strain atcc 23877  (ATCC)

Journal: PLOS Biology

Article Title: Prophage induction can facilitate the in vitro dispersal of multicellular Streptomyces structures

doi: 10.1371/journal.pbio.3002725

Figure Lengend Snippet: ( A ) Pairwise comparison of S . ambofaciens ATCC 23877 and DSM 40697 chromosomes. The gray line indicates the position of genes whose order is perfectly conserved between strains. The blue dots indicate areas of synteny break identified using Synteruptor . The red circles correspond to genomic islands containing at least 20 CDS in one of the strains. The blue arrows indicate the position of xSAM1-pSAM2 integrated elements as well as the genomic island of interest harboring Samy prophage, detailed in the panels ( B ) and ( C ). The central compartment (delimitated by the distal rrn operons) , the arms (defined as terminal regions devoid of core genes), the terminal inverted repeats (TIRs), and the position of the origin of replication ( oriC ) in S . ambofaciens ATCC 23877 chromosome are indicated. The level of gene order conservation between both strains as well as the GC percent content calculated in a window of 50 kb are indicated below the dot-plot. The original plot generated by Synteruptor is available on the “S_ambofaciens_close2” database ( https://bioi2.i2bc.paris-saclay.fr/synteruptor/explore_db.php ). The data and scripts underlying the lower panel can be found in and , respectively. ( B ) Schematic representation of the genomic island containing the Samy prophage. The regions in which gene order is perfectly conserved in S . ambofaciens ATCC 23877 and DSM 40697 chromosomes are defined as the left and right synteny blocks (gray). The coordinates of the genomic island borders in S . ambofaciens ATCC 23877 strain are indicated in blue. The genomic island is composed of 2 regions: a remnant integrative element and the Samy prophage. These regions are separated by a short intergenic region (represented by an asterisk; 305 bp located from 6,589,473 to 6,589,777 bp) present in both strains. Two serine integrase coding sequences (red) have been predicted. The prophage also contains 4 tRNA encoding genes (pink). ( C ) Annotation of Samy sequence and comparison with the genome of the phylogenetically related PhiC31 phage. Gene functions are color-coded as detailed in the legend. The annotation of PhiC31 (Lomovskayavirus C31) genes was previously reported in and . Black vertical lines indicate the nine Samy genes identified as overexpressed compared to the entire Samy phage genome across all non- or poorly induced conditions . The genome comparison was performed using Easyfig software (e-value <10 −3 ). The percentage identity between DNA homologous sequences in Samy and PhiC31 is shown in shades of gray. Other abbreviations: DNK, Deoxynucleoside monophosphate kinase; MCP, Major capsid protein; MTP, Major tail protein; RDF, Recombination directionality factor; TSS, Terminal small subunit; TLS, Terminal large subunit; TMP, Tape measure protein.

Article Snippet: In addition, both strains presented different phenotypes in the following days of growth in BM medium: While the strain ATCC 23877 remained relatively dispersed at a density that did not drop much, strain DSM 40697 formed large pellets ( ).

Techniques: Comparison, Generated, Sequencing, Software

Journal: PLOS Biology

Article Title: Prophage induction can facilitate the in vitro dispersal of multicellular Streptomyces structures

doi: 10.1371/journal.pbio.3002725

Figure Lengend Snippet: ( A ) Impact of medium composition on Samy phage production, final pH, and antibacterial activity of S . ambofaciens ATCC 23877 supernatants. The supernatants of S . ambofaciens ATCC 23877 grown in different media (composition summarized below the graph and detailed in ) were harvested after 4 d and 0.2 μm-filtered. Phage titer was determined by qPCR after DNAse treatment. The pH of the medium and the antibacterial activity against Micrococcus luteus of the supernatant after 4 d of growth are indicated on the top. All media had an initial pH of 7.3 (±0.1), except MP5 and MP5 devoid of MOPS, which had a pH of 7.5 (±0.1). All the boxplots represent the first quartile, median, and third quartile. The upper whisker extends from the hinge to the largest value no further than 1.5 * the interquartile range (IQR, i.e., distance between the first and third quartiles). The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. Each dot represents an independent experiment. The p -values of two-sided Wilcoxon rank sum tests with continuity correction is indicated for each comparison to the viral titer observed in HT condition. The data and scripts underlying this panel can be found in and , respectively. ( B ) Kinetics of phage production. S . ambofaciens ATCC 23877 was grown in BM medium. The viral titer of supernatants filtered and DNAse-treated were determined by qPCR. Each color represents an independent experiment. The boxplot is plotted as described in the legend of the panel ( A ). The p -value of two-sided Wilcoxon rank sum tests with continuity correction is 0.0294 when comparing the viral titers at 24 h and 48 h, and 1 (no difference) when comparing the evolution of the titer on the following days. The data and scripts underlying this panel can be found in and , respectively. ( C ) Imaging of Samy phage produced by transmission electron microscopy. S . ambofaciens ATCC 23877 was grown during 4 d in BM medium. The supernatant was concentrated by CsCl-gradient ultracentrifugation. Viral particles were negatively stained with uranyl acetate (also see ). Scale bar: 100 nm.

