DNA‐Interacting characteristics of the archaeal Rudiviral protein SIRV2_Gp1

Whereas the infection cycles of many bacterial and eukaryotic viruses have been characterized in detail, those of archaeal viruses remain largely unexplored. Recently, studies on a few model archaeal viruses such as SIRV2 (Sulfolobus islandicus rod‐shaped virus) have revealed an unusual lysis mechanism that involves the formation of pyramidal egress structures on the host cell surface. To expand understanding of the infection cycle of SIRV2, we aimed to functionally characterize gp1, which is a SIRV2 gene with unknown function. The SIRV2_Gp1 protein is highly expressed during early stages of infection and it is the only protein that is encoded twice on the viral genome. It harbours a helix‐turn‐helix motif and was therefore hypothesized to bind DNA. The DNA‐binding behavior of SIRV2_Gp1 was characterized with electrophoretic mobility shift assays and atomic force microscopy. We provide evidence that the protein interacts with DNA and that it forms large aggregates, thereby causing extreme condensation of the DNA. Furthermore, the N‐terminal domain of the protein mediates toxicity to the viral host Sulfolobus. Our findings may lead to biotechnological applications, such as the development of a toxic peptide for the containment of pathogenic bacteria, and add to our understanding of the Rudiviral infection cycle.Publisher PDFPeer reviewe

Prevalence of Pf1-like (pro)phage genetic elements among Pseudomonas aeruginosa isolates

Pf1-like bacteriophages (family Inoviridae) of Pseudomonas aeruginosa can contribute to bacterial short term evolution and virulence. Here we examine Pf1-like (pro)phage diversity and prevalence among different P. aeruginosa isolates. Pf1-like prophages in sequenced genomes of P. aeruginosa were analyzed and grouped into four clades: Pf4, Pf5, Pf7 and Pf-LES. P. aeruginosa strains (n=241) were screened for the presence of universal (primers PfUa and PfUb) and specific Pf1-like genetic elements (Pf1, Pf4 and Pf5). More than half of the strains contained at least one Pf1-like genetic element (60%); universal elements were detected in 56% of the strains, Pf4 in 22%, Pf1 in 18% and Pf5 in 7%. Infectivity experiments confirmed that strains yielding PCR products with either universal or Pf4 specific primers can release infective virions. Based on the high prevalence of Pf1-like (pro)phages, it is necessary to further examine their involvement in P. aeruginosa virulence.publisher: Elsevier articletitle: Prevalence of Pf1-like (pro)phage genetic elements among Pseudomonas aeruginosa isolates journaltitle: Virology articlelink: http://dx.doi.org/10.1016/j.virol.2015.04.008 content_type: article copyright: Copyright © 2015 Elsevier Inc. All rights reserved.status: publishe

A Protein Interaction Map of the Kalimantacin Biosynthesis Assembly Line

The antimicrobial secondary metabolite kalimantacin (also called batumin) is produced by a hybrid polyketide/non-ribosomal peptide system in Pseudomonas fluorescens BCCM_ID9359. In this study, the kalimantacin biosynthesis gene cluster is analyzed by yeast two-hybrid analysis, creating a protein-protein interaction map of the entire assembly line. In total, 28 potential interactions were identified, of which 13 could be confirmed further. These interactions include the dimerization of ketosynthase domains, a link between assembly line modules 9 and 10, and a specific interaction between the trans-acting enoyl reductase BatK and the carrier proteins of modules 8 and 10. These interactions reveal fundamental insight into the biosynthesis of secondary metabolites. This study is the first to reveal interactions in a complete biosynthetic pathway. Similar future studies could build a strong basis for engineering strategies in such clusters.status: publishe

A protein interaction map of the kalimantacin biosynthesis assembly line

The antimicrobial secondary metabolite kalimantacin is produced by a hybrid polyketide/ non-ribosomal peptide system in Pseudomonas fluorescens BCCM_ID9359. In this study, the kalimantacin biosynthesis gene cluster is analyzed by yeast two-hybrid analysis, creating a protein-protein interaction map of the entire assembly line. In total, 28 potential interactions were identified, of which 13 could be confirmed further. These interactions include the dimerization of ketosynthase domains, a link between assembly line modules 9 and 10, and a specific interaction between the trans-acting enoyl reductase BatK and the carrier proteins of modules 8 and 10. These interactions reveal fundamental insight into the biosynthesis of secondary metabolites.This study is the first to reveal interactions in a complete biosynthetic pathway. Similar future studies could build a strong basis for engineering strategies in such clusters

