UFV-P2 as a member of the Luz24likevirus genus: a new overview on comparative functional genome analyses of the LUZ24-like phages

  • Monique R Eller1,

    Affiliated with

    • Pedro M P Vidigal2,

      Affiliated with

      • Rafael L Salgado3,

        Affiliated with

        • Maura P Alves1,

          Affiliated with

          • Roberto S Dias4,

            Affiliated with

            • Cynthia C da Silva4, 6,

              Affiliated with

              • Antônio F de Carvalho1,

                Affiliated with

                • Andrew Kropinski7, 8 and

                  Affiliated with

                  • Sérgio O De Paula4, 5Email author

                    Affiliated with

                    BMC Genomics201415:7

                    DOI: 10.1186/1471-2164-15-7

                    Received: 16 July 2013

                    Accepted: 26 December 2013

                    Published: 3 January 2014

                    Abstract

                    Background

                    Phages infecting spoilage microorganisms have been considered as alternative biocontrol agents, and the study of their genomes is essential to their safe use in foods. UFV-P2 is a new Pseudomonas fluorescens-specific phage that has been tested for its ability to inhibit milk proteolysis.

                    Results

                    The genome of the phage UFV-P2 is composed of bidirectional modules and presented 75 functionally predict ORFs, forming clusters of early and late transcription. Further genomic comparisons of Pseudomonas-specific phages showed that these viruses could be classified according to conserved segments that appear be free from genome rearrangements, called locally collinear blocks (LCBs). In addition, the genome organization of the phage UFV-P2 was shown to be similar to that of phages PaP3 and LUZ24 which have recently been classified as a Luz24likevirus.

                    Conclusions

                    We have presented the functional annotation of UFV-P2, a new Pseudomonas fluorescens phage. Based on structural genomic comparison and phylogenetic clustering, we suggest the classification of UFV-P2 in the Luz24likevirus genus, and present a set of shared locally collinear blocks as the genomic signature for this genus.

                    Background

                    According to the International Committee of Virus Taxonomy (ICTV) classification scheme based on morphology, biological characteristics and genome organization (http://​www.​ictvonline.​org/​virusTaxonomy.​asp), the bacteriophage family Podoviridae contains two subfamilies and 11 genera, and the Luz24likevirus genus comprises the Pseudomonas-infecting bacteriophages PaP3 [1] and LUZ24 [2]. Beyond PaP3 and LUZ24, the phages tf [3], MR299-2 [4], PaP4(KC294142), vB_PaeP_p2-10_Or1 (HF543949) and vB_PaeP_C1-14_Or (HE983844) have similar genomic compositions and should be classified to this genus.

                    Pseudomonas fluorescens bacteriophage UFV-P2 [5], is a virus with a high ability to reduce casein proteolysis in milk. Milk proteolysis is caused by thermo-resistant enzymes produced by psychrotrophs and is responsible for serious losses in the dairy industry due to negative effects on the quality and reduced shelf life of dairy products. In this environment, Pseudomonas spp. are prevalent contaminants [68], mainly P. fluorescens[9, 10]. The use of phages in biocontrol has been suggested as an alternative to the use of chemicals. For example, P. fluorescens-specific phages had been studied to control Pseudomonas population and as sanitation agents to efficiently remove bacterial biofilms on stainless steel surfaces similar to those used in food industries, where these contaminants are common [1113]. However, they must be used with caution. In addition to proteolysis reduction and biofilm inhibition studies and, their host range determination, it is necessary to understand phages’ genome and proteome to make possible their use as biocontrol agents.

                    To expand our understanding about the P. fluorescens-specific phage UFV-P2, we present in detail the analysis of its structural genome and its comparisons to other phage genomes.

                    Methods

                    Sampling

                    The phage UFV-P2 was isolated from wastewater of a dairy industry in Minas Gerais, Brazil, and propagated at 30°C in LB medium in a strain of P. fluorescens 07A, courtesy of Laboratory of Food Microbiol, located at the Federal University of Viçosa, Brazil.

                    Genome extraction and composition

                    Phages were propagated in LB medium containing the bacteria in exponential phase. After incubation at 30°C for 8 h, particle assemble was induced with mitomicin and the virions were recovered by centrifugation and filtration. Phage suspensions were incubated with 75 μg/mL of proteinase K in the presence of 0.01% SDS at 56°C for 90 min. Proteins were removed by extraction with phenol, phenol:chloroform (1:1), followed by chloroform. Genetic material was concentrated with an equal volume of isopropanol and resuspended in 30 μL of distilled water. For analysis of viral genome composition, 5 μL of the genomic extracts were submitted to digestion assays with enzymes DNase I (50 μg/mL) or RNaseA (100 μg/mL) for 60 min at 37°C, followed by 1% agarose gel electrophoresis and visualization by staining with GelRed (Biotium, USA).

