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  • Short genome report
  • Open Access

Complete genome sequence of lytic bacteriophage RG-2014 that infects the multidrug resistant bacterium Delftia tsuruhatensis ARB-1

  • 1, 4,
  • 1, 5,
  • 2,
  • 1,
  • 2, 3 and
  • 1Email author
Standards in Genomic Sciences201712:82

https://doi.org/10.1186/s40793-017-0290-y

©  The Author(s). 2017

  • Received: 23 May 2017
  • Accepted: 24 November 2017
  • Published:

Abstract

A lytic bacteriophage RG-2014 infecting a biofilm forming multidrug resistant bacterium Delftia tsuruhatensis strain ARB-1 as its host was isolated from a full-scale municipal wastewater treatment plant. Lytic phage RG-2014 was isolated for developing phage based therapeutic approaches against Delftia tsuruhatensis strain ARB-1. The strain ARB-1 belongs to the Comamonadaceae family of the Betaproteobacteria class. RG-2014 was characterized for its type, burst size, latent and eclipse time periods of 150 ± 9 PFU/cell, 10-min, <5-min, respectively. The phage was found to be a dsDNA virus belonging to the Podoviridae family. It has an isometric icosahedrally shaped capsid with a diameter of 85 nm. The complete genome of the isolated phage was sequenced and determined to be 73.8 kbp in length with a G + C content of 59.9%. Significant similarities in gene homology and order were observed between Delftia phage RG-2014 and the E. coli phage N4 indicating that it is a member of the N4-like phage group.

Keywords

  • Bacteriophage
  • Delftia tsuruhatensis
  • Multidrug resistant
  • Biofouling
  • Biofilm
  • Genome
  • Podoviridae

Introduction

The occurrence and spread of antibiotic resistant bacteria in the environment are regarded as environmental challenges of highest concern in the twenty-first century. ARB bacteria are becoming common, and the Centers for Disease Control and Prevention in the United States estimates more than 23,000 patients die annually due to ARB infections in the US alone [1]. With diminishing opportunities to discover new drugs to combat ARB infections, there is an urgent need to develop alternative therapeutic methods. Phage therapy has been regarded as an alternative to the need of synthesizing new antibiotics [2].

The Delftia genus resides in the Comamonadaceae family of the Betaproteobacteria class and is a Gram negative, short rod-shaped bacterium. Delftia species are widely distributed in the environment and have significant biodegradation capability [3, 4]. A recently described species, closely related to Delftia acidovorans , Delftia tsuruhatensis , has been reported to cause biofouling of bioreactor membranes [5], reverse osmosis membrane filters [6] and heating systems [7]. In addition, D. tsuruhatensis has been reported to be the causative agent of catheter-related nosocomial human infections [8, 9]. Previously, we isolated a multi-drug resistant D. tsuruhatensis strain ARB-1 from a municipal wastewater treatment plant along with the lytic bacteriophage. We demonstrated phage based therapy to combat biofouling caused by D. tsuruhatensis strain ARB-1 with the newly isolated lytic phage as the therapeutic agent [10].

Here, we report the complete genome sequence of the lytic phage specific to D. tsuruhatensis ARB-1 that we named RG-2014 (it does not infect Delftia Cs1–4 or Delftia acidovorans SPH-1 (our unpublished results) [10]. The RG-2014 sequence is annotated and analyzed in order to explore its potential application as an anti-biofilm bio-agent. The host of RG-2014 is multi-drug resistant, using it as a control agent can be an especially appropriate application. The present study is not part of a larger genomic survey.

Organism information

Classification and features

The lytic bacteriophage RG-2014 belongs to the Podoviridae family in the order Caudovirales. It is a double-stranded DNA virus that forms 1-2 mm diameter clear plaques when infecting the multidrug resistant bacterium Delftia tsuruhatensis strain ARB-1.

A sample of sludge was obtained from a local wastewater treatment plant, the Central Valley Water Reclamation Facility in Salt Lake City UT, USA. A lytic phage infecting D. tsuruhatensis ARB-1 was isolated from this sample following a previously described protocol [11, 12]. To remove bacteria and debris the sample was sequentially filtered through 0.45 and 0.2 μm filter membranes [10]. The resulting phage-containing liquid was spotted (without further concentration) on an R2A agar (0.5 g/L protease peptone, 0.5 g/L yeast extract, 0.3 g/L K2HPO4, 0.05 g/L MgSO4·7H2O, pH 7) plate containing a lawn of D. tsuruhatensis ARB-1 [10]. Following incubation of the plates at 37 °C overnight, a clear plaque was picked, followed by the isolation of a second well-separated single plaque on a fresh D. tsuruhatensis ARB-1 lawn.

