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. 2017 Mar 3;17:51. doi: 10.1186/s12866-017-0940-7

Functional and comparative genome analysis of novel virulent actinophages belonging to Streptomyces flavovirens

A Sharaf 1,2,✉, F Mercati 3, I Elmaghraby 4, R M Elbaz 5, E M Marei 6
PMCID: PMC5336643  PMID: 28257628

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.

Keywords: Bacteriophage, Biological stability, Whole genome sequence, NGS, Comparative genomics

Background

Bacteriophages (phages), natural viral predators of bacteria, are engaged in a constant evolutionary arms race with their hosts [1], playing major roles in the ecological balance of microbial life and in microbial diversity.

Most double-stranded DNA (dsDNA) phages share the same gene pool [2]; however, sequence comparisons reveal a widespread horizontal exchange of sequences among genomes, mediated by both non-homologous and homologous recombination. High frequency exchange among phages occupying similar ecological niches results in a high rate of mosaic diversity in local populations [3]. Studies confirm that phage genomes are mosaics and represent a large common genetic pool due to horizontal exchange [4, 5].

The screening of microbial natural products continues to constitute an important route to the discovery of chemicals for developing new therapeutic agents and evaluating the therapeutic potential of bacterial taxa [6–8]. In this respect, actinomycetes are a group of microorganisms mostly used in biotechnology for handling bioactive compounds. [9, 10]. Moreover, bacteriophages can be used to detect antiviral compound production by actinomycetes. Finally, actinophages are isolated and investigated because they can influence antibiotic production in bacterial strains, causing problems in the pharmaceutical industry. The vast majority of actinophages were isolated from sediments, but direct isolation from soil generally yields extremely low titers [11, 12]. However, although it is difficult to grow bacteriophages from soil without enrichment, a wide range of counts has been reported [13, 14].

Recently, there has been expanding interest in bacteriophages that infect Streptomyces species, since the phages can support the development of cloning vectors [15]. Such vectors could open the way for genetic manipulation as an important tool for Streptomyces improvement. Moreover, the mechanisms of the system for phage infection and multiplication could be useful in the fermentation industry and lead to the development of phage cloning vectors [16]. To date, no studies on phages isolated from S. flavovirens, an important source for several pharmaceutical drugs, such as actinomycin complex, mureidomycin and pravastatin [17, 18], have been carried out.

The development of high-throughput NGS (Next Generation Sequencing) technologies [19, 20] and the possibility to sequence entire genomes or transcriptomes more efficiently and economically than with first generation sequencing strategies permitted the collection of large amounts of information and the analysis of sequences from hundreds of thousands of species. Therefore, the dawn of next generation sequencing technologies has opened up exciting possibilities for whole genome sequencing in a wide range of organisms and the bacterial viruses have not been excluded from this revolution, despite the fact that their genomes are orders of magnitude smaller in size compared with bacteria and other organisms.

The Actinophage Sequence Databases (http://phagesdb.org/) currently include 5861 genomes from putative actinophages, 120 of which infect Streptomyces species and sixty-five of which are sequenced, but no genomes of phages isolated form S. flavovirens are currently available. The NCBI genome database contains around 600 Caudovirales genomes to date but the number of complete bacteriophage genomes published is growing slowly [21].

Until now, no phages belonging to S. flavovirens have been sequenced and relatively little is known about S. flavovirens phage genomics. In the present work, we report the first whole genome sequencing study and annotation of two S. flavovirens virulent phages. The results will provide an important genomic resource for future investigations in the bacteriophages related to S. flavovirens and for phage evolution studies.

Methods

Source of lytic actinophages

Two isolates of Streptomyces flavovirens phages, named Sf1 and Sf3, were obtained from the virology lab, Agric. Microbiology Department, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. Phages were isolated from soil and the morphological properties were analyzed by standard methodology and reported in Marei and Elbaz (2013) [22].

Purification of lytic actinophages

The high titer phage suspension of each isolated phage was prepared using a liquid culture enrichment technique. The high titer phage suspension of each phage was ultra-centrifuged at 30000 rpm for 90 min. at 4 °C in a Beckman L7-35 ultracentrifuge. The pellet was gently resuspended in 0.5 ml of 0.2 M phosphate buffer pH 7.2 [23].

Adsorption rate and one-step growth experiments

The adsorption experiments were carried out with two isolated phage suspensions added to spores of their indicator host (S. flavovirens). Suspensions of each phage were incubated at 30 °C with gentle shaking. Samples were withdrawn at regular intervals after inoculation.

The mycelial fragments of the indicator strain were removed by centrifugation and the concentration of phage remaining in the supernatant was counted. The adsorption rates of the two phages were determined by measuring residual plaque-forming ability in membrane-filtered samples of an attachment mixture [24] and the adsorption rate constant k (mL/min) was calculated [25]. The one-step growth experiment was performed as described by Dowding (1973) [24].

Physiochemical stability

To evaluate the phages’ stability three different chemicals (NaCl, MgCl2.6H2o and ZnCl2), were used. Five concentrations (0.1, 0.2, 0.3, 0.4 and 0.5 mM) for each salt were employed [26]. To test the effect of different treatments phage solutions for both tested strains with final concentrations of 107 PFU/ml were utilized. The mixture was incubated for 10 min at room temperature (RT). The number of plaques was determined using the double layer method (plaque assay test) [27]. A control test was prepared by mixing bacterial suspension with phage without the tested chemicals.