                    Genomic DNA sequencing and assembly

                    UFV-P2 genome was sequenced using an Illumina Genome Analyzer II by CD Genomics (New York, USA) and was assembled and analyzed using CLC Genomics Workbench version 5.1 (CLC bio, Cambridge, MA, USA). The sequence reads were assembled into contigs using stringent parameters, in which 90% of each read had to cover the other read with 90% identity. The data are available in GenBank database under accession number JX863101.

                    Bioinformatics analysis

                    The genome of phage UFV-P2 was oriented to be collinear with that of the type species, Pseudomonas phage LUZ24, and manually annotated using Kodon (Applied Maths, Austin, TX, USA.) [14]. The GenBank flat file (gbk) file was exported from Kodon and converted to FASTA-formatted protein sequences using gbk2faa (http://​lfz.​corefacility.​ca/​gbk2faa/​). The latter were screened for viral homologs using the BLASTP feature of Geneious R6.1 (Biomatters Ltd., Auckland, New Zealand); and, for protein motifs, using TMHMM [15], Phobius [16] and Batch Web CD-Search Tool [17] at http://​www.​ncbi.​nlm.​nih.​gov/​Structure/​bwrpsb/​bwrpsb.​cgi.

                    Putative promoters were identified using the Kodon sequence similarity search feature employing TTGACA(N15-18)TATAAT and allowing for a 2 bp mismatch. Rho-independent terminators were tentatively identified using ARNold [18, 19] at http://​rna.​igmors.​u-psud.​fr/​toolbox/​arnold/​index.​php.

                    For comparative purposes at the genomic level EMBOSS Stretcher [20] and progressive Mauve [21] were employed; while at the proteomic level we used CoreGenes [22, 23]. Seventeen genomic reference sequences of phages were downloaded from GenBank (Table 1) and compared to UFV-P2 genome.
                    Table 1

                    Pairwise comparisons of phage UFV-P2 and others phage genomes

                        

                    Phage UFV-P2

                    Phage

                    GenBank accession

                    GC content (%)

                    Genome density (genes/kbp)

                    Identities

                    %

                    UFV-P2

                    JX863101

                    51.5

                    1.65

                    -

                    -

                    vB_PaeP_p2-10_Or1

                    HF543949

                    52.0

                    1.32

                    27,253

                    57.46

                    vB_PaeP_C1-14_Or

                    HE983844

                    52.0

                    1.41

                    27,672

                    57.31

                    LUZ24

                    NC_010325

                    52.2

                    1.49

                    27,510

                    56.80

                    PaP4

                    KC294142

                    52.5

                    1.59

                    27,015

                    56.73

                    PaP3

                    NC_004466

                    52.2

                    1.56

                    27,358

                    56.20

                    MR299-2

                    JN254801

                    52.0

                    1.52

                    27,192

                    56.19

                    Tf

                    NC_017971

                    53.2

                    1.51

                    23,750

                    49.55

                    Phi-2

                    NC_013638

                    58.9

                    1.00

                    22,672

                    46.73

                    phiKMV

                    NC_005045

                    62.3

                    1.13

                    22,392

                    46.43

                    phiIBB-PF7A

                    NC_015264

                    56.3

                    1.27

                    22,150

                    46.41

                    Bf7

                    NC_016764

                    58.4

                    1.15

                    21,825

                    46.16

                    PaP2

                    NC_005884

                    45.4

                    1.32

                    22,523

                    46.11

                    119X

                    NC_007807

                    44.9

                    1.29

                    22,434

                    46.03

                    T7 ( Enterobacteria )

                    NC_001604

                    48.4

                    1.50

                    21,602

                    45.66

                    gh-1

                    NC_004665

                    57.4

                    1.12

                    21,218

                    45.35

                    F116

                    NC_006552

                    63.2

                    1.07

                    27,102

                    41.20

                    LUZ7

                    NC_013691

                    53.2

                    1.54

                    29,160

                    38.74

                    Phylogenetic clustering

                    For clustering UFV-P2 phage in an evolutionary way, a phylogenetic hypothesis was inferred by Bayesian inference (BI) using MrBayes v3.2.2 [24]. The genomic sequences of phages were aligned using ClustalW [25], and a pairwise distance matrix was calculated MEGA version 5 [26] (Table 1). The alignment was manually inspected, and the sites with gaps were excluded. To expedite the construction of phylogenetic trees, a model of nucleotide substitution was estimated using the jModelTest 2 program [27]. The GTR + G substitution model was selected as the best DNA evolution model for genomic sequences, according to the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC).

