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Prophage induction can facilitate the in vitro dispersal of multicellular Streptomyces structures [1]

['Hoda Jaffal', 'Université Paris-Saclay', 'Cea', 'Cnrs', 'Institute For Integrative Biology Of The Cell', 'Gif-Sur-Yvette', 'Mounia Kortebi', 'Pauline Misson', 'Inrae', 'Agroparistech']

Date: 2024-07 

Streptomyces are renowned for their prolific production of specialized metabolites with applications in medicine and agriculture. These multicellular bacteria present a sophisticated developmental cycle and play a key role in soil ecology. Little is known about the impact of Streptomyces phage on bacterial physiology. In this study, we investigated the conditions governing the expression and production of “Samy,” a prophage found in Streptomyces ambofaciens ATCC 23877. This siphoprophage is produced simultaneously with the activation of other mobile genetic elements. Remarkably, the presence and production of Samy increases bacterial dispersal under in vitro stress conditions. Altogether, this study unveiled a new property of a bacteriophage infection in the context of multicellular aggregate dynamics.

Data Availability: The RNA-seq data generated during this study have been deposited in the NCBI Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/ ) under the accession code GSE232795 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE232795 ). The virome sequencing data are available in the bioproject PRJNA974565 on SRA ( https://www.ncbi.nlm.nih.gov/bioproject/974565 ). We used RNA-seq data available under the accession code GSE162865 ( https://www-ncbi-nlm-nih-gov.insb.bib.cnrs.fr/geo/query/acc.cgi?acc=GSE232795 ). Samy phage complete sequence is available on GenBank under the following accession number: OR263580.1. The Bioproject accession number of this study is PRJEB62744. The numeric data used to generate Figs 1A , 2A–2B , 3A–3B , 4A, 4D, 4F, 4G , S2 , S3A–S3C , S4 and S11 are available in S1 Data . The scripts used to generate these figures, along with the script used to run the SARTools DESeq2-based R pipeline, are provided in S2 Data . The SARTools statistical report is included in S3 Data .

In this study, we investigated the (pro)phage biology of Streptomyces ambofaciens, a strain valued for its spiramycin production. Although the central chromosome region is considered as a hotspot for integrative element insertion [ 29 ], this strain is predicted to harbor a complete prophage in its terminal chromosome compartment. We previously reported that in exponential phase, this compartment is less active transcriptionally compared to the central region and exhibits higher levels of antisense-oriented transcription, especially in the predicted prophage region [ 30 ]. Here, we characterize the condition of production of this novel temperate phage from S. ambofaciens ATCC 23877, which we named “Samy.” This prophage is induced under metabolic stress and activated at the same time as other mobile genetic elements. We observed that this induction promotes bacterial dispersal under in vitro stress conditions. This study reports a new property resulting from phage–bacteria interaction in the context of multicellularity.

The last few years have been marked by a sustained effort in the isolation and sequencing of Streptomyces phages from environmental samples [ 12 , 19 – 21 , 22 – 25 ]. Moreover, the Streptomyces genome can constitute per se a viral genetic reservoir, through the genomes of temperate prophages that they may host. Indeed, prophage-like sequences are detected in about half of the Actinobacteria [ 26 , 27 ], including 62.4% of Streptomyces genomes [ 26 ]. While experimental validation is necessary to assess the functionality of these regions, this suggests a significant role for temperate phages in shaping the population dynamics of these bacteria. The prophage state is, perhaps, one of the most important stages in the interaction between phage and host genomes. Indeed, the lysogenic cycle represents an opportunity for phages to confer new properties to their host notably through the expression of moron genes that may contribute to bacterial fitness and environmental niche expansion [ 28 ].

By causing lysis of their host, some Streptomyces phages are likely to contribute to the death process of a part of the colony and/or to premature termination of antibiotic production. Accordingly, they can lead to industrial fermentation failure [ 11 ]. Moreover, Streptomyces–phage interplay may also encompass specific traits linked to the complex life cycle of these bacteria. For instance, some phages can induce the release of specialized metabolites [ 12 , 13 ]. Reciprocally, some bioactive compounds produced by Streptomyces can act as antiphage defenses [ 14 – 16 ]. Furthermore, some Streptomyces phages encode homologs of sporulation and antibiotic production regulators [ 17 ] and can impact the developmental cycle of Streptomyces [ 13 ]. Reciprocally, susceptibility to phage infection varies along this cycle [ 13 , 18 ].