DNA isolation, library preparation and whole genome sequencing

Genomic DNA was isolated from the propagated phages according to the procedure described by Kieser et al. [28]. DNA quality was assessed using a Nanodrop Bioanalyzer ND1000 (ThermoScientific). Sequencing libraries were prepared by shearing 1 μg of DNA in blunt-ended fragments by linking the Ion adapters using an Ion XpressTM Plus Fragment Library Kit (Life Technologies, Carlsbad, USA) according to the manufacturer’s specifications. The sized and ligated fragments were amplified by emulsion-PCR using the Ion OneTouch 200 Template kit (Life Technologies, Carlsbad, USA). Quality and insert size distribution were assessed using an Agilent Bioanalyzer DNA 1000 chip. Libraries were sequenced on an Ion Torrent PGM semiconductor sequencer (Life Technologies, Carlsbad, USA) using the 200 bp protocol and an Ion Torrent 314 chip following the manufacturer instructions (Life Technologies, Carlsbad, USA).

Assembly and bioinformatics analyses

Raw reads resulting from Sf1 and Sf3 sequencing were trimmed using Trimmomatic with single end mode (no quality encoding was specified to allow the program to determine it automatically [29]) and assembled separately using the gsAssembler (Roche Applied Science, Indianapolis, IN); the Graphical User Interface (GUI) version was used with the default parameters. The collected contigs were visualized and validated using Hawkeye [30]. Resulting contigs for each phage showed approximately 60-fold sequence read coverage. The expected sequence accuracy was 95% with a statistical error of less than 1 in 10,000 bp. Sequence homologies were determined by using BLASTn against the actinophage database to assign the phages to a cluster [31].

Open reading frame (ORF) analysis and gene prediction

Open reading frames (ORFs) were identified and the genome sequences of each phage were annotated as described previously in Dobbins et al., 2004 by using DNA Master (J. G. Lawrence) (http://cobamide2.bio.pitt.edu) software and visual inspection [32]. For a genome-wide viewpoint an association with the annotation refinement, functional analysis and other explorations was developed using Phamerator. Protein sequence relationships and conserved domains within genes were also studied. Gene products were grouped into “Phamilies” generally referred to as “Phams”, or groups of proteins with a high degree of similarity to one another. The pairwise alignment scores and significant rate were determined using BLASTp and ClustalW [33].

Genomic comparisons between the sequenced and the close related phages

Sequence comparisons were performed by using the BLAST algorithm available at NCBI [34] and Mauve software [35]. A comparison map among Sf1 and Sf3 Streptomyces phages and closely related phages (VWB and SV1) with available genomes in the National Center for Biotechnology Information (NCBI) nucleotide database (https://www.ncbi.nlm.nih.gov/) was generated by Circoletto (http://tools.bat.infspire.org/circoletto/) [34, 36]. For pictogram construction, bit-score values were used to describe the quality of the alignment at a given point. The bit-score is a normalized version of the score value returned by the BLAST searches, expressed in bits [37].

The phylogenetic tree of Major Capsid Protein (MCP) genes from two new isolated phages (Sf1 and Sf3) and 20 related Streptomyces phages available in the NCBI database was constructed with Geneious software version (R8) (http://www.geneious.com) [38] based on the Neighbor-Joining (NJ) algorithm.

Results and discussion

Adsorption rate constant and growth characteristics of isolated phages

Adsorption of Sf1 and Sf3 was determined using S. flavovirens cells grown in phage medium to the early exponential phase of growth (15-h cultures). About 82 and 85% of all infective Sf1 and Sf3 particles, respectively, were adsorbed within 20 min of contact. The adsorption reached a maximum after 30 min. for both phages. The adsorption constant K was 3.66 pL/min for Sf1 and 3.80 pL/min for Sf3, determined by the Adams’s formula [27]. The phages adsorption rates were higher than other actinophages [39], which was probably due to the strong specificity of the Sf1 and Sf3 phages to their host.

The production of Sf1 and Sf3 phages were determined in a one-step growth experiment at 30 °C. Results revealed that the latent periods of Sf1 and Sf3 were approximately 15 and 30 mins, respectively. After 30 and 45 mins the maximum rise period was shown and the burst sizes were 1.95 and 2.49 PFU/mL for Sf1 and Sf3, respectively (Fig. 1). The present results are in agreement with the data obtained from a study on 24 actinophages [40], underlining that under controlled cultural conditions the infection of isolated Streptomycetes cells by phages was varied.

Fig. 1.

Fig. 1

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One-step growth experiment for Sf1 and Sf3 phages development on S. flavovirens at 30 °C

Physiochemical stability of isolated actinophages

Sodium and magnesium chloride treatments yielded a significant increase in both phages’ activity for all concentrations used compared with the control, while zinc chloride application with concentrations > 0.3 mM caused a significant decrease of activity for Sf1 and Sf3 (Fig. 2). Similar results were reported in previous studies [41–43]. Absence of calcium and magnesium ions prevents adsorption and the lysis cycle, while their presence stimulates a significant increase in phage activity, probably due to the increase of adsorption and penetration rates. On the contrary, zinc and aluminum chloride showed significant loss of infectivity in both phages. This is in accordance with the experiments performed by Robert and Charles, which suggested that aluminum caused viral inactivation related to the dissociation of viral capsid proteins [44].

