validates the use of these tools across several environmentally and biotechnologically relevant strains to expand the possibilities of genetic manipulation in the Pseudomonas genus. IMPORTANCE The Pseudomonas genus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol, and synthetic biology. Though several strains have been identified with beneficial properties, chromosomal manipulations to further improve these strains for commercial applications have been limited due to the lack of efficient genetic tools that have been tested across this genus. Here, we test the recombineering efficiencies of five phage-derived recombinases across three biotechnologically relevant Pseudomonas strains: P. putida KT2440, P. protegens Pf-5, and P. protegens CHA0. These results demonstrate a method to generate targeted mutations quickly and efficiently across these strains, ideally introducing a method that can be implemented across the Pseudomonas genus and a strategy that may be applied to develop analogous systems in other nonmodel bacteria.

Project description:BackgroundStreptococcus uberis is one of the main causative agents of ovine mastitis, however little is known about this global, environmental pathogen and its genomic mechanisms of disease. In this study, we performed genomic analysis on 46 S. uberis isolates collected from mastitis-infected sheep in Sardinia (Italy).ResultsGenomes were assigned into lineage clusters using PopPUNK, which found 27 distinct isolate clusters, indicating considerable genetic variability consistent with environmental isolates. Geographic trends were identified including regional linkage of several isolate clusters. Multi-locus Sequence Typing (MLST) performed poorly and provided no new insights. Genomes were then screened for antimicrobial resistance genes, which were compared to phenotypic resistance profiles. Isolates showed consistent phenotypic resistance to aminoglycosides with variable resistance to novobiocin and tetracycline. In general, identification of antimicrobial resistance genes did not correlate with phenotypic resistance profiles, indicating unknown genetic determinants. A multi-antimicrobial resistance cassette (aminoglycoside, lincosamide and streptogramin) was identified in the chromosome of three genomes, flanked by vestigial phage recombinases. This locus appears to have spread horizontally within discrete S. uberis populations within a 40 km radius (Sassari region). Genomes were screened for putative virulence factors, which identified 16 genes conserved between sheep and cow isolates, with no host-specific genes shared uniformly across all host-specific isolates. Pangenomic analysis was then performed to identify core genes which were putatively surface-exposed, for identification of potential vaccine targets. As all genomes encoded sortase, core genes were screened for the sortase cleavage motif. Of the 1445 core S. uberis genes, 64 were putative sortase substrates and were predominantly adhesins, permeases and peptidases, consistent with compounds found within ruminant milk such as xanthine, fibronectin and lactoferrin.ConclusionsThis study demonstrated the importance of whole genome sequencing for surveillance of S. uberis and tracking horizontal acquisition of antimicrobial resistance genes, as well as providing insight into genetic determinants of disease, which cannot be inferred from the MLST schemes. Future mastitis surveillance should be informed by genomic analysis.

Dataset Information

Phage ORF family recombinases: conservation of activities and involvement of the central channel in DNA binding.

Publications

Phage ORF family recombinases: conservation of activities and involvement of the central channel in DNA binding.

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