As shown in Fig. 1(a) the head of phage RG-2014 virion has a diameter of 85 nm and displays a hexagonal outline implying that it likely possesses icosahedral symmetry. It can also be seen from this transmission electron micrograph, that the virion has a very short tail, indicating that it is a member of the Podoviridae class of viruses. Figure 1(b) shows a micrograph with RG-2014 phage particles attached to a D. tsuruhatensis bacterial cell pili; it is not known if such pili may serve as receptor for this phage. Table 1 gives the classification and general features of RG-2014 phage. The genome of the phage is linear double-stranded DNA (dsDNA) that is about 70 kb in length as measured by its mobility during pulsed-field gel electrophoresis (Fig. 1(c)).
Fig. 1
Fig. 1

Negative strain transmission electron micrographs of (a) RG-2014 virions (scale bar represents 100 nm), (b) RG-2014 infecting D. tsuruhatensis ARB1 (scale bar represents 1 μm) and (c) Pulsed field electrophoresis gel strained with acridine orange; Lane 1, Molecular weight marker (numbers shown are in kbp); Lane 2, 2 μg of DNA from phage RG-2014 virions; lane 3, same as lane 2 with 0.5 μg of phage DNA

Table 1

Classification and general features of Delftia tsuruhatensis ARB-1 bacteriophage RG-2014

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Viruses

TAS [40]

Kingdom Viruses

TAS [40]

  

Phylum: unassigned

TAS [40]

Class: dsDNA viruses, no RNA phase

TAS [40]

Order: Caudovirales

TAS [40]

  

Family: Podoviridae

TAS [40]

  

Genus: N4likevirus

TAS [40]

  

Species: unassigned

 

(Type) strain: RG-2014 (KM879221.2)

 

Gram stain

Not applicable

TAS [40]

Virion shape

Icosahedral

IDA

Motility

non-motile

IDA

Sporulation

Not reported

IDA

Temperature range

20–38 °C

IDA

Optimum Temperature

37 °C

IDA

pH range; Optimum

6.5–7.6

IDA

Carbon Source

Host cell

IDA

MIGS-6

Habitat

Wastewater

IDA

MIGS-6.3

Salinity

Not reported

 

MIGS-22

Oxygen

Facultative aerobic

IDA

MIGS-15

Biotic relationship

Obligate intracellular parasite of D. tsuruhantensis ARB-1

IDA

MIGS-14

Pathogenicity

Infective phage of D. tsuruhantensis ARB-1

IDA

MIGS-4

Geographic location

Central Valley Water Reclamation Facility, UT, USA

IDA

MIGS-5

Sample collection time

02/01/2011, 11:00 AM

IDA

MIGS-4.1

Latitude

40.7056

IDA

MIGS-4.2

Longitude

111.913953

IDA

MIGS-4.3

Depth

Surface

IDA

MIGS-4.4

Altitude

0 m

 

aEvidence codes - IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature)

A one step growth curve was performed with the phage RG-2014 following previously described protocols [10]. The burst size, latent and eclipse period were found to be 150 ± 9 PFU/cell, 10-min, and <5-min, respectively, at 37 °C [10].

The complete genome sequence of the phage RG-2014 was determined. The analysis of the genome clearly shows that it is a member of the N4-like phage group (see below). Grose and Casjens [11] showed that the major capsid proteins (MCPs) of virulent tailed phages parallel the evolution of the nucleotide sequence of the whole phage genome. Phylogeny of the MCPs of selected N4-like phages and other tailed phages shows that the phage RG-2014’s major capsid protein (identified by its similarity that of E. coli phage N4, accession no. EF056009) falls robustly within the N4-like phage group (Fig. 2).
Fig. 2
Fig. 2

Phylogenetic tree highlighting the position of major coat protein of phage RG-2014 relative to major coat proteins of other hosts. The corresponding GenBank accession numbers for each phage coat protein is indicated in parenthesis. Eleven other types of Podoviridae are included below the N4-like group for comparison. The tree construction used MUSCLE model to align the protein sequences by MEGA (v.5), and the Maximum-likelihood algorithm to construct a distance matrix based on alignment model positions using bootstrap method with 1000 bootstrap replications

Genome sequencing information

Genome project history

Phage RG-2014 was isolated in February of 2011, with D. tsuruhatensis strain ARB-1 as its host, The genome sequencing and analysis of phage RG-2014 was completed in December of 2016. It is the first genome sequence reported for a lytic phage infecting D. tsuruhatensis . The purified phage DNA was sequenced with a MiSeq Bench-top DNA sequencer (Illumina, CA) in the High-throughput Genomic Core Facility at the University of Utah. A summary of the phage RG-2014 genome sequencing information is presented below and in the Table 2.
Table 2

Project information of Delftia tsuruhatensis ARB-1 bacteriophage RG-2014

MIGS ID

Property

Term

MIGS-31

Finishing quality

Closed

MIGS-28

Libraries used

N/A

MIGS-29

Sequencing platforms

Illumina MiSeq Benchtop

MIGS-31.1

Fold coverage

20×

MIGS-30

Assemblers

CLC genomics workbench v. 7.0.3

MIGS-32

Gene calling method

GeneMarkS

Locus Tag

RG2014

 

Genome database release

Genbank

 

Genbank ID

KM879221.2

 

Genbank Date of Release

Oct, 8, 2014; Mar, 17, 2017 (Corrected genome release date)