Fig. 2.

Fig. 2

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Effect of three different chemicals on the Sf1 and Sf3 infectivity

Genome organization of phages Sf1 and Sf3

Genome sequencing generated 69,719 and 107,273 reads for each phage with around 60-fold coverage and 43,150 bp, and 60,934 bp assembled sequences for Sf1 and Sf3, respectively. The pair-wise alignment [45] revealed that the genomes of Sf1 and Sf3 shared an overall high level of similarity, with conserved regions of high identity (100% identity) interspersed between regions with high variability (ranging from 23.9% to 87.5%) (Fig. 3a). A similar mosaic genome structure has been observed in most other phage genomes, indicating extensive horizontal genetic exchange among phages [46–49]. No close relatives (Singleton) from modeling of both genome construction were revealed (Fig. 1).

Fig. 3.

Fig. 3

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Genomic organization of Sf1 and Sf3 phages. Phages were mapped using Phamerator; the purple lines between phages underline the regions with high similarity, while the ruler corresponds to genome base pairs. The predicted genes are shown as boxes either above or below the genome (ruler), depending on whether are rightwards or leftwards transcribed, respectively. Gene numbers are shown within each box; pink boxes refereed to the genes with high similarity between two phages while the blue boxes refereed to the genes that show low similarity. a The phages maps showed by cluster conservation. b The phages maps showed by phams; genes are colored according to their function categories “phams”

Annotation of Sf1 and Sf3 genomes revealed 52 and 91 putative ORFs, respectively. According to their homology, 19 out of 52 ORFs (36.5%) from Sf1 and 28 out of 91 (30.8%) from the Sf3 genome have been assigned functions compared to known conserved domains [50] (Tables 1 and 2). Isolated genes were mainly involved in DNA replication and repair, nucleotide metabolism, lysis, phage structural proteins and other enzymes. The results obtained are in agreement with other bacteriophage studies [51–53]. Phage Sf1 showed 52 ORFs (Table 1), named gp1 - gp52, while 91 ORF were identified from Phage Sf3, from gp1 to gp91 (Table 2). The majority of members of identified families are bacteriophage proteins, while others (75%) have unknown function [54, 55].

Table 1.

Overview of Sf1 phage ORFs, summary of homology searches and annotations

ORF Product Strand Begin End AA Motif Predicted functions Homology score E-value
ORF 1 gp1 + 126 650 175 pfam05119 Terminase_4 superfamily 65,72 1.77e-14
ORF 2 gp2 + 643 2349 569 pfam03354 Terminase_1 superfamily 243 3.32e-73
ORF 3 gp3 + 2376 3785 470 pfam05133 Phage portal protein 147 3.13e-39
ORF 4 gp4 + 3796 4842 349 cd13126 (MATE) proteins 36,9 5.07e-03
ORF 5 gp5 + 4857 5480 208 pfam09787 Golgin subfamily A5 35,58 6.35e-03
ORF 6 gp6 + 5535 6479 315 PHA00665 major capsid protein 42,17 9.33e-05
ORF 7 gp7 + 6661 7194 178 - - - -
ORF 8 gp8 + 7191 7532 114 - - - -
ORF 9 gp9 + 7532 7783 84 PRK14573 bifunctional D-alanyl-alanine synthetase 34,02 2.66e-03
ORF 10 gp10 + 7783 8178 132 - - - -
ORF 11 gp11 + 8168 8740 191 - - - -
ORF 12 gp12 + 8843 9109 89 - - - -
ORF 13 gp13 + 9145 9489 115 - - - -
ORF 14 gp14 + 9493 12630 1046 pfam10145 Phage-related minor tail protein 88,94 1.30e-19
ORF 15 gp15 + 12631 12837 69 - - - -
ORF 16 gp16 + 12896 15148 751 - - - -
ORF 17 gp17 + 15163 16281 373 pfam13550 Putative phage tail protein 43,42 1.33e-05
ORF 18 gp18 + 16281 16538 86 - - - -
ORF 19 gp19 + 16563 17216 218 - - - -
ORF 20 gp20 - 17411 17518 36 - - - -
ORF 21 gp21 + 17716 18126 137 - - - -
ORF 22 gp22 + 18141 19799 553 - - - -
ORF 23 gp23 + 19824 21251 476 pfam05133 Phage portal protein 129 4.91e-33
ORF 24 gp24 + 21244 22044 267 - - - -
ORF 25 gp25 + 22107 22856 250 - - - -
ORF 26 gp26 + 22870 23265 132 pfam02924 Bacteriophage lambda head decoration protein D 47,27 2.61e-08
ORF 27 gp27 + 23280 24326 349 pfam03864 Phage major capsid protein E 62,35 3.13e-11
ORF 28 gp28 + 24323 24646 108 - - - -
ORF 29 gp29 + 24652 25095 148 pfam09355 Phage protein Gp19 33,61 5.91e-03
ORF 30 gp30 + 25092 25445 118 - - - -
ORF 31 gp31 + 25442 25726 95 - - - -
ORF 32 gp32 + 25726 26127 134 - - - -
ORF 33 gp33 + 26200 26865 222 - - - -
ORF 34 gp34 + 26969 27292 108 - - - -
ORF 35 gp35 + 27337 27765 143 - - - -
ORF 36 gp36 + 27772 31344 1191 cd00254 Lytic Transglycosylase (LT) 56,65 1.82e-09
ORF 37 gp37 + 31349 32239 297 - - - -
ORF 38 gp38 + 32239 33384 382 - - - -
ORF 39 gp39 + 33386 34312 309 - - - -
ORF 40 gp40 + 34326 34943 206 - - - -
ORF 41 gp41 + 34953 36977 675 pfam12708 Pectate lyase_3 superfamily protein 73,24 8.54e-15
ORF 42 gp42 + 37059 37886 276 cd06583 Peptidoglycan recognition proteins (PGRPs) 58,45 4.03e-11
ORF 43 gp43 + 37933 38184 84 - - - -
ORF 44 gp44 + 38228 38560 111 COG4467 YabA 34,76 9.25e-04
ORF 45 gp45 - 38602 39129 176 - - - -
ORF 46 gp46 + 39475 40689 405 cd00093 Helix-turn-helix XRE-family like proteins 45,24 1.45e-06
ORF 47 gp47 + 40777 41046 90 - - - -
ORF 48 gp48 + 41043 41237 65 - - - -
ORF 49 gp49 + 41234 41752 173 - - - -
ORF 50 gp50 + 41901 42383 161 - - - -
ORF 51 gp51 + 42383 42481 33 - - - -
ORF 52 gp52 + 42474 42923 150 cd00075 Histidine kinase-like ATPases 36,09 4.50e-04
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Table 2.

