Low Efficiency of Homology-Facilitated Illegitimate Recombination during Conjugation in Escherichia coli

Homology-facilitated illegitimate recombination has been described in three naturally competent bacterial species. It permits integration of small linear DNA molecules into the chromosome by homologous recombination at one end of the linear DNA substrate, and illegitimate recombination at the other end. We report that homology-facilitated illegitimate recombination also occurs in Escherichia coli during conjugation with small non-replicative plasmids, but at a low frequency of 3×10−10 per recipient cell. The fate of linear DNA in E. coli is either RecBCD-dependent degradation, or circularisation by ligation, and integration into the chromosome by single crossing-over. We also report that the observed single crossing-overs are recA-dependent, but essentially recBCD, and recFOR independent. This suggests that other, still unknown, proteins may act as mediator for the loading of RecA on DNA during single crossing-over recombination in E. coli

CAGO: A Software Tool for Dynamic Visual Comparison and Correlation Measurement of Genome Organization

CAGO (Comparative Analysis of Genome Organization) is developed to address two critical shortcomings of conventional genome atlas plotters: lack of dynamic exploratory functions and absence of signal analysis for genomic properties. With dynamic exploratory functions, users can directly manipulate chromosome tracks of a genome atlas and intuitively identify distinct genomic signals by visual comparison. Signal analysis of genomic properties can further detect inconspicuous patterns from noisy genomic properties and calculate correlations between genomic properties across various genomes. To implement dynamic exploratory functions, CAGO presents each genome atlas in Scalable Vector Graphics (SVG) format and allows users to interact with it using a SVG viewer through JavaScript. Signal analysis functions are implemented using R statistical software and a discrete wavelet transformation package waveslim. CAGO is not only a plotter for generating complex genome atlases, but also a platform for exploring genome atlases with dynamic exploratory functions for visual comparison and with signal analysis for comparing genomic properties across multiple organisms. The web-based application of CAGO, its source code, user guides, video demos, and live examples are publicly available and can be accessed at http://cbs.ym.edu.tw/cago

Architecture, flexibility and performance of a special electron linac dedicated to Flash radiotherapy research: electronFlash with a triode gun of the centro pisano flash radiotherapy (CPFR)

The FLASH effect is a radiobiological phenomenon that has garnered considerable interest in the clinical field. Pre-clinical experimental studies have highlighted its potential to reduce side effects on healthy tissues while maintaining isoeffectiveness on tumor tissues, thus widening the therapeutic window and enhancing the effectiveness of radiotherapy. The FLASH effect is achieved through the administration of the complete therapeutic radiation dose within a brief time frame, shorter than 200 milliseconds, and, therefore, utilizing remarkably high average dose rates above at least 40 Gy/s. Despite its potential in radiotherapy, the radiobiological mechanisms governing this effect and its quantitative relationship with temporal parameters of the radiation beam, such as dose-rate, dose-per-pulse, and average dose-rate within the pulse, remain inadequately elucidated. A more profound comprehension of these underlying mechanisms is imperative to optimize the clinical application and translation of the FLASH effect into routine practice. Due to the aforementioned factors, the undertaking of quantitative radiobiological investigations becomes imperative, necessitating the utilization of sophisticated and adaptable apparatus capable of generating radiation beams with exceedingly high dose-rates and dose-per-pulse characteristics. This study presents a comprehensive account of the design and operational capabilities of a Linear Accelerator (LINAC) explicitly tailored for FLASH radiotherapy research purposes. Termed the “ElectronFlash” (EF) LINAC, this specialized system employs a low-energy configuration (7 and 9 MeV) and incorporates a triode gun. The EF LINAC is currently operational at the Centro Pisano FLASH Radiotherapy (CPFR) facility located in Pisa, Italy. Lastly, this study presents specific instances exemplifying the LINAC’s adaptability, enabling the execution of hitherto unprecedented experiments. By enabling independent variations of the temporal parameters of the radiation beam implicated in the FLASH effect, these experiments facilitate the acquisition of quantitative data concerning the effect’s dependence on these specific parameters. This novel approach hopefully contributes to a more comprehensive understanding of the FLASH effect, shedding light on its intricate radiobiological behavior and offering valuable insights for optimizing its clinical implementation

Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome

<p><u>(A) Circular map of the <i>E</i>. <i>coli</i> chromosome</u>: <i>oriC</i>, <i>dif</i> and <i>terD</i> to <i>terB</i> sites are indicated. Numbers refer to the chromosome coordinates (in kb) of MG1655. (<u>B) Linear map of the terminus region:</u> chromosome coordinates are shown increasing from left to right, as in the marker frequency panels (see Figure 1C for example), therefore in the opposite direction to the circular map. In addition to <i>dif</i> and <i>ter</i> sites, the positions of the <i>parS</i>_pMT1 sites used for microscopy experiments are indicated. (<u>C) MFA analysis of terminus DNA loss in the <i>recB</i> mutant</u>: sequence read frequencies of exponential phase cells normalized to the total number of reads were calculated for each strain. Ratios of normalized reads in isogenic wild-type and <i>recB</i> mutant are plotted against chromosomal coordinates (in kb). The profile ratio of the terminus region is enlarged and the profile of the corresponding entire chromosomes is shown in inset. Original normalized profiles used to calculate ratios are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007256#pgen.1007256.s005" target="_blank">S1 Fig</a>. The position of <i>dif</i> is indicated by a red arrow. The <i>ter</i> sites that arrest clockwise forks (<i>terC</i>, <i>terB</i>, green arrow) and counter-clockwise forks (<i>terA</i>, <i>terD</i>, blue arrow) are shown. <u>(D) Schematic representation of focus loss in the <i>recB</i> mutant:</u> Time-lapse microscopy experiments showed that loss of a focus in the <i>recB</i> mutant occurs concomitantly with cell division in one of two daughter cells, and that the cell that keeps the focus then generates a focus-less cell at each generation. The percentage of initial events was calculated as the percentage of cell divisions that generate a focus-less cell, not counting the following generations. In this schematic representation, two initial events occurred (generations #2 and #7) out of 9 generations, and focus loss at generation #2 is heritable. Panels shown in this figure were previously published in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007256#pgen.1007256.ref019" target="_blank">19</a>] and are reproduced here to introduce the phenomenon.</p

SIGffRid: A tool to search for sigma factor binding sites in bacterial genomes using comparative approach and biologically driven statistics

<p>Abstract</p> <p>Background</p> <p>Many programs have been developed to identify transcription factor binding sites. However, most of them are not able to infer two-word motifs with variable spacer lengths. This case is encountered for RNA polymerase Sigma (<it>σ</it>) Factor Binding Sites (SFBSs) usually composed of two boxes, called -35 and -10 in reference to the transcription initiation point. Our goal is to design an algorithm detecting SFBS by using combinational and statistical constraints deduced from biological observations.</p> <p>Results</p> <p>We describe a new approach to identify SFBSs by comparing two related bacterial genomes. The method, named SIGffRid (SIGma Factor binding sites Finder using R'MES to select Input Data), performs a simultaneous analysis of pairs of promoter regions of orthologous genes. SIGffRid uses a prior identification of over-represented patterns in whole genomes as selection criteria for potential -35 and -10 boxes. These patterns are then grouped using pairs of short seeds (of which one is possibly gapped), allowing a variable-length spacer between them. Next, the motifs are extended guided by statistical considerations, a feature that ensures a selection of motifs with statistically relevant properties. We applied our method to the pair of related bacterial genomes of <it>Streptomyces coelicolor </it>and <it>Streptomyces avermitilis</it>. Cross-check with the well-defined SFBSs of the SigR regulon in <it>S. coelicolor </it>is detailed, validating the algorithm. SFBSs for HrdB and BldN were also found; and the results suggested some new targets for these <it>σ </it>factors. In addition, consensus motifs for BldD and new SFBSs binding sites were defined, overlapping previously proposed consensuses. Relevant tests were carried out also on bacteria with moderate GC content (i.e. <it>Escherichia coli</it>/<it>Salmonella typhimurium </it>and <it>Bacillus subtilis</it>/<it>Bacillus licheniformis </it>pairs). Motifs of house-keeping <it>σ </it>factors were found as well as other SFBSs such as that of SigW in <it>Bacillus </it>strains.</p> <p>Conclusion</p> <p>We demonstrate that our approach combining statistical and biological criteria was successful to predict SFBSs. The method versatility autorizes the recognition of other kinds of two-box regulatory sites.</p