While Streptomyces is the largest prokaryotic genus with over 900 species (Genome Taxonomy Database, Release 08-RS214), relatively few Streptomyces phages have been characterized to date. They represent less than 2.5% of the bacterial viruses listed by the International Committee on Taxonomy of Viruses (ICTV) (release 21–221122_MSL37) or the National Center for Biotechnology Information (NCBI) and only 7.5% of the Actinobacteriophage Database [ 6 ]. The Streptomyces phages identified so far are double stranded DNA viruses belonging to the Caudoviricetes class or Tectiviridae family, with the possible exception of an RNA virus detected in a Streptomyces transcriptome [ 7 ]. Nevertheless, the mechanisms by which bacteriophages recognize, attach to, multiply into, and eventually propagate within multicellular mycelia remain largely unknown. The research on phage diversity and impact on Streptomyces physiology and ecology is still in its infancy, with only a few examples to date [ 8 ]. Exploring how the multicellular nature and complex differentiation of Streptomyces can influence the viral cycle, potentially leading to partial and/or transient resistance to phages, is a promising avenue for research [ 9 , 10 ].

Streptomyces are among the most prolific producers of specialized metabolites, with applications in medicine and agriculture [ 1 , 2 ]. These filamentous gram-positive bacteria are widely distributed in the environment and play a key role in soil ecology [ 3 , 4 ]. Streptomyces are a rare example of multicellular bacteria capable of forming hyphae with connected compartments. They present a complex life cycle that involves uni- to multicellular transitions, sporulation, metabolic differentiation, programmed cell death, and exploration [ 3 , 5 ].

Results

Identification of Samy, a complete prophage, in the S. ambofaciens ATCC 23877 chromosome The comparison of S. ambofaciens ATCC 23877 genome to the closely related strain DSM 40697 led to the identification of a genomic island (≈71 kb, 113 genes; Fig 1A), which is remarkably large. Indeed, only 12% of S. ambofaciens ATCC 23877 genomic islands exceed 45 genes [30]. This region exhibits the lowest GC content (≈66%) and is predicted to contain a complete prophage (Fig 1A), as previously reported [26,30]. This latter, further named “Samy,” is exclusively present in the ATCC 23877 strain and stands as the sole complete prophage identified in its genome. The ATCC 23877 and DSM 40697 strains share 99.04% of average nucleotide identity calculated by using the BLASTn algorithm (ANIb) [31], indicating that they belong to the same species. The absence of Samy in the DSM 40697 strain indicates that the infection is relatively recent, i.e., occurred after speciation. The identification of a direct repeat (“TCGGGTGTGTCG”) in front of a serine integrase gene and approximately 61 kb downstream prompted us to propose this sequence as belonging to the left (attL) and right (attR) attachment sites of the Samy prophage (6,589,778 to 6,650,857 bp position in the S. ambofaciens ATCC 23877 chromosome). Immediately upstream, a region of approximately 10 kb contains only a few phage genes, including one coding for a putative integrase, indicating that it may correspond to a remnant prophage (Fig 1B). The mosaic composition of the genomic island suggests that it is located in a region prone to integration and/or fixation of exogenous sequences. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Samy prophage identification by comparative genomics. (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 [32]. 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) [30], 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 [32] 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 S1 and S2 Data, 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 [33] and [34]. 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 (S3 Table). 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. https://doi.org/10.1371/journal.pbio.3002725.g001 Samy prophage presents a typical GC content (66.53%) and size (61.07 kb, 102 genes) compared to other Streptomyces phages (S1 Fig and S1 Table). Annotation of Samy sequence allowed identification of most structural genes that are essential to form a complete phage (for instance, encoding head, neck and tail proteins, base plate and tail fiber proteins) as well as replication proteins, endolysin, several nucleases, transcriptional regulators, integrase, and recombination directionality factor (Fig 1C and S2 Table). Altogether, these analyses suggested that this prophage may be able to produce viral particles. This led us to investigate the conditions for its expression.

Samy production increases in alkaline conditions In order to determine the key compounds responsible for Samy induction, and to set up monitoring of its production in liquid medium, we tested variants of the HT liquid medium by eliminating one or more of its constituents. The simplest version of a medium associated with high viral titers, as quantified by qPCR, was named “BM” (for “Bacteriophage production Medium”) (Fig 3A). We noted a fairly high inter-experimental variability in Samy titer in BM medium (8.106 up to 2.1010 phage/ml, depending on the experiment), which might reflect its stochastic induction within the cell population. However, production kinetics is the same in all experiments: Samy particles are released between 24 and 48 h of growth in BM medium, the titer being almost constant over the following days (Fig 3B). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Samy phage production and morphology. (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 S6 Table) 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 S1 and S2 Data, 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 S1 and S2 Data, 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 S5A and S5B Fig). Scale bar: 100 nm. https://doi.org/10.1371/journal.pbio.3002725.g003 Interestingly, we observed a positive correlation between viral titer and pH, which is statistically significant (Spearman rank correlation rho = 0.73, p-value = 0.016). Indeed, adding buffering compounds (for instance, MOPS or dextrin) in BM medium strongly decreased phage production (Fig 3A). These results indicate that alkalinity constitutes an additional signal triggering Samy production and/or increasing virion stability in the supernatant.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002725

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