Overview of Sf3 phage, ORFs, summary of homology searches and annotations

ORF no. Product Strand Begin End AA Motif Predicted functions Homology score E-value
ORF 1 gp1 + 16 426 137 - - - -
ORF 2 gp2 + 441 2099 553 - - - -
ORF 3 gp3 + 2124 3551 476 pfam05133 Phage portal protein_Gp6 129 4.91e-33
ORF 4 gp4 + 3544 4344 267 - - - -
ORF 5 gp5 + 4407 5156 250 - - - -
ORF 6 gp6 + 5176 5565 130 pfam02924 Bacteriophage lambda head decoration protein D 46,5 5.02e-08
ORF 7 gp7 + 5580 6626 349 pfam03864 Phage major capsid protein E 62,35 3.13e-11
ORF 8 gp8 + 6623 6946 108 - - - -
ORF 9 gp9 + 6952 7395 148 pfam09355 Phage protein Gp19 33,61 5.91e-03
ORF 10 gp10 + 7392 7745 118 - - - -
ORF 11 gp11 + 7748 8026 93 - - - -
ORF 12 gp12 + 8026 8427 134 - - - -
ORF 13 gp13 + 8500 9165 222 - - - -
ORF 14 gp14 + 9269 9592 108 - - - -
ORF 15 gp15 + 9637 10065 143 - - - -
ORF 16 gp16 + 10072 13644 1191 pfam03864 Phage major capsid protein E 62,35 3.13e-11
ORF 17 gp17 + 13649 14539 297 - - - -
ORF 18 gp18 + 14539 15684 382 - - - -
ORF 19 gp19 + 15686 16612 309 - - - -
ORF 20 gp20 + 16626 17243 206 - - - -
ORF 21 gp21 + 17253 19277 675 pfam12708 Pectate lyase superfamily protein 73,24 8.54e-15
ORF 22 gp22 + 19359 20186 276 cd06583 Peptidoglycan recognition proteins (PGRPs) 58,45 4.03e-11
ORF 23 gp23 + 20233 20484 84 - - - -
ORF 24 gp24 + 20528 20860 111 COG4467 YabA 34,76 9.25e-04
ORF 25 gp25 + 20908 21546 213 PHA03169 hypothetical protein; Provisional 35,72 5.62e-03
ORF 26 gp26 + 21757 22989 411 cd00093 Helix-turn-helix XRE-family like proteins. 45,24 1.33e-06
ORF 27 gp27 + 23077 23346 90 - - - -
ORF 28 gp28 + 23343 23537 65 - - - -
ORF 29 gp29 + 23534 24052 173 - - - -
ORF 30 gp30 + 24201 24683 161 - - - -
ORF 31 gp31 + 24668 24781 38 - - - -
ORF 32 gp32 + 24774 25223 150 cd00075 Histidine kinase-like ATPases 36,09 4.50e-04
ORF 33 gp33 - 25247 25363 39 - - - -
ORF 34 gp34 + 25319 25381 21 - - - -
ORF 35 gp35 + 25382 26221 280 pfam00730 HhH-GPD superfamily base excision DNA repair protein 47,36 3.71e-07
ORF 36 gp36 + 26181 27680 500 - - - -
ORF 37 gp37 + 27677 28327 217 cd01672 Thymidine monophosphate kinase (TMPK) 112 1.37e-30
ORF 38 gp38 + 28324 28755 144 cd04683 the Nudix hydrolase superfamily 153 3.17e-48
ORF 39 gp39 - 29387 30592 402 - - - -
ORF 40 gp40 - 30712 30963 84 - - - -
ORF 41 gp41 + 30962 31105 48 - - - -
ORF 42 gp42 - 31162 31524 121 cd00093 Helix-turn-helix XRE-family like proteins. 41 1.99e-06
ORF 43 gp43 + 32113 32337 75 - - - -
ORF 44 gp44 + 32425 32778 118 - - - -
ORF 45 gp45 + 32771 33139 123 - - - -
ORF 46 gp46 + 33136 33678 181 - - - -
ORF 47 gp47 + 33675 33947 91 - - - -
ORF 48 gp48 + 33944 34774 277 pfam12705 PD-(D/E)XK nuclease superfamily 34,99 5.68e-03
ORF 49 gp49 + 34777 35838 354 - - - -
ORF 50 gp50 + 35835 36647 271 cd06127 DEDDh 3’–5’ exonuclease domain family 111 4.30e-30
ORF 51 gp51 + 36644 37099 152 - - - -
ORF 52 gp52 + 37096 37713 206 cd00529 Holliday junction resolvases (HJRs) 38,38 3.22e-04
ORF 53 gp53 + 37710 37985 92 - - - -
ORF 54 gp54 + 37991 38428 146 - - - -
ORF 55 gp55 + 38425 38775 117 - - - -
ORF 56 gp56 + 38788 39564 259 - - - -
ORF 57 gp57 + 39567 40202 212 - - - -