GOLD ID

Go0332698

BIOPROJECT

PRJNA287956

MIGS 13

Source Material Identifier

Personal culture collection

Project relevance

Virulence, Bacteriophage based biocontrol

Growth conditions and genomic DNA preparation

Phage RG-2014 virions were purified from infected D. tsuruhatensis ARB-1 lysates. Briefly, 0.5 L of cells were grown to 1 × 108 cells per mL in R2A medium at 37 °C with shaking at 150 RPM [10]. The culture was then infected with five RG-2014 phages per cell, followed by incubation for 12 h. After clear cell lysis was observed leading to a cleared culture (the cells lysed), cell debris was removed by centrifugation for 30 mins at 5500×g. Phage virions were then pelleted by centrifugation overnight (>12 h) at 8890×g at 4 °C, and the pellet was re-suspended in SM buffer with Gelatin (5.8 g/L NaCl, 2.0 g/L, MgSO4.7H2O, 50 mL/L of 1 M Tris-HCl pH 7.5 and 5.0 mL/L of a 5% solution of gelatin). Purified phage virions were obtained by CsCl step gradient centrifugation as described by Earnshaw et al. [12]. The purified phages were stored in SM buffer with gelatin until further use.

The purified RG-2014 virion preparation was used for phage DNA extraction according to the protocol described by Casjens and Gilcrease [13]. Briefly, 400 μL of the CsCl purified phage particles was mixed with 75 μL of lysis buffer (5 μL of 20% SDS, 50 μL 1 M Tris. Cl, 20 μL 0.5 M EDTA, pH = 8) and incubated at 65 °C for 15 min. 50 μL of 5 M potassium acetate was added to the sample and incubated on ice for 1 h. The sample was then centrifuged at 8000×g for 15 min at 4 °C, and the supernatant was carefully transferred into a new 1.5 mL micro-centrifuge tube. After adding 0.9 mL of absolute ethanol to the supernatant and inverting several times, the DNA precipitate was collected by winding it onto the tip of a sterile Pasteur pipette. The DNA precipitate was transferred into a new micro-centrifuge tube, washed with 70% ethanol by inverting a few times, and subsequently pelleted by centrifugation in a microfuge. The DNA pellet was allowed to dry at room temperature for 10–20 min and resuspended in 100 μL of TE buffer (10 mM Tris-Cl pH 7.5 and 1 mM EDTA pH 8.0). About 0.1 μg of the phage DNA was mixed with 5 μL of loading dye and separated by 1% agarose pulsed-field gel electrophoresis (PFGE), with a 1–25-s pulse ramp, a voltage of 6.0 V/cm with an angle of 120° for 24 h at a constant temperature of 14 °C on a CHEF DR III system (Bio-Rad, USA). After completion of electrophoresis the gel was stained with ethidium bromide (Molecular Probes, USA) and visualized under CHEM DOC gel documentation system (Bio-Rad, USA).

Genome sequencing and assembly

Approximately 8 million paired-end reads with an average length of 300 bp were generated using a MiSeq Bench-top DNA sequencer (Illumina, CA). The reads were interleaved and trimmed based on a Phred score of 28 and a minimum post-trimming average length of 290 bp on the CLC Genomics Workbench 7.0.4 (CLC Bio, Denmark). The trimmed reads were de novo assembled on the CLC Genomics Workbench 7.0.4 with the following criteria: word size, 20 bp; automatic bubble size, 50 bp; minimum contig length, 200 bp as described in Bhattacharjee et al. [10].

The termini of the virion chromosome were determined by dideoxynucleotide Sanger sequencing [14] using the virion DNA as a template using the following primers which direct sequencing runs off the two ends as follows; right end, 5′-TGCTTCATGATCTTCAGTCC-3′ and left end, 5′-GAAGGCATCAGCATGTTCAG-3′.

Genome annotation

Glimmer [15] was used to identify the open reading frames and GeneMarkS [16] for predicting genes. The predicted genes were used to search the NCBI non-redundant database, the conserved domain database, the Cluster of Orthologous Groups database and the InterPro database and were annotated using Blast2GO 2.5.0 [17]. Automated annotation performed by Blast2GO 2.5.0 was manually curated by individually analyzing each predicted gene using BLAST against NCBI nr database with minimum e-value cut off of 10−3 [18]. ARAGORN [19] and tRNAScanSE [20] were used for detection of transfer RNA genes. The complete annotated genome sequence is available in Genbank under the accession number KM879221.

