The properties of staphylococcal phages from the Siphoviridae, Podoviridae, and Myoviridae families were monitored using capillary electrophoretic methods on fused-silica capillaries with different morphology of surface roughness. Isoelectric points of the examined phages were determined by capillary isoelectric focusing in the original, smooth fused-silica capillary, and they ranged from 3.30 to 3.85. For capillary electrophoresis of phages, fused-silica capillaries with the "pock" and "cone" roughened surface types were prepared by etching a part of the capillary with supercritical water. The best resolution of the individual phages (to range from 3.2 to 4.6) was achieved with the "cone" surface-type fused-silica capillary. Direct application of phage K1/420 at the infection site, represented by human plasma or full blood spiked with Staphylococcus aureus, was on-line monitored by micellar electrokinetic chromatography. The phage particles were dynamically adhered onto the roughened surface of the capillary from 10 μL of the prepared sample at the optimized flow rate of 6.5 μL min-1. The limit of detection was determined to be 104 phage particles. The linearity of the calibration lines was characterized by the regression coefficient, R2 = 0.998. The relative standard deviation (RSD) of the peak area, calculated from ten independent measurements, was (±) 2%. After analysis, viability of the detected phages was verified by the modified "double-layer drop assay" method, and collected phage fractions were simultaneously off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Graphical abstract.
BACKGROUND: Next Generation Sequencing (NGS) technologies provide exciting possibilities for whole genome sequencing of a plethora of organisms including bacterial strains and phages, with many possible applications in research and diagnostics. No Streptomyces flavovirens phages have been sequenced to date; there is therefore a lack in available information about S. flavovirens phage genomics. We report biological and physiochemical features and use NGS to provide the complete annotated genomes for two new strains (Sf1 and Sf3) of the virulent phage Streptomyces flavovirens, isolated from Egyptian soil samples. RESULTS: The S. flavovirens phages (Sf1 and Sf3) examined in this study show higher adsorption rates (82 and 85%, respectively) than other actinophages, indicating a strong specificity to their host, and latent periods (15 and 30 min.), followed by rise periods of 45 and 30 min. As expected for actinophages, their burst sizes were 1.95 and 2.49 virions per mL. Both phages were stable and, as reported in previous experiments, showed a significant increase in their activity after sodium chloride (NaCl) and magnesium chloride (MgCl2.6H2O) treatments, whereas after zinc chloride (ZnCl2) application both phages showed a significant decrease in infection. The sequenced phage genomes are parts of a singleton cluster with sizes of 43,150 bp and 60,934 bp, respectively. Bioinformatics analyses and functional characterizations enabled the assignment of possible functions to 19 and 28 putative identified ORFs, which included phage structural proteins, lysis components and metabolic proteins. Thirty phams were identified in both phages, 10 (33.3%) of them with known function, which can be used in cluster prediction. Comparative genomic analysis revealed significant homology between the two phages, showing the highest hits among Sf1, Sf3 and the closest Streptomyces phage (VWB phages) in a specific 13Kb region. However, the phylogenetic analysis using the Major Capsid Protein (MCP) sequences highlighted that the isolated phages belong to the BG Streptomyces phage group but are clearly separated, representing a novel sub-cluster. CONCLUSION: The results of this study provide the first physiological and genomic information for S. flavovirens phages and will be useful for pharmaceutical industries based on S. flavovirens and future phage evolution studies.
- MeSH
- bakteriofágy genetika izolace a purifikace patogenita fyziologie MeSH
- biologická evoluce MeSH
- chlorid hořečnatý farmakologie MeSH
- chlorid sodný farmakologie MeSH
- chloridy farmakologie MeSH
- DNA virů izolace a purifikace MeSH
- fylogeneze MeSH
- genom virový genetika fyziologie MeSH
- genomika MeSH
- hostitelská specificita MeSH
- otevřené čtecí rámce genetika MeSH
- půda MeSH
- půdní mikrobiologie MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie nukleových kyselin MeSH
- sloučeniny zinku farmakologie MeSH
- Streptomyces virologie MeSH
- virion MeSH
- virové geny MeSH
- virové plášťové proteiny genetika MeSH
- virové proteiny genetika MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Egypt MeSH