ORF 58 gp58 + 40199 40402 68 - - - -
ORF 59 gp59 + 40399 40926 176 - - - -
ORF 60 gp60 + 40923 41120 66 - - - -
ORF 61 gp61 + 41153 41506 118 - - - -
ORF 62 gp62 + 41503 42369 289 - - - -
ORF 63 gp63 + 42366 42692 109 - - - -
ORF 64 gp64 + 42689 42814 42 pfam10969 Protein of unknown function (DUF2771) 35,51 1.35e-04
ORF 65 gp65 + 42811 43368 186 - - - -
ORF 66 gp66 + 43466 44308 281 - - - -
ORF 67 gp67 - 44375 44590 72 pfam02604 Antitoxin Phd_YefM 30,73 5.22e-03
ORF 68 gp68 + 44674 46254 527 - - - -
ORF 69 gp69 + 46345 47619 425 - - - -
ORF 70 gp70 + 47651 47743 31 - - - -
ORF 71 gp71 + 47817 47996 60 - - - -
ORF 72 gp72 + 48073 48510 146 cd00397 DNA breaking-rejoining enzymes 40,54 2.27e-05
ORF 73 gp73 - 49011 49772 254 - - - -
ORF 74 gp74 + 49506 49766 87 - - - -
ORF 75 gp75 + 49841 49996 52 - - - -
ORF 76 gp76 + 49993 50292 100 cd00085 HNH nucleases 38,22 1.45e-05
ORF 77 gp77 + 50587 51171 195 COG4983 Uncharacterized protein 79,98 9.19e-18
ORF 78 gp78 + 51278 51349 24 - - - -
ORF 79 gp79 - 51714 52163 150 - - - -
ORF 80 gp80 - 52167 53399 411 - - - -
ORF 81 gp81 - 53709 54161 151 - - - -
ORF 82 gp82 + 54647 55672 342 pfam06381 Protein of unknown function (DUF1073) 39,22 1.07e-03
ORF 83 gp83 - 55695 55889 65 - - - -
ORF 84 gp84 + 55948 56844 299 TIGR01641 phage putative head morphogenesis protein 59,7 1.40e-11
ORF 85 gp85 - 56884 57198 105 PRK13502 transcriptional activator RhaR 32,72 8.64e-03
ORF 86 gp86 + 57346 57642 99 - - - -
ORF 87 gp87 + 57698 57847 50 - - - -
ORF 88 gp88 + 58134 58682 183 - - - -
ORF 89 gp89 - 58679 59956 426 COG1783 Phage terminase_3 161 6.32e-45
ORF 90 gp90 - 60233 60691 153 - - - -
ORF 91 gp91 - 60684 60932 83 - - - -
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Phage structure and assembly genes

Several genes code for terminase subunit proteins, such as gp1 and 2 which code for terminase_4 (pfam05119) and terminase_1 (pfam03354) super-families, respectively. The gp3 and gp23 genes encode for the phage portal protein (pfam05133), an important protein involved in DNA transport during its packaging and ejection. Another relevant gene is gp6 which, together with gp27,codes for the major capsid protein (PHA00665) [56] and the major capsid protein E domain (pfam03864) [57], respectively, involved in the stabilization of the condensed form of DNA in phage heads. Some genes involved in tail development, gp14 (pfam10145) and gp17 (pfam13550), were also identified.

In Sf3we found a gene (gp3) encoding phage portal protein (pfam05133), crucial for DNA migration and building the junction between head and tail proteins [58], and others, such as gp7 and gb16, that encode for the major capsid protein E domain (pfam03864) [57] or for lyase (gp21), like pectate lyase_3 superfamily protein (pfam12708). A phage putative head morphogenesis protein (TIGR01641) of 110 amino acids found exclusively in phage-related proteins, was encoded by gp84. Putaive head morphogenesis proteins such as gp85, which encodesthe transcriptional activator RhaR (PRK13502), and gp89, involved in the phage terminase_3 (COG1783) synthesis, are activated during the beginning of double-stranded viral DNA packaging [59].

