Open Access

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

BMC MicrobiologyBMC series – open, inclusive and trusted201717:51

https://doi.org/10.1186/s12866-017-0940-7

Received: 26 August 2016

Accepted: 24 January 2017

Published: 3 March 2017

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 [68]. 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

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 [4143]. 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

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 [4649]. No close relatives (Singleton) from modeling of both genome construction were revealed (Fig. 1).
Fig. 3

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 [5153]. 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

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

-

-

-

-

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

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

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

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.

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

Declarations

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.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Genetic Department, Faculty of Agriculture, Ain Shams University
(2)
Institute of Parasitology, Biology Centre, Czech Academy of Sciences
(3)
Institute of Biosciences and Bioresources (IBBR), National Research Council (CNR) of Italy
(4)
Central Lab. of Organic Agriculture, Agricultural Research Center
(5)
Botany and Microbiology Department, Faculty of Science, Helwan University
(6)
Microbiology Department, Faculty of Agriculture, Ain Shams University

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