Genome properties

The lytic phage RG-2014’s complete genome size was found to be 73,882 bps that includes 450 bp direct terminal repeats (we note that, when it has been examined, the genomes of other N4-like phages invariably have several hundred bp terminal repeats)with a G + C content of 59.9%. The annotation includes 88 putative protein coding ORFs and no tRNAs (Table 3). Predicted proteins were classified in COG functional categories [21, 22] using the WebMGA web server for metagenome analysis [23]. The number of predicted genes and the relative percentage of phage genes associated with the 25 general functional COG categories are described in Table 4. Twenty-eight (31.8%) of the 88 genes in the RG-2014 phage genome were assigned a putative function based on significant sequence similarity to genes of known functionality in the NCBI database. Twenty-one (23.8%) genes encode putative proteins that were assigned to the conserved hypothetical protein category. Additionally, 40 predicted genes (44.3%) had no similarity to genes in the current database, and their products were classified as hypothetical proteins (Table 5). Annotation using the CDD on the NCBI server was also performed and is presented in Table 6.
Table 3

Genome statistics

Attribute

Value

% of Totala

Genome size (bp)

73,882

100.00

DNA Coding (bp)

69,793

93.90

DNA G + C (bp)

44,247

59.90

DNA scaffold

0

0.00

Total genes

88

100.00

Protein-coding genes

88

100.00

RNA genes

0

0.00

Pseudo genes

0

0.00

Genes in internal clusters

0

0.00

Genes with function prediction

21

23.86

Genes assigned to COGs

10

9.09

Genes with Pfam domains

12

13.64

Genes with signal peptides

2

2.27

Genes with transmembrane helices

13

14.77

CRISPR repeats

0

0.00

aThe total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome

Table 4

Number of genes associated with the 25 general COG functional categories

Code

Value

% agea

Description

J

0

0

Translation

A

0

0

RNA processing and modification

K

2

2.27

Transcription

L

2

2.27

Replication, recombination and repair

B

0

0

Chromatin structure and dynamics

D

0

0

Cell cycle control, mitosis and meiosis

Y

0

0

Nuclear structure

V

0

0

Defense mechanisms

T

0

0

Signal transduction mechanisms

M

1

1.14

Cell wall/membrane biogenesis

N

1

1.14

Cell motility

Z

0

0

Cytoskeleton

W

0

0

Extracellular structures

U

0

0

Intracellular trafficking and secretion

O

0

0

Posttranslational modification, protein turnover, chaperones

C

0

0

Energy production and conversion

G

0

0

Carbohydrate transport and metabolism

E

0

0

Amino acid transport and metabolism

F

2

2.27

Nucleotide transport and metabolism

H

0

0

Coenzyme transport and metabolism

I

0

0

Lipid transport and metabolism

P

0

0

Inorganic ion transport and metabolism

Q

0

0

Secondary metabolites biosynthesis, transport and catabolism

R

2

2.27

General function prediction only

S

1

1.14

Function unknown

–

77

87.5

Not in COGs

aThe total is based on the total number of protein coding genes in the annotated genome

Table 5

Delftia phage RG-2014 gene prediction

Gene

Strand

Number of codons

Predicted function

Organism with best match

N4 genea

Gene accession no.

% Idb

E-valueb

1

+

101

Conserved hypothetical protein

Erwinia phage Ea9–2

–

AIU44254

32

0.002

2

+

139

Conserved hypothetical protein

Achromobacter phage JWdelta

2

AHC56518

36

2e-21

3

+

121

Hypothetical protein

–

–

–

–

–

4

+

122

Conserved hypothetical protein

Roseovarius sp. phage 1

14

CBW47037

57

3e-45

5

+

109

Hypothetical protein

–

–

–

–

–

6

+

115

Hypothetical protein

–

–

–

–

–

7

–

104

Hypothetical protein

–

–

–

–

–

8

+

105

Hypothetical protein

–

–

–

–

–

9

+

50

Hypothetical protein

–

–

–

–

–

10

+

69

Hypothetical protein

–

–

–

–

–

11

+

186

Conserved Hypothetical protein

Pithovirus sibericum

–

YP 009001006

32

3e-22

12

+

285

Conserved hypothetical protein

Achromobacter sp.