DNA replication and metabolic genes

The gp44 gene encodes YabA (COG4467), a protein that interacts with the DnaA initiator and the DnaN sliding clamp and drives the control of DNA replication initiation [60, 61]. gp46 and gp52 encode for helix-turn-helix XRE-family like proteins (cd00093) [62] and histidine kinase-like ATPases (cd00075) [63], respectively, two important binding proteins with roles in the replication, repair, storage and modification of DNA. gp4 encodes a protein belonging to the MATE family (cd13126), which functions as a translocase for lipopolysaccharides [64], while gp5 codes for the golgin subfamily protein A5, a protein responsible for maintaining Golgi structure in intra-Golgi retrograde transport [65].

ORFs with the same biological roles were also identified in Sf3 phage. Indeed gp35 encodes for a HhH-GPD superfamily base excision DNA repair protein (pfam00730). This group includes endonuclease III, 8-oxoguanine DNA glycosylases and DNA-3-methyladenine glycosylase II [66]. Other members include different types of DNA and RNA exonucleases such as RNase T, oligoribonuclease, and RNA exonuclease (REX) [67]; Holliday junction resolvases (HJRs) (cd00529), endonucleases structurally similar to RNase H and Hsp70, which specifically resolve Holliday junction DNA intermediates during homologous recombination was encoded by gp52 [68]. Gp76 encodes for HNH nucleases (cd00085), an endonuclease signature which is found in viral, prokaryotic and eukaryotic proteins [69].

Cell lysis genes

Crucial genes implicated in lysis activities, such as the cell wall degradation process in bacteria during host infection, were identified in the Sf1 genome. Indeed, gp36 encodes for the lytic transglycosylase (LT) (cd00254) that catalyzes the cleavage of the beta-1,4-glycosidic bond between N-acetylmuramic acid and N-acetyl-D-glucoseamine, similar to “goose-type” lysozymes. gp42 encodespeptidoglycan recognition proteins (PGRPs) (cd06583), namely receptors that bind and hydrolyze peptidoglycans of bacterial cell walls, and contains two conserved histidines and a cysteine, typical residues of zinc binding sites [70].

While gp21 is included in the pectate lyase superfamily (pfam12708), proteins with a beta helical structure like pectate lyase and most closely related to glycosyl hydrolase family and gp22 encodes to Peptidoglycan recognition proteins (PGRPs) (cd06583) [70], were identified in Sf3 genome.

Both phage genomes show up to bring a modular organization, with genes of related function clustered together (Fig. 3a and b). DNA sequences of the first 13 kb in Sf3 are highly similar to the last DNA sequences in Sf1 and encode for DNA packaging structural proteins (Fig. 3b).

On the basis of the amino acid sequence similarity between the gene products, the conserved pfam05133 motif and the gene locations, orf3 is predicted to encode a portal protein in both phages. No small terminase-encoding gene could be identified in either genome. The largest gene in Sf1 genome is located in orf36 (3.5 kb) encoding the lytic transglycosylase (LT), while the largest one in Sf3 genome with the same length is orf16, encoding the major capsid protein E domain. [48, 71, 72]. A possible lyase gene is positioned distinctively in both phage genomes (orf41 for Sf1 and orf21 for Sf3). Those genes located downstream in both phage genomes encode proteins involved in DNA synthesis, metabolism and repair (Fig. 3b).

Evolutionary relationship of Sf1 and Sf3

Sf1 and Sf3 phages show 30 phams, where 29 out of 30 phams contain two members (Table 3), while three members belong to pham number 12. Ten phams (33.3%) were assigned with known functionality; the others are unknown. Therefore, some of these phams are informative and can be used in evolutionary studies. Indeed, as reported for mycobacteriophages [73], single, ubiquitous, semi-conserved genes can be utilized for cluster prediction, useful when the whole genome sequence is unavailable. The 30 identified phams, which include important genes (see below), underline a close phylogenetic relationship between the two isolated phages and provide important information that can be used in future evolutionary relationship studies by comparing the genes identified in the Streptomyces flavovirens phages and homologous genes in other bacteriophages.

Table 3.