SIGffRid: A tool to search for sigma factor binding sites in bacterial genomes using comparative approach and biologically driven statistics

<p>Abstract</p> <p>Background</p> <p>Many programs have been developed to identify transcription factor binding sites. However, most of them are not able to infer two-word motifs with variable spacer lengths. This case is encountered for RNA polymerase Sigma (<it>σ</it>) Factor Binding Sites (SFBSs) usually composed of two boxes, called -35 and -10 in reference to the transcription initiation point. Our goal is to design an algorithm detecting SFBS by using combinational and statistical constraints deduced from biological observations.</p> <p>Results</p> <p>We describe a new approach to identify SFBSs by comparing two related bacterial genomes. The method, named SIGffRid (SIGma Factor binding sites Finder using R'MES to select Input Data), performs a simultaneous analysis of pairs of promoter regions of orthologous genes. SIGffRid uses a prior identification of over-represented patterns in whole genomes as selection criteria for potential -35 and -10 boxes. These patterns are then grouped using pairs of short seeds (of which one is possibly gapped), allowing a variable-length spacer between them. Next, the motifs are extended guided by statistical considerations, a feature that ensures a selection of motifs with statistically relevant properties. We applied our method to the pair of related bacterial genomes of <it>Streptomyces coelicolor </it>and <it>Streptomyces avermitilis</it>. Cross-check with the well-defined SFBSs of the SigR regulon in <it>S. coelicolor </it>is detailed, validating the algorithm. SFBSs for HrdB and BldN were also found; and the results suggested some new targets for these <it>σ </it>factors. In addition, consensus motifs for BldD and new SFBSs binding sites were defined, overlapping previously proposed consensuses. Relevant tests were carried out also on bacteria with moderate GC content (i.e. <it>Escherichia coli</it>/<it>Salmonella typhimurium </it>and <it>Bacillus subtilis</it>/<it>Bacillus licheniformis </it>pairs). Motifs of house-keeping <it>σ </it>factors were found as well as other SFBSs such as that of SigW in <it>Bacillus </it>strains.</p> <p>Conclusion</p> <p>We demonstrate that our approach combining statistical and biological criteria was successful to predict SFBSs. The method versatility autorizes the recognition of other kinds of two-box regulatory sites.</p

Isolation, in vitro characterization and efficacy assessment in Galleria mellonella larvae of four bacteriophages targeting Aeromonas salmonicida

The Gram-negative bacteria Aeromonas (A.) salmonicida is a primary fish pathogen that causes furunculosis in salmonids as well as septicemia in a variety of fish. In one hand because this disease is responsible for significant losses in salmonid production worldwide and in the other hand because of the frightening tendency of this bacteria to exhibit antimicrobial (multi-)resistances, phage therapy could represent a leading alternative to treat this infection in aquaculture. The aims of this study were to create a collection of A. salmonicida strains, isolate phages targeting these strains, phenotypically and genomically characterize these newly isolated phages and finally assess their potential for phage therapy in the preliminary in vivo model of Galleria (G.) mellonella larvae. Four new phages active against A. salmonicida were isolated from water samples collected in fish farms and natural aquatic environments in southern Belgium. Genomic analysis showed that 3 of these phages, named vB_AsaM_ULASA2 (170,823bp), vB_AsaM_ULASA3 (164,381bp) and vB_AsaM_ULASA4 (171,205bp), belong to the Straboviridae family while vB_AsaM_ULASA1 (47,813bp) stay in the unclassified part of the Caudoviricetes class. All 4 presented a myovirus morphotype. Four-day efficacy experiments in the preliminary in vivo model of G. mellonella larvae showed that 3 of these 4 phages were responsible for a significant extension in the larval survival time at the 2 treatment doses tested (MOI 10 and 100). In light of these results, these phages targeting A. salmonicida could represent potential new candidates for the development of anti-furunculosis phage treatments in aquaculture

Isolation, in vitro characterization and efficacy assessment in Galleria mellonella larvae of four bacteriophages targeting Aeromonas salmonicida