Article Snippet: In addition, both strains presented different phenotypes in the following days of growth in BM medium: While the strain ATCC 23877 remained relatively dispersed at a density that did not drop much, strain DSM 40697 formed large pellets ( ).

Techniques: Activity Assay, Whisker Assay, Comparison, Imaging, Produced, Transmission Assay, Electron Microscopy, Staining

Journal: PLOS Biology

Article Title: Prophage induction can facilitate the in vitro dispersal of multicellular Streptomyces structures

doi: 10.1371/journal.pbio.3002725

Figure Lengend Snippet: ( A ) Growth and pH in BM and MP5 media. Each symbol represents an independent experiment. Due to the presence of large aggregates (see panel B ), the pseudo-opacimetry (OD 600 nm ) was not exploitable for DSM 40697 strain after 48 h of growth in BM medium. ( B ) Dispersed versus aggregated growth of S . ambofaciens strains in BM media. The results are representative of the appearance of the most frequently observed cultures, as the quantity and size of cell clusters may vary from one experiment to the other. The S . ambofaciens ATCC 23877 Δ Samy strain corresponds to clone #3, deleted from at least the phage integrase by a CRIPSR-based approach ( and ). Scale bar: 1.5 cm. ( C ) Microscopy of Streptomyces colonies after 4 d growth in BM medium. S . ambofaciens ATCC 23877 and its derivate CRISPR-deleted of Samy prophage (clone #3) were grown in BM medium and imaged using differential interference contrast microscopy (also see ). Scale bar: 10 μm. ( D ) Colony-forming units after 4 d of growth in BM medium. The S . ambofaciens ATCC 23877 Δ Samy strains correspond to 3 independent mutants (#1, #3, and #4) we obtained by a CRIPSR-based approach . The boxplot is plotted as described in the legend of . Each dot represents an independent experiment per condition. The p -values of two-sided Wilcoxon rank sum tests with continuity correction are indicated for each comparison. Please note that this number is the result of both cell survival and mycelium dispersal (propensity to form separate colonies). ( E ) Pattern of Samy MCP-mCherry expression in a S . ambofaciens colony. Bacteria encoding a MCP-mCherry fusion were inoculated from plates and grown 4 d in BM medium before red fluorescence imaging. The overlay of red fluorescence and bright field images is shown. Additional images and controls are presented in . Scale bar: 10 μm. ( F ) Correlation between Samy phage production and colony-forming units by diverse S . ambofaciens strains. S . ambofaciens strains obtained from distinct collections were grown during 4 d in BM medium, before counting colony-forming units and phage production in the supernatants. Even strains ATCC 23877 and DSM 40697 were cultured for this experiment from stocks produced by another laboratory. Each dot represents an independent experiment per strain. The correlation was analyzed by a Spearman rank correlation test. Results of 2 independent experiments. ( G ) Bioassay performed with the supernatants of S . ambofaciens ATCC 23877 WT strain and its derivatives deleted in Samy regions (clones #1, #3, and #4; and ). Then 50 μL of filtered supernatant from S . ambofaciens culture after 4 d in BM medium was deposited on a Micrococcus luteus mat, as previously described . The size of the M . luteus growth inhibition halo after 24 h incubation at 37°C was measured. The boxplot is plotted as described in the legend of . The p -values of two-sided Wilcoxon rank sum tests with continuity correction are indicated. ( H ) Model of the stress- and phage-induced dispersed growth of Streptomyces in liquid medium. After germination of the spore, the multicellular bacteria form successively primary and secondary mycelia. In the absence of prophage, the S . ambofaciens DSM 40497 tends to form particularly large and dense pellets in response to metabolic stress encountered in BM medium (nitrogen unbalance, translation inhibition, basic pH). Under the same conditions, phage production by S . ambofaciens ATCC 23877 is correlated with dispersed growth in sparse clumps and filaments. We propose that phage-induced cell death within the secondary mycelium leads to a dislocation of multicellular aggregates. The subsequent increase in the number of small colonies promotes strain dispersal under stressful conditions. The data and scripts underlying the A, D, F, and G panels can be found in and , respectively.

Article Snippet: In addition, both strains presented different phenotypes in the following days of growth in BM medium: While the strain ATCC 23877 remained relatively dispersed at a density that did not drop much, strain DSM 40697 formed large pellets ( ).