Related Conserved Domains (CD) to the detected Phamilies using Phamerator

Pham Conserves Domains (CD) Number of members Mean translation length Phage Sf1 Phage Sf3
1 - 2 136 ORF 21 ORF 1
2 - 2 552 ORF 22 ORF 2
3 Phage portal protein 2 475 ORF 23 ORF 3
4 - 2 266 ORF 24 ORF 4
5 - 2 249 ORF 25 ORF 5
6 Bacteriophage lambda head decoration protein D 2 130 ORF 26 ORF 6
7 Phage major capsid protein E 2 348 ORF 27 ORF 7
8 - 2 107 ORF 28 ORF 8
9 Phage protein Gp19 2 147 ORF 29 ORF 9
10 - 2 117 ORF 30 ORF 10
11 - 2 93 ORF 31 ORF 11
12 Terminase_4 superfamily 3 132,3333 ORF 1, ORF 32 ORF 12
13 - 2 221 ORF 33 ORF 13
14 - 2 107 ORF 34 ORF 14
15 - 2 142 ORF 35 ORF 15
16 - 2 296 ORF 37 ORF 17
17 - 2 381 ORF 38 ORF 18
18 - 2 308 ORF 39 ORF 19
19 - 2 205 ORF 40 ORF 20
20 Pectate lyase superfamily protein 2 674 ORF 41 ORF 21
21 Peptidoglycan recognition proteins (PGRPs) 2 275 ORF 42 ORF 22
22 - 2 83 ORF 43 ORF 23
23 YabA 2 110 ORF 44 ORF 24
24 Helix-turn-helix XRE-family like proteins 2 407 ORF 46 ORF 26
25 - 2 89 ORF 47 ORF 27
26 - 2 64 ORF 48 ORF 28
27 - 2 172 ORF 49 ORF 29
28 - 2 160 ORF 50 ORF 30
29 - 2 34,5 ORF 51 ORF 31
30 Histidine kinase-like ATPases 2 149 ORF 52 ORF 32
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orf27 (Sf1) and orf7 (Sf3) as members of pham n.7 were assigned as phage major capsid protein (MCP) E domains; this important class of genes was also used as a single gene prediction system for the mycobacteriophage clusters analysis [73]. orf23 (Sf1) and orf3 (Sf3), members of pham n. 3, were classified as phage portal proteins. These proteins were used in some previous investigations as a marker of diversity indicating, in some cases, the connections between habitat properties, microbial community structure and phage community composition [74]. orf29 (Sf1) and orf9 (Sf3) are the members of pham n.9, were assigned to phage protein gp19, an important tail component. Most of the proteins forming the phage tail components as well as other needle-like assemblies (e.g. secretion systems and bacteriocins) have a common origin from a single protein module [74]. This evidence emphasizes the importance of phage protein diversification and specialization in the evolution of different and complex bacterial systems and in bacterial adaptation, developing new functions and providing a distinct selective advantage [74].

As expected, the virulent phages developed phams involved in lysogenic pathways. Indeed, orf41 (Sf1) and orf21 (Sf3), grouped in pham n.20, showed high homology to the pectate lyase superfamily protein that can modify the properties of polysaccharides. Since the pectinolytic protein family is commonly represented in prokaryotic and eukaryotic microorganisms and, in plants, is involved in remodelling cell walls, it is clear that the divergence from the ancestral protein over time has allowed different micro-organisms to target a range of pectin-like substrates while the overall structure has been maintained [75]. orf42 (Sf1) and orf22 (Sf3) are members of pham n.21 and classified as peptidoglycan recognition proteins (PGRPs), an innate class of immunity molecules present in insects, mollusks, echinoderms, and vertebrates that by interacting with peptidoglycan in the cell wall, rather than permeabilizing bacterial membranes, kills bacteria. These proteins were reported, at least in one carboxy-terminal domain, as homologous in bacteriophage and bacteria [76]. orf46 (Sf1) and orf26 (Sf3) are grouped in pham n.24 and were identified as helix-turn-helix (HTH) XRE-family-like proteins, one of the early studied regulatory DNA-binding proteins involved in metabolic regulation in bacteria. This class of genes encodes components to process environmental metabolites (e.g. lactose) and to produce interacting constituents in the development of a lytic or lysogenic pathway in phages. A common ancestor for all DNA-binding domains was suggested and, through its duplication and divergence, the diversity of transcription regulators that drive bacterial and phage genes was generated. The HTH fold investigations confirmed the significance of this module in DNA–protein interactions across a wide phylogenetic spectrum including a wide variety of phages [77].

orf26 (Sf1) and orf6 (Sf3), members of pham n. 6, were classified as bacteriophage lambda head decoration protein D. Since the protein allows for the display of many copies of a foreign protein, which is advantageous for displaying weak ligands for affinity selection, a useful platform for phage polypeptide display was recently developed [78]. Interestingly, orf32 in Sf1 and orf12 in Sf3 were not assigned functions previously, although they belong to the pham n. 12 together with orf 1 (Sf1) which is classified as terminase_4.

A standard Nucleotide NCBI BLAST (blastn) search was developed using both Sf1 and Sf3 phage whole genome sequences as a query against a non-redundant nucleotide sequences database. Starting from a whole phage dataset (https://www.ncbi.nlm.nih.gov/) the available phage genomes with the best identity percentages (VWB and SV1) were chosen and a pictogram was developed (Fig. 4). Seventy-eight percent identity for both S. flavovirens phages compared to the complete genome of bacteriophage VWB, isolated from S. venezuelae strain ETH 14630 (AY320035.2), was exhibited (with 29% and 36% of coverage for Sf1 and Sf3, respectively), while 75% of identity for both studied phages with S. venezuelae phage SV1 (JX182371.1) was reported, but with low query coverage (11% for Sf1 and 14% for Sf3), probably due to the phylogenetic distance between the compared phages.

Fig. 4.