–

CYTR01000018

38

2e-26

13

+

108

Conserved hypothetical protein

Escherichia phage phAPEC8

3

YP_007348409

29

3e-04

14

+

137

Hypothetical protein

–

–

–

–

–

15

+

89

Hypothetical protein

–

–

–

–

–

16

+

44

Hypothetical protein

–

–

–

–

–

17

+

77

Hypothetical protein

–

–

–

–

–

18

+

142

Conserved hypothetical protein

Achromobacter phage øAxp-3

–

YP_009148381

55

3e-37

19

+

193

Hypothetical protein

–

–

–

–

–

20

+

76

Conserved hypothetical protein

Pseudomonas phage PPpw-3 c

–

YP_008873216

40

5e-09

21

+

217

Hypothetical protein

–

–

–

–

–

22

+

272

RNA polymerase I subunit

Erwinia vB EamP Rexella

15

ANJ65251

54

1e-102

23

+

432

RNA polymerase II subunit

Erwinia phage Ea9–2

16

AAL71577

47

1e-135

24

+

181

Virion decoration protein

Achromobacter phage øAxp-3

17

YP_009208670

36

1e-10

25

+

157

Hypothetical protein

–

–

–

–

–

26

+

155

Hypothetical protein

–

–

–

–

–

27

+

122

Hypothetical protein

–

–

–

–

–

28

+

82

Hypothetical protein

–

–

–

–

–

29

+

80

Hypothetical protein

–

–

–

–

–

30

+

115

Hypothetical protein

–

–

–

–

–

31

+

242

Hypothetical protein

–

–

–

–

–

32

+

209

Hypothetical protein

–

–

–

–

–

33

+

359

Conserved hypothetical protein

Erwinia phage Ea9–2

24

AHI60096

46

7e-104

34

+

127

Conserved hypothetical protein

Achromobacter phage øAxp-3

–

YP009208682

41

7e-11

35

+

92

Hypothetical protein

–

–

–

–

–

36

+

405

Conserved hypothetical protein

Escherichia phage N4

25

ABK54394

39

1e-86

37

+

170

dCTP deaminase

Escherichia phage Bp4

26

AHN83412

51

4e-53

38

+

78

Hypothetical protein

–

–

–

–

–

39

+

124

Hypothetical protein

–

–

–

–

–

40

+

140

Hypothetical protein

–

–

–

–

–

41

+

169

Hypothetical protein

–

–

–

–

–

42

+

121

Hypothetical protein

–

–

–

–

–

43

+

103

Hypothetical protein

–

–

–

–

–

44

+

73

Hypothetical protein

–

–

–

–

–

45

+

317

Thymidylate synthase

Salmonella phage SEGD1 c

–

KU726251

48

2e-101

46

+

104

Conserved hypothetical protein

Escherichia phage N4

35

YP_950513

59

1e-36

47

+

135

Conserved hypothetical protein

Paenibacillus phage PG1 c

–

YP_008129928

66

5E-54

48

+

197

Nucleotide pyrophospho-hydrolase

Pseudomonas phage PaMx74 c

–

YP_009199508

33

3e-13

49

+

436

DNA helicase

Achromobacter phage JWdelta

37

AHC56567

48

4e-137

50

+

172

Conserved hypothetical protein

Achromobacter phage JWalpha

38

YP_009004756

34

2e-27

51

+

884

DNA polymerase

Escherichia phage N4

39

ABK54408

60

0.0

52

+

127

Hypothetical protein

–

–

–

–

–

53

+

286

Conserved Hypothetical protein

Nitrincola phage 1 M3–16

–

YP 009037286

47

1e-12

54

+

327

Conserved hypothetical protein

Escherichia phage G7C

41

AEL79653

45

7e-97

55

+

724

DNA primase

Achromobacter phage øAxp-3

42

ALA45517

62

0.0

56

+

249

Conserved hypothetical protein

Escherichia phage N4

43

ABK54413

57

3e-100

57

+

253

Single-stranded DNA-binding protein

Erwinia phage S6

44

AEJ81593

38

5e-37

58

+

372

Conserved hypothetical protein

Salmonella phage FSL_SP-076

–

YP_008240188

43 4

4e-24

59

+

61

Hypothetical protein

–

–

–

–

–

60

+

65

Hypothetical protein

–

–

–

–

–

61

+

235

Hypothetical protein

–

–

–

–

–

62

+

102

Hypothetical protein

–

–

–

–

–

63

+

59

Hypothetical protein

–

–

–

–

–

64

+

98

Conserved Hypothetical protein

Bacillus phage SP-10

–

YP 007003301

40

3e-10

65

+

288

Possible transcriptional regulator

Burkholderia phage AH2 c

 