Four-year clinical evaluation of a self-adhesive luting agent for ceramic inlays

Objectives: The aim of this randomized controlled clinical trial was to evaluate the 4-year clinical performance of a self-adhesive resin cement, RelyX Unicem (3M ESPE), used for cementation of ceramic inlays. In addition, the influence of selectively acid-etching enamel prior to luting on the clinical performance of the restorations was assessed. Methods: Sixty-two IPS Empress 2 inlays/onlays were placed in 31 patients by two experienced clinicians. The restorations were luted with RelyX Unicem with (=experimental group: E) or without (=control group: NE) prior enamel etching with phosphoric acid. At baseline, 6 months, 1, 2 and 4 years after placement, the restorations were assessed by two calibrated investigators using modified USPHS criteria. Ten selected samples of each group were investigated under SEM regarding morphological changes at the cement-inlay interface. Results: The recall rate at 4 years was 97%. Two restorations (1 E, 1 NE) were lost, and one (E) had to be replaced due to inlay and tooth fracture resulting in a survival rate of 95%. No significant differences between the experimental and control group were noticed regarding all criteria (McNemar, p<0.05). An obvious deterioration in marginal integrity was observed after 4 years as only 5% (E=7%; NE=3%) of the restorations exhibited an excellent marginal adaptation. In 90% of the restorations small, still clinically acceptable marginal deficiencies were observed. SEM of the luting gap showed an increased wear of the RelyX Unicem cement over the 4-year period. Conclusions: The self-adhesive luting cement RelyX Unicem can be recommended for bonding of ceramic inlays/onlays. Additional selective enamel etching does not improve the clinical performance of the restorations within the 4-year period.status: publishe

A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for <i>Pseudomonas </i>with efficient vector curing

The development of CRISPR-Cas-based engineering technologies has revolutionized the microbial biotechnology field. Over the years, the Class II Type II CRISPR-Cas9 system has become the gold standard for genome editing in many bacterial hosts. However, the Cas9 system does not allow efficient genomic integration in Pseudomonas putida, an emerging Synthetic Biology host, without the assistance of lambda-Red recombineering. In this work, we utilize the alternative Class I Type I-C CRISPR-Cas3 system from Pseudomonas aeruginosa as a highly efficient genome editing tool for P. putida and P. aeruginosa. This system consists of two vectors, one encoding the Cas genes, CRISPR array and targeting spacer, and a second Standard European Vector Architecture vector, containing the homologous repair template. Both vectors are Golden Gate compatible for rapid cloning and are available with multiple antibiotic markers, for application in various Gram-negative hosts and different designs. By employing this Cas3 system, we successfully integrated an 820-bp cassette in the genome of P. putida and performed several genomic deletions in P. aeruginosa within a week, with an efficiency of &gt;83% for both hosts. Moreover, by introducing a universal self-targeting spacer, the Cas3 system rapidly cures all helper vectors, including itself, from the host strain in a matter of days. As such, this system constitutes a valuable engineering tool for Pseudomonas, to complement the existing range of Cas9-based editing methods, and facilitates genomic engineering efforts of this importan

Omics analyses of potato plant materials using an improved one-class classification tool to identify aberrant compositional profiles in risk assessment procedures

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Impact of phage predation on P. aeruginosa adhered to human airway epithelium: major transcriptomic changes in metabolism and virulence-associated genes

Phage therapy is a promising adjunct therapeutic approach against bacterial multidrug-resistant infections, including Pseudomonas aeruginosa-derived infections. Nevertheless, the current knowledge about the phage-bacteria interaction within a human environment is limited. In this work, we performed a transcriptome analysis of phage-infected P. aeruginosa adhered to a human epithelium (Nuli-1 ATCC® CRL-4011). To this end, we performed RNA-sequencing from a complex mixture comprising phagebacteriahuman cells at early, middle, and late infection and compared it to uninfected adhered bacteria. Overall, we demonstrated that phage genome transcription is unaltered by bacterial growth and phage employs a core strategy of predation through upregulation of prophage-associated genes, a shutdown of bacterial surface receptors, and motility inhibition. In addition, specific responses were captured under lung-simulating conditions, with the expression of genes related to spermidine syntheses, sulphate acquisition, biofilm formation (both alginate and polysaccharide syntheses), lipopolysaccharide (LPS) modification, pyochelin expression, and downregulation of virulence regulators. These responses should be carefully studied in detail to better discern phage-induced changes from bacterial responses against phage. Our results establish the relevance of using complex settings that mimics in vivo conditions to study phage-bacteria interplay, being obvious the phage versatility on bacterial cell invasion.The work was supported by the Fundação para a Ciência e a Tecnologia [SFRH/BD/133193/2017]; Fundação para a Ciência e a Tecnologia [UIDB/04469/2020]; Fundação para a Ciência e a Tecnologia [LA/P/ 0029/2020]; H2020 European Research Council [819800]; H2020 European Research Council [[819800]]; Portuguese Foundation for Science and Technology (FCT) [EXPL/EMD-EMD/1142/2021]; “la Caixa” Foundation [LCF/PR/HP21/52310017].info:eu-repo/semantics/publishedVersio