Fig. 4

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Sequence similarities among Sf1, Sf3, VWB and SV1 phages. The picture shows the results of the BLAST local alignments using Sf1 and Sf3 as a query against the VWB and SV1 phages sequences. The different colours (blue, green, orange and red) represent the overall quality of the aligned segments along the phage sequences, evaluated on the basis of the bit-score values from the worst to the best score (blue to red). The bit-score is a normalized version of the score value obtained by BLAST searches, expressed in bits. The height of the coloured bars in the histogram shows how many times each colour hits a specific fragment of the other phage sequences. A twist in a ribbon indicates that the local alignment is inverted (query and database sequence on opposite strands)

The alignment of both Sf1 and Sf3 genomes against the sequences of VWB phage, carried out by Mauve software, revealed that most hits occurred around a 13Kb region (Fig. 4). The approximate location of this region were (18000–31000) within the Sf1 genome, (1–13000) in the Sf3 genome and (23000–36000) in the VWB genome. On the contrary, the alignment of both S. flavovirens phage genomes versus the sequences of SV1 showed only a short region (~1Kb) with moderate bit score ranging from 9691–10707 and 10300–11208 in the genomes of Sf1 and Sf3, respectively, consistent with the low sequence coverage obtained.

The MCPs diversity between Sf1, Sf3 and 20 related Streptomyces phages, due to a combination of illegitimate and homologous recombination [79] and mutational drift, was also evaluated. The current investigation highlighted the hybrid generation between phage genera [80] or phage families [81]. Twenty-two Streptomyces phages were grouped in five main branches (Fig. 5). The Lannister MCP shared a close evolutionary relationship with the Izzy, Aaronocolus, and Caliburn sequences, demonstrating that phages may undergo genetic exchange by horizontal gene transfer from a large shared pool [4] and that horizontal gene transfer between phages is a component of their evolution. Numerous gene exchanges within each major clade and core phage functions do not appear to have co-evolved with specific hosts [82].

Fig. 5.

Fig. 5

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Phylogenetic analysis of studied phages and other members (20) of the Streptomyces phages group based on MCPs. Bootstrap values indicate the number of times a node was supported in 1000 resampling replications

Our phylogenetic analysis is useful for further studies, since both Sf1 and Sf3 were recovered in a clade that included phages that infect Streptomyces species but most of these phages (Maih, YDN12, Xkcd426 and TP1604) were members of the BG phage cluster; this clustering does not represent a phylogenetic or taxonomic grouping but rather provides a framework for reflecting their overall genome relationships and for identifying genes that have been recently exchanged and their genomic context [83, 84]. Moreover, Sf1 and Sf3 grouped in a separate branch, indicating that isolated phages belong to the BG phage cluster but represent a different sub-cluster.

Conclusion

Recently, large advances have occurred in phage genomics; nevertheless,the full extent of phage diversity and evolutionary pathways are yet unknown. With the advent of NGS technologies a much greater volume of transcriptome and genome sequences is available and we can therefore expect an increased flow of new data in upcoming years. Current assessment suggests that more than 1031 phages exist on earth, representing more than ten million phage “species”. Of these, less than 6000 have been observed using electron microscopy and fewer than 1000 genomes have been sequenced. The available sequences show that the majority of phages analyzed are tailed phages belonging to the family Siphoviridae, but less is known about the degree of their genetic diversity. The genomic characterization of phages is necessary to evaluate their important ecological impact. In spite of their ubiquity, phages have not yet been characterized for many bacterial genera. In the present study, biological, physiochemical and genome sequences of two new virulent Streptomyces phages are presented, representing the first genomic report of S. flavovirens phages which may represent a new sub-cluster of the BG Streptomyces phage cluster.

Acknowledgement

The authors would like to thank the Center for Research in Agricultural Genomics (CRAG) service laboratory, Barcelona, Spain for providing the sequencing instruments and reagents used in the study, Ezio Fontana, IBBR-CNR, Palermo, Italy for his advice and discussion about the whole genome data analysis and Heather Esson, Biology Center ASCR, Institute of Parasitology, Czech Republic for assistance with language editing.

Funding

This work does not obtained any fund.

Availability of data and materials

The complete genome sequences of Sf1 and Sf3 phages were deposited in the National Center for Biotechnology Information (NCBI) GenBank under accession numbers (KT221033 and KT221034), respectively.

Authors’ contributions

All authors conceived and designed the experiments; AS carried out the experiments and performed the bioinformatics and statistical analysis; AS and FM compiled the results and drafted the manuscript. All the authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.

Abbreviations

dsDNA

Double-stranded DNA

HJRs

Holliday junction resolvases

HTH

Helix-turn-helix

LT

Lytic transglycosylase

MCP

Major capsid protein

NCBI

National Center for Biotechnology Information

NGS

New Generation Sequencing

ORFs

Open reading frames

PGRPs

Peptidoglycan recognition proteins

Phages

Bacteriophages

REX

RNA exonuclease

TDP

Thymidine diphosphate

TMP

Thymidine monophosphate

TMPK

Thymidine monophosphate kinase

TTP

Thymidine triphosphate

Contributor Information

A. Sharaf, Phone: 00420-777313025, Email: sharaf@paru.cas.cz

F. Mercati, Email: francesco.mercati@ibbr.cnr.it

I. Elmaghraby, Email: ibrahim_elmaghraby@yahoo.com

R. M. Elbaz, Email: d.reham@hotmail.com

E. M. Marei, Email: wel_memo@yahoo.com

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The complete genome sequences of Sf1 and Sf3 phages were deposited in the National Center for Biotechnology Information (NCBI) GenBank under accession numbers (KT221033 and KT221034), respectively.

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