AEY69538

38

5e-44

66

+

110

Hypothetical protein

–

–

–

–

–

67

+

172

Hypothetical protein

Deftia phage øW-14 3

–

YP_003359016

39 e

1e-10

68

–

3413

Virion RNA polymerase

Achromobacter phage øAxp-3

50

ALA45523

42

0.0

69

–

712

Lysozyme-like domain virion structural protein

Escherichia phage ECBP1

51

AFR52010

25

5e-18

70

–

135

Conserved hypothetical protein

Achromobacter phage JWdelta

–

AHC56583

75

2e-38

71

–

921

Conserved hypothetical protein

Achromobacter phage øAxp-3

53

ALA45526

36

1e-168

72

–

300

Virion structural protein

Escherichia phage N4

54

AAO24827

50

2e-101

73

–

265

Conserved hypothetical protein

Achromobacter phage øAxp-3

55

ALA45528

38

3e-47

74

–

411

Major capsid protein

Achromobacter phage øAxp-3

56

ALA45529

66

0.0

75

–

281

Conserved hypothetical protein

Escherichia phage IME11

57

AFV29058

38 d

7e-42

76

–

116

Hypothetical protein

Erwinia phage S6

58

YP_007005822

71 e

0.006

77

–

138

Conserved Hypothetical protein

Roseovarius sp. 217 phage 1

–

CBW47064

28

0.002

78

–

766

Portal protein

Erwinia phage Frozen

59

ANJ65209

59

0.0

79

–

170

Lysis / possible Rz-like spanin

Achromobacter phage JWalpha

60

AHC94031

40

4e-21

80

–

201

Lysis / N-acetylmuramidase

Escherichia phage G7C

61

AEL79672

52

7e-71

81

–

108

Conserved hypothetical protein

Escherichia phage N4

63

ABK54424

34

1e-17

82

–

416

Conserved hypothetical protein

Achromobacter phage øAxp-3

64

ALA45537

64

0.0

83

–

1388

Tail sheath and receptor binding virion protein

Achromobacter phage øAxp-3

65

ALA45538

51

0.0

84

–

140

Hypothetical protein

–

–

–

–

–

85

–

234

Possible virion appendage protein

Erwinia phage Ea9–2

66

AHI60147

44 d

2e-67

86

–

536

Large terminase subunit

Escherichia phage ECBP1

68

AFR52033

61

0.0

87

–

228

Conserved hypothetical protein

Escherichia phage N4

69

ABK54430

46

2e-61

88

–

340

Conserved hypothetical protein

Achromobacter phage øAxp-3

49

ALA45543

34

1e-17

a E. coli phage N4 is the best characterized and therefore the prototypical member of this phage group

b% identity and e-value determined by BLASTp at NCBI web site; unless otherwise noted, values are listed if the patch of similarity includes ≥60% of the protein

cAll phages in this column are in the N4-like group except AH2, øW-14, SEGD1, PG1, PaMx74 and PPpw-3

dSequence similarity only in N-terminal region

eSequence similarity only in C-terminal region

Table 6

Delftia phage RG 2014 annotation using conserved domain database*

Gene

Evidence

E value

Bit Score

Accession

4

cl10259 superfamily

2.22E-55

167.72

Cl10259

12

MTTB superfamily

0.004977

36.9774

Cl15385

15

MDR superfamily

0.0037317

33.0936

Cl16912

22

Pha00452

1.96E-05

44.2438

Pha00452

23

RNA_pol superfamily

4.77E-09

56.9554

Cl20211

24

Big_2

0.00242

34.2896

Pfam02368

24

Big_2 superfamily

3.49E-07

39.6898

Cl02708

24

Cog5492

3.72E-09

53.2664

Cog5492

33

Aaa

6.72E-05

41.3627

Cd00009

33

ABC_atpase superfamily

6.72E-05

41.3627

Cl21455

36

Vwfa

0.0009673

38.3158

Cd00198

36

Vwfa superfamily

1.20E-20

85.5169

Cl00057

36

DUF2201_N superfamily

9.26E-31

117.611

Cl16157

37

Trimeric_dutpase

3.53E-13

60.5857

Cd07557

37

Trimeric_dutpase superfamily

2.62E-23

89.0534

Cl00493

45

TS_Pyrimidine_hmase

5.70E-91

268.76

Cd00351

45

TS_Pyrimidine_hmase superfamily

5.17E-137

387.525

Cl19097

48

NTP-ppase superfamily

0.002418

35.1816

Cl16941

49

ABC_atpase superfamily

2.77E-17

78.3824

Cl21455

49

Uvrd_C_2

8.48E-08

47.1547

Pfam13538

49

Uvrd_C_2 superfamily

8.48E-08

47.1547

Cl22491

49

Aaa_30

2.77E-17

78.3824

Pfam13604

49

Cog1112

9.34E-05

43.4113

Cog1112

51

DNA_pol_A superfamily

1.80E-26

110.198

Cl02626

51

DNAq_like_exo superfamily

0.0005505

40.4172

Cl10012

55

Prict_1

1.28E-07

47.6526

Pfam08708

55

Prict_1 superfamily

1.28E-07

47.6526

Cl07362

56

ABC_atpase superfamily

0.0005674

38.4072

Cl21455

63

Prk14085

0.0005556

34.1837

Prk14085

64

DUF2829

1.18E-16

66.5176

Pfam11195

64

DUF2829 superfamily

1.18E-16

66.5176

Cl12744

65

Parbc

0.0004658

37.3039

Pfam02195

65

Parbc superfamily

0.0003729

37.2839

Cl02129

66

DUF1178

0.0021343

34.0766

Pfam06676

67

Extradiol_Dioxygenase_3B_like superfamily

0.0057676

34.7714

Cl00599

69

Lt_gewl

1.36E-18

80.5286

Cd00254

69

Lysozyme_like superfamily

1.36E-18

80.5286

Cl00222

70

Polyadenylate-binding_protein_3

0.0067594

34.0122

Tigr01628

72

DUF3584

0.0060894

36.9891

Pfam12128

74

Hypothetical_protein

5.26E-76

237.638

Tigr04387

74

P22_coatprotein superfamily

5.26E-76

237.638

Cl22542

78

Cog4913

0.001198

41.1603

Cog4913

79

Prk09039

0.000734

37.6381

Prk09039

80

Glyco_hydro_108 superfamily

9.31E-23

86.0288

Cl09583

80

PG_binding_3 superfamily

0.0001066

38.2277

Cl09627

86

COG5362 superfamily

3.02E-08

51.3532

Cl02216

88

Phage_gp49_66

2.28E-21

85.3759

Pfam13876

88

Phage_gp49_66 superfamily

2.28E-21

85.3759

Cl10351

*Evidence of gene functions provided by blast analysis using conserved domain database (e-value ≤10−5)