The Gram-negative bacteria Aeromonas (A.) salmonicida is a primary fish pathogen that causes furunculosis in salmonids, carp and perch, as well as septicemia in a variety of fish. This species is considered as one of the main bacterial pathogens responsible for important economic losses in aquaculture industry. Large amounts of antibiotics are used to treat this infection, which highly contributes to the emergence of antibiotic-resistant strains. The application of bacteriophages (phages) in aquaculture seems to be a promising solution to control pathogenic bacteria in this field. The aims of this work were to isolate new phages active against A. salmonicida, characterize them in vitro and assess their potential use for phage therapy in a preliminary in vivo model. Four new phages were isolated from water samples collected in fish farms and natural aquatic environments in southern Belgium. Transmission electron microscopy allowed to classify these four phages in the Caudoviricetes class which includes tailed-phages with dsDNA and an icosahedral capsid. Genomic analysis showed that three of these phages, named vB_AsaM_ULASA2 (170,823bp), vB_AsaM_ULASA3 (164,381bp) and vB_AsaM_ULASA4 (171,205bp), belong to the Straboviridae family while vB_AsaM_ULASA1 (47,813bp) stay in the unclassified part of the Caudoviricetes class. Four strains of A. salmonicida were tested for virulence on Galleria mellonella larvae and two of them, named ATCC7965 and Asa-CER1, were selected for further experiments. Inoculation doses were determined as 102 CFU/10 µl and 104 CFU/10 µl, respectively. The safety and efficacy of the four phages at MOI 10 and 100 in this model are currently assessed

In vitro characterization and preliminary efficacy assessment in galleria mellonella larvae of four newly isolated bacteriophages targeting aeromonas salmonicida

The Gram-negative bacteria Aeromonas (A.) salmonicida is a primary fish pathogen that causes furunculosis in salmonids as well as septicemia in a variety of fish. In one hand because this disease is responsible for significant losses in salmonid production worldwide and in the other hand because of the frightening tendency of this bacteria to exhibit antimicrobial (multi-)resistances, phage therapy could represent a leading alternative to treat this infection in aquaculture. The aims of this study were to create a collection of A. salmonicida strains, isolate phages targeting these strains, phenotypically and genomically characterize these newly isolated phages and finally assess their potential for phage therapy in the preliminary in vivo model of Galleria (G.) mellonella larvae. Four new phages active against A. salmonicida were isolated from water samples collected in fish farms and natural aquatic environments in southern Belgium. Genomic analysis showed that 3 of these phages, named vB_AsaM_ULASA2 (170,823bp), vB_AsaM_ULASA3 (164,381bp) and vB_AsaM_ULASA4 (171,205bp), belong to the Straboviridae family while vB_AsaM_ULASA1 (47,813bp) stay in the unclassified part of the Caudoviricetes class. All 4 presented a myovirus morphotype. Four-day efficacy experiments in the preliminary in vivo model of G. mellonella larvae showed that 3 of these 4 phages were responsible for a significant extension in the larval survival time at the 2 treatment doses tested (MOI 10 and 100). In light of these results, these phages targeting A. salmonicida could represent potential new candidates for the development of anti-furunculosis phage treatments in aquacultur

Genomic insight to understand the persistence of Listeria monocytogenes strains in processing environments of pork products

Listeria monocytogenes (L. monocytogenes) is an ubiquitous bacterium that causes a severe foodborne illness. It is established that the contamination of food production facilities over long time period, are potentially one of the major food product contamination sources. L. monocytogenes persistence was observed in almost all food sectors and particularly in pork production facilities. The characterization of such L. monocytogenes contamination is therefore crucial to improve the food safety and prevent outbreaks. These strains are called persistent but this trait remains loosely defined, and no genetic determinants have been firmly associated with it. This study aims at identifying molecular markers associated with the persistence. A panel of 13 presumed persistent (PP) strains, versus 9 strains not exposed to food processing environment (NEP) strains, was constructed from the databases of the French Institute for Pig and Pork Industry (Ifip), the French National Reference Laboratory (NRL) and the European Union Reference Laboratory (EURL), the last two are hosted by Anses. The genome sequences obtained in the present study were compared to 180 genomes of the Anses strains reference collection and 340 genomes publicly available. Two analysis were performed on the genomes, (i) an allele diversity analysis of 14 loci gathered from a review of significant functions potentially involved in the persistence capacity and (ii) a whole genome variant calling analysis to detect single nucleotide polymorphisms (SNPs) and insertions, deletions specific of persistent strains. The preliminary results were obtained on 4 strains. The comparison of these whole genome sequencing (WGS) data with those of the whole strain panel is ongoing.</p