                    The BI phylogenetic tree was calculated using the Bayesian Markov Chain Monte Carlo (MCMC) method, in two runs with 5,000,000 generations. The convergence of the parameters was analyzed in TRACER v1.5.0 (http://​beast.​bio.​ed.​ac.​uk/​tracer), and the chains reached a stationary distribution after 50,000 generations. Then, a total of 1% of the generated trees was burned to produce the consensus tree. To root the phylogenetic tree, the Enterobacteria phage T7 (NC_001604) was selected as outgroup taxa.

                    Results and Discussion

                    Transmission electron microscopy of the UFV-P2 virions (data not shown) showed that this virus has isometric capsids and very short tails, with morphological similarity to the P. aeruginosa phages Pap3 and MR299-2. Thus, UFV-P2 can be inserted in the Podoviridae family, order Caudovirales.

                    Functional genomic organization

                    The viral genome was extracted and sequenced

                    The phage UFV-P2 has a linear 45,517 bp DNA genome with a GC content of 51.5%, and was sequenced with coverage of 30,655 fold. One of the interesting characteristics of members of the Luz24likevirus genus is the presence of localized single-stranded breaks associated with the consensus sequence TACTRTGMC [28]. Fourteen of these sequences were found in the top strand of the tf DNA, while the genome of UFV-P2 contains 15.

                    At first, bioinformatics analyses had showed that the UFV-P2 genome has a bidirectional organization with 92 predicted open reading frames (ORFs) larger than 100 bp, but only 41 ORFs (44.75%) could be identified as coding sequences (CDS) by similarity searches against known proteins in the GenBank and UniProt databases [5]. However, we propose a new annotation of the genome of this virus based on different tools, which were able to functionally predict 75 ORFs also bidirectionally oriented and forming clusters of early and late transcription (Figure 1 and Table 2).
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-15-7/MediaObjects/12864_2013_5641_Fig1_HTML.jpg
                    Figure 1

                    The genomic organization of the phage UFV-P2. The colored squares in the background correspond to the conserved locally collinear blocks (LCB) found for the UFV-P2 in relation to the other members of the Luz24likevirus genus (Figure 2). The detailed annotation is shown in Table 2.

                    Table 2

                    Functional genomic annotation of phage UFV-P2

                    Gene

                    Predicted protein

                    Genomic coordinates

                    Strand

                    Protein mass (Da)

                    Protein pI

                    AA residues

                    Homologs (a) & Motifs

                    E-value

                    ORF01

                    Conserved hypothetical protein

                    1205..1483

                    +

                    10486

                    10.5

                    92

                    NP_955002 hypothetical protein PaP3p71 [Pseudomonas phage PaP3]

                    7.34E-09

                    ORF02

                    Conserved hypothetical protein

                    1501..1983

                    +

                    17752

                    9.4

                    160

                    YP_006382463 hypothetical protein tf_02 [Pseudomonas phage tf]; protein motifs: cl10333 PHA01782

                    3.22E-37

                    ORF03

                    Conserved hypothetical protein

                    2046..2216

                    +

                    6515

                    7.8

                    56

                    YP_007183264 hypothetical protein BN425_ORF_56 [Pseudomonas phage vB_PaeP_p2-10_Or1]

                    7.83E-02

                    ORF04

                    Hypothetical protein

                    2231..2368

                    +

                    5276

                    9.8

                    45

                    -

                    -

                    ORF05

                    Hypothetical protein

                    2365..2556

                    +

                    7171

                    9.2

                    63

                    -

                    -

                    ORF06

                    Hypothetical membrane protein

                    2553..2687

                    +

                    4708

                    9.5

                    44

                    protein motifs: one transmembrane domain discovered using TMHMM and Phobius

                     

                    ORF07

                    Conserved hypothetical protein

                    2677..2898

                    +

                    8374

                    9.8

                    73

                    YP_006382470 hypothetical protein tf_10 [Pseudomonas phage tf]

                    2.09E-04

                    ORF08

                    Conserved hypothetical protein

                    2898..3020

                    +

                    5178

                    9.3

                    40

                    AGC35239 hypothetical protein PaP4_008 [Pseudomonas phage PaP4]