Insights from the genome sequence

The phylogenetic tree of MCPs in Fig. 2 indicates that phage RG-2014 is most closely related to the group of phages typified by Escherichia coli phage N4 (NC_008720) [13, 24–28]. In addition their hosts, E. coli K-12 and D. tsuruhatensis strain ARB-1 belong to the same phylum Proteobacteria . Table 1 summarizes the classification and general features of the phage RG-2014. BLAST searches using the Delftia phage RG-2014 genome as a probe was undertaken to confirm this notion. Genome comparisons with E. coli phage N4 (NC_008720) were performed, and significant similarities in gene homology and order were observed between phages RG-2014 and N4 (Table 5 and Fig. 3). The phage RG-2014 genome shows mosaicism that is typical of tailed phages, with (for example) some regions displaying close relatedness to phage N4 (Fig. 3). Mosaicism in bacteriophage genomes is a well-known phenomenon wherein regions of high similarity are interspersed with less related or unrelated regions. These mosaic patterns in bacteriophage genomes corroborate the theory that horizontal gene transfer plays a significant role in phage evolution [29–31].
Fig. 3
Fig. 3

Whole genome comparison of Delftia phage RG-2014 (KM872991.2) phage to E. coli phage N4 (NC_008720). The Figure was generated with Easyfig [38]. Genomes were aligned using Easyfig [38]. The functions of genes in phage N4 are shown above and predicted functions of RG-2014 genes are indicated below the maps

E. coli phage N4 does not depend upon its host’s RNA polymerase to transcribe its early and middle genes. But encodes its own set of two RNAPs. These are encoded by three genes, one for the early RNAP and the two subunits of the middle gene transcribing RNAP [28, 32]. The host’s RNAP transcribes the N4 late genes. A striking and unique feature of this type of phage is that a unique single-subunit vRNAP is carried in the virion. vRNAP is encoded by N4 gene 50 and is injected into the host cell with the DNA where it transcribes the phage’s early genes. The RNAPII that transcribes the middle genes and is encoded by the two N4 genes 15 and 16. The RG-2014 genome harbors three genes that are homologues of the N4 RNAP genes, 68, 22 and 23, respectively. The closest relatives of these RG-2014 genes are present in N4-like phages Achromobacter phage øAxp-3, Erwina phage Frozen, and Erwina phage Ea9–2, respectively (Table 5).

Most of the N4 like phages have been shown to harbor between 1 and 3 genes encoding tRNA. Paepe et al. [33] and Bailey-Bechet et al. [34] suggesting, virulent phages harbor more tRNA genes than temperate phages to ensure optimal translation leading to faster replication. However, the phage RG-2014 genome lacks transfer RNA genes, suggesting that the phage is highly adapted to its host D. tsuruhatensis ARB-1, with regard to codon usage, allowing it to translate its genes efficiently without the need of synthesizing its own tRNAs [24]. To support our finding average codon usage bias was calculated for the phage RG-2014 and D. tsuruhatensis CM13 (NZ_CP017420), a close representative of the host D. tsuruhatensis ARB-1. The average codon usage bias calculation was performed using CodonO web server (http://sysbio.cvm.msstate.edu/CodonO/) [35]. D. tsuruhatensis CM13 (NZ_CP017420) and phage RG-2014 had similar average codon usage bias of 0.440141 and 0.406048, respectively, suggested the phage was adapted to its host.

There are two known types of virion assembly gene arrangements in the N4-like phages. First, those like phage N4 that have a single contiguous gene cluster that encodes all of the known structural genes and lysis proteins except the head decoration protein (N4 gene 17). Second, typified by Pseudomonas phage LIT1 in which several tail genes are present inside the replication gene cluster [25, 36]. Phage RG-2014 carries a set of homologous genes, including the separate decoration protein gene (RG-2014 gene 24), that have the phage N4 type organization. By homology to those of N4 [36], RG-2014 genes 24, 68, 69, 71–78, 83 and 85 encode virion structural proteins.

Phage RG-2014 makes clear plaques and carries no genes that encode proteins (such as integrase or protelomerase) that might suggest a temperate lifestyle. In addition, we also recently showed that the database of bacterial genome sequences has grown to a point where relatives of essentially all known temperate phages can be found as prophages present in the reported genome sequences of their hosts [37]. Thus, absence of closely related homologous genes (the MCP gene was used in that study) in closely related host genomes of the same bacterial family is strong evidence that a phage is virulent; related prophages would be found to encode such a gene if the phage in question were temperate. In fact no genes that are closely related to MCP of the phage RG-2014 are present in the current bacterial sequence database. The closest MCP gene relatives in prophages are from the distantly related bacterial genera Mesorhizobium , Pantoea and Acinetobacter whose encoded homologous proteins are only 47–56% identical to the amino acid sequence of phage RG-2014 MCP. The latter gene matches are found (when the sequence contigs are sufficiently large for such a determination) to be present in rather distantly related prophages that have other similarities to the N4-like phages including a prophage encoded vRNAP, suggesting that there are currently undescribed temperate phages that are very distantly related to the N4-like phage group (our unpublished observation). Nonetheless, among the 143 currently available genomes from the Comamonadaceae bacterial family (including eight Delftia genomes) the best-encoded protein matches have only 22% identity to the phage RG-2014 MCP. We conclude that phage RG-2014 is virulent.