Techniques: Microscopy, CRISPR, Comparison, Expressing, Bacteria, Fluorescence, Imaging, Cell Culture, Produced, Bioassay, Clone Assay, Inhibition, Incubation

Journal: PLOS Biology

Article Title: Prophage induction can facilitate the in vitro dispersal of multicellular Streptomyces structures

doi: 10.1371/journal.pbio.3002725

Figure Lengend Snippet: ( A ) Pairwise comparison of S . ambofaciens ATCC 23877 and DSM 40697 chromosomes. The gray line indicates the position of genes whose order is perfectly conserved between strains. The blue dots indicate areas of synteny break identified using Synteruptor . The red circles correspond to genomic islands containing at least 20 CDS in one of the strains. The blue arrows indicate the position of xSAM1-pSAM2 integrated elements as well as the genomic island of interest harboring Samy prophage, detailed in the panels ( B ) and ( C ). The central compartment (delimitated by the distal rrn operons) , the arms (defined as terminal regions devoid of core genes), the terminal inverted repeats (TIRs), and the position of the origin of replication ( oriC ) in S . ambofaciens ATCC 23877 chromosome are indicated. The level of gene order conservation between both strains as well as the GC percent content calculated in a window of 50 kb are indicated below the dot-plot. The original plot generated by Synteruptor is available on the “S_ambofaciens_close2” database ( https://bioi2.i2bc.paris-saclay.fr/synteruptor/explore_db.php ). The data and scripts underlying the lower panel can be found in and , respectively. ( B ) Schematic representation of the genomic island containing the Samy prophage. The regions in which gene order is perfectly conserved in S . ambofaciens ATCC 23877 and DSM 40697 chromosomes are defined as the left and right synteny blocks (gray). The coordinates of the genomic island borders in S . ambofaciens ATCC 23877 strain are indicated in blue. The genomic island is composed of 2 regions: a remnant integrative element and the Samy prophage. These regions are separated by a short intergenic region (represented by an asterisk; 305 bp located from 6,589,473 to 6,589,777 bp) present in both strains. Two serine integrase coding sequences (red) have been predicted. The prophage also contains 4 tRNA encoding genes (pink). ( C ) Annotation of Samy sequence and comparison with the genome of the phylogenetically related PhiC31 phage. Gene functions are color-coded as detailed in the legend. The annotation of PhiC31 (Lomovskayavirus C31) genes was previously reported in and . Black vertical lines indicate the nine Samy genes identified as overexpressed compared to the entire Samy phage genome across all non- or poorly induced conditions . The genome comparison was performed using Easyfig software (e-value <10 −3 ). The percentage identity between DNA homologous sequences in Samy and PhiC31 is shown in shades of gray. Other abbreviations: DNK, Deoxynucleoside monophosphate kinase; MCP, Major capsid protein; MTP, Major tail protein; RDF, Recombination directionality factor; TSS, Terminal small subunit; TLS, Terminal large subunit; TMP, Tape measure protein.

Article Snippet: Remarkably, the number of CFUs was around 2-logs higher in the phage-producing ATCC 23877 strain than in its phage-deleted derivative or the DSM 40697 strain ( ).

Techniques: Comparison, Generated, Sequencing, Software

Journal: PLOS Biology

Article Title: Prophage induction can facilitate the in vitro dispersal of multicellular Streptomyces structures

doi: 10.1371/journal.pbio.3002725

Figure Lengend Snippet: ( A ) Impact of medium composition on Samy phage production, final pH, and antibacterial activity of S . ambofaciens ATCC 23877 supernatants. The supernatants of S . ambofaciens ATCC 23877 grown in different media (composition summarized below the graph and detailed in ) were harvested after 4 d and 0.2 μm-filtered. Phage titer was determined by qPCR after DNAse treatment. The pH of the medium and the antibacterial activity against Micrococcus luteus of the supernatant after 4 d of growth are indicated on the top. All media had an initial pH of 7.3 (±0.1), except MP5 and MP5 devoid of MOPS, which had a pH of 7.5 (±0.1). All the boxplots represent the first quartile, median, and third quartile. The upper whisker extends from the hinge to the largest value no further than 1.5 * the interquartile range (IQR, i.e., distance between the first and third quartiles). The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. Each dot represents an independent experiment. The p -values of two-sided Wilcoxon rank sum tests with continuity correction is indicated for each comparison to the viral titer observed in HT condition. The data and scripts underlying this panel can be found in and , respectively. ( B ) Kinetics of phage production. S . ambofaciens ATCC 23877 was grown in BM medium. The viral titer of supernatants filtered and DNAse-treated were determined by qPCR. Each color represents an independent experiment. The boxplot is plotted as described in the legend of the panel ( A ). The p -value of two-sided Wilcoxon rank sum tests with continuity correction is 0.0294 when comparing the viral titers at 24 h and 48 h, and 1 (no difference) when comparing the evolution of the titer on the following days. The data and scripts underlying this panel can be found in and , respectively. ( C ) Imaging of Samy phage produced by transmission electron microscopy. S . ambofaciens ATCC 23877 was grown during 4 d in BM medium. The supernatant was concentrated by CsCl-gradient ultracentrifugation. Viral particles were negatively stained with uranyl acetate (also see ). Scale bar: 100 nm.