The N4-like phage group is clearly well separated from the other known tailed bacteriophages [11, 28], but the taxonomic status of different phages within the group remains less understood. Unlike some other tailed phage types, the N4-like phages include members that infect a wide range of bacterial hosts in the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria classes [25, 28]. Fig. 4 shows a dotplot of a diverse sample of N4-like phage genomes that illuminates several aspects of the phages in this group (no diagonal lines are present when comparison is with other tailed phage types, data not shown). First, phage RG-2014 is not particularly closely related to any of the other currently known N4-like phages; its closest, but nonetheless rather distant, relatives are Achromobacter phages JWDelta, JWAlpha and øAxp-1. We note that these four phages infect members of the Βetap roteobacteria . A second conclusion that can be drawn from fig. 4 is that genome similarity within this group of phages generally parallels the relatedness of their hosts. The various subtypes of the N4-like phage group (separated by thick red lines in the figure) are usually restricted to single genus; the one current exception to this rule is the relatively close relationship between Vibrionaceae phage VPB47 and Pseudoalteromonadaceae phage pYD6-A. It thus appears that recent “jumping” of these phages between taxonomically distant hosts is not common. On the other hand, more than one N4-like phage subtype can infect a given host genus; for example, Escherichia and Erwinia N4-like phages are clearly present as two subtypes (e.g. the Escherichia N4/EcP1 and Erwinia Ea9–2/S6 pairs). More distant host relationships are complex. Very weak diagonal similarity lines are present when the Escherichia (phage N4 subtype), Erwinia and Achromobacter N4-like phages are compared. These could tentatively correspond to members of the proposed Enquatravirinae subfamily [28].
Fig. 4
Fig. 4

Dotplot of N4-like phage genomes. Phage genomes were arranged in the same orientation and a dot plot was constructed by Gephard [39] with a word length setting of 11. The phages in the figure include the current extant diversity among the N4-like phages; those that are not included are very similar to one of the phages that is included (their sequences are all in GenBank and can be retrieved by searching with their names). In the plot thin red lines separate the phage genomes, and thick red lines separate the most clearly delineated subtypes. At the right, the genus (red text), family (black text) and class (blue text) of each phage’s host bacteria are indicated; vertical very thick red lines on the right indicate phages that infect the same host genus, and very thick blue lines mark host families

Conclusions

The D. tsuruhatensis infecting phage RG-2014 belongs to the Podoviridae viral family. The phage RG-2014 genome sequence shows significant synteny and sequence similarity to E. coli bacteriophage N4 and other members of the N4-like group of tailed phages; this clearly demonstrates phage RG-2014’s membership in this group. Our analysis confirms that phages in the virulent N4-like group are widely present in the wild. The members of the N4-like group infect bacterial hosts in several classes within the Proteobacteria phylum. Their virulent nature, widespread distribution and efficient infection suggest that members of this group will be useful in many bacterial control situations.

Abbreviations

ARB: 

Antibiotic Resistant Bacteria

COG: 

Cluster of Orthologous Groups

ORF: 

Open reading frame

RNAP: 

RNA polymerase

Declarations

Acknowledgements

This research was conducted under the CAREER funding mechanism of NSF. This project was awarded to Dr. Ramesh Goel. Mr. Eddie B. Gilcrease was supported under a NIH funded project with Dr. Sherwood Casjens as the principal investigator. Any opinions expressed in this paper are those of the authors and do not necessarily reflect the views of the agency; therefore, no official endorsement should be inferred. Any mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Funding

This research was partially supported by National Science Foundation (NSF) Grant 1,055,786 and 58,501,574 to RG and NIH grant GM114817 to SRC. The views and opinions expressed in this manuscript are those of authors and do not necessarily relate to the funding agency.

Authors’ contributions

ASB design the study, performed the experiments, analyzed data, and wrote the manuscript. AMM helped with the experiments and writing of the manuscript. EBG helped with the PCR assays for finishing up the genome. SRC participated in the experiments, helped in analyzing the data, and writing the manuscript. MII reviewed the manuscripts and provided valuable comments. RG helped in designing the study, coordinated the project and assisted in drafting the manuscript. All authors have read and approved the final manuscript.

Competing interests

The authors declare that they have no financial and non-financial competing interests.

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Authors’ Affiliations

(1)
Department of Civil and Environmental Engineering, University of Utah, Salt Lake City, UT, USA
(2)
Division of Microbiology and Immunology, Pathology Department, University of Utah School of Medicine, Salt Lake City, UT, USA
(3)
Department of Biology, University of Utah, Salt Lake City, UT, USA
(4)
Bigelow Laboratory for Ocean Science, 60 Bigelow Dr., East Boothbay, ME, USA
(5)
Department of Civil, Environmental, and Construction Engineering, University of Central Florida, 12800 Pegasus Dr., Room 340, Orlando, FL, USA

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