Article Snippet: Remarkably, the number of CFUs was around 2-logs higher in the phage-producing ATCC 23877 strain than in its phage-deleted derivative or the DSM 40697 strain ( ).

Techniques: Activity Assay, Whisker Assay, Comparison, Imaging, Produced, Transmission Assay, Electron Microscopy, Staining

Journal: PLOS Biology

Article Title: Prophage induction can facilitate the in vitro dispersal of multicellular Streptomyces structures

doi: 10.1371/journal.pbio.3002725

Figure Lengend Snippet: ( A ) Growth and pH in BM and MP5 media. Each symbol represents an independent experiment. Due to the presence of large aggregates (see panel B ), the pseudo-opacimetry (OD 600 nm ) was not exploitable for DSM 40697 strain after 48 h of growth in BM medium. ( B ) Dispersed versus aggregated growth of S . ambofaciens strains in BM media. The results are representative of the appearance of the most frequently observed cultures, as the quantity and size of cell clusters may vary from one experiment to the other. The S . ambofaciens ATCC 23877 Δ Samy strain corresponds to clone #3, deleted from at least the phage integrase by a CRIPSR-based approach ( and ). Scale bar: 1.5 cm. ( C ) Microscopy of Streptomyces colonies after 4 d growth in BM medium. S . ambofaciens ATCC 23877 and its derivate CRISPR-deleted of Samy prophage (clone #3) were grown in BM medium and imaged using differential interference contrast microscopy (also see ). Scale bar: 10 μm. ( D ) Colony-forming units after 4 d of growth in BM medium. The S . ambofaciens ATCC 23877 Δ Samy strains correspond to 3 independent mutants (#1, #3, and #4) we obtained by a CRIPSR-based approach . The boxplot is plotted as described in the legend of . Each dot represents an independent experiment per condition. The p -values of two-sided Wilcoxon rank sum tests with continuity correction are indicated for each comparison. Please note that this number is the result of both cell survival and mycelium dispersal (propensity to form separate colonies). ( E ) Pattern of Samy MCP-mCherry expression in a S . ambofaciens colony. Bacteria encoding a MCP-mCherry fusion were inoculated from plates and grown 4 d in BM medium before red fluorescence imaging. The overlay of red fluorescence and bright field images is shown. Additional images and controls are presented in . Scale bar: 10 μm. ( F ) Correlation between Samy phage production and colony-forming units by diverse S . ambofaciens strains. S . ambofaciens strains obtained from distinct collections were grown during 4 d in BM medium, before counting colony-forming units and phage production in the supernatants. Even strains ATCC 23877 and DSM 40697 were cultured for this experiment from stocks produced by another laboratory. Each dot represents an independent experiment per strain. The correlation was analyzed by a Spearman rank correlation test. Results of 2 independent experiments. ( G ) Bioassay performed with the supernatants of S . ambofaciens ATCC 23877 WT strain and its derivatives deleted in Samy regions (clones #1, #3, and #4; and ). Then 50 μL of filtered supernatant from S . ambofaciens culture after 4 d in BM medium was deposited on a Micrococcus luteus mat, as previously described . The size of the M . luteus growth inhibition halo after 24 h incubation at 37°C was measured. The boxplot is plotted as described in the legend of . The p -values of two-sided Wilcoxon rank sum tests with continuity correction are indicated. ( H ) Model of the stress- and phage-induced dispersed growth of Streptomyces in liquid medium. After germination of the spore, the multicellular bacteria form successively primary and secondary mycelia. In the absence of prophage, the S . ambofaciens DSM 40497 tends to form particularly large and dense pellets in response to metabolic stress encountered in BM medium (nitrogen unbalance, translation inhibition, basic pH). Under the same conditions, phage production by S . ambofaciens ATCC 23877 is correlated with dispersed growth in sparse clumps and filaments. We propose that phage-induced cell death within the secondary mycelium leads to a dislocation of multicellular aggregates. The subsequent increase in the number of small colonies promotes strain dispersal under stressful conditions. The data and scripts underlying the A, D, F, and G panels can be found in and , respectively.

Article Snippet: Remarkably, the number of CFUs was around 2-logs higher in the phage-producing ATCC 23877 strain than in its phage-deleted derivative or the DSM 40697 strain ( ).

Techniques: Microscopy, CRISPR, Comparison, Expressing, Bacteria, Fluorescence, Imaging, Cell Culture, Produced, Bioassay, Clone Assay, Inhibition, Incubation