                    1.19E-05

                    ORF09

                    Hypothetical protein

                    3021..3275

                    +

                    9120

                    4.1

                    84

                    -

                    -

                    ORF10

                    Hypothetical protein

                    3368..3568

                    +

                    7493

                    9.6

                    66

                    -

                    -

                    ORF11

                    Hypothetical protein

                    3555..3869

                    +

                    11403

                    5.0

                    104

                    -

                    -

                    ORF12

                    Hypothetical protein

                    3869..4063

                    +

                    7139

                    9.0

                    64

                    -

                    -

                    ORF13

                    Conserved hypothetical protein

                    4060..4458

                    +

                    14906

                    9.4

                    132

                    YP_006382473 hypothetical protein tf_13 [Pseudomonas phage tf]

                    9.55e-15

                    ORF14

                    Hypothetical membrane protein

                    4539..4736

                    +

                    7618

                    9.1

                    65

                    protein motifs: one to two transmembrane domains discovered using TMHMM and Phobius

                     

                    ORF15

                    Conserved hypothetical protein

                    4884..5714

                    +

                    29824

                    8.5

                    276

                    AGC35249 transposase fusion protein [Pseudomonas phage PaP4]; protein motifs: pfam01145 Band_7, & cl02525 Band_7

                    1.15E-113

                    ORF16

                    Conserved hypothetical protein

                    5727..6017

                    +

                    10708

                    9.1

                    96

                    YP_006659979 hypothetical protein tf_14 [Pseudomonas phage tf]

                    2.29E-11

                    ORF17

                    Conserved hypothetical protein

                    6021..6149

                    +

                    4783

                    11.0

                    42

                    NP_775206 hypothetical protein PaP3p51 [Pseudomonas phage PaP3]

                    1.32E-03

                    ORF18

                    Hypothetical protein

                    6159..6374

                    +

                    7803

                    5.0

                    71

                    -

                    -

                    ORF19

                    Conserved hypothetical protein

                    6441..7289

                    +

                    31291

                    9.2

                    282

                    YP_006382477 hypothetical protein tf_17 [Pseudomonas phage tf]

                    1.75E-03

                    ORF20

                    Conserved hypothetical protein

                    7296..8159

                    +

                    32906

                    4.9

                    287

                    YP_006382478 hypothetical protein tf_18 [Pseudomonas phage tf]

                    2.06E-07

                    ORF21

                    Conserved hypothetical protein

                    8171..8968

                    +

                    29423

                    5.0

                    265

                    YP_006382479 hypothetical protein tf_19 [Pseudomonas phage tf]; protein motifs: PF14395.1 COOH-NH2_lig

                    8.75E-112

                    ORF22

                    Conserved hypothetical protein

                    8965..9525

                    +

                    21203

                    7.5

                    186

                    NP_775210 hypothetical protein PaP3p47 [Pseudomonas phage PaP3]

                    2.53E-40

                    ORF23

                    Amidoligase

                    9501..10658

                    +

                    43407

                    5.5

                    385

                    YP_001671891 hypothetical protein [Pseudomonas phage LUZ24]; protein motifs: PF12224.3 Amidoligase_2

                    4.46E-96

                    ORF24

                    HNH endonuclease

                    10667..11071

                    +

                    15340

                    9.6

                    134

                    YP_002003475 gp2.8 [Enterobacteria phage BA14]; protein motifs: PF13392.1 HNH_3

                    2.71E-20

                    ORF25

                    Glutamine amidotransferase

                    11084..12544

                    +

                    54036

                    5.9

                    486

                    YP_006382482 glutamine amidotransferase [Pseudomonas phage tf]; protein motifs: PF13522.1 GATase_6, & cd00352 Gn_AT_II

                    2.72E-151

                    ORF26

                    Conserved hypothetical protein

                    12547..12765

                    +

                    8166

                    4.8

                    72

                    NP_775213 hypothetical protein PaP3p44 [Pseudomonas phage PaP3]

                    2.25E-20

                    ORF27

                    Hypothetical protein

                    12805..12909

                    +

                    3505

                    5.3

                    34

                    -

                    -

                    ORF28

                    ATP-grasp enzyme

                    12906..13781

                    +

                    31797

                    7.7

                    291

                    YP_006382486 hypothetical protein tf_24 [Pseudomonas phage tf]

                    2.60E-96

                    ORF29

                    Conserved hypothetical protein

                    13774..14169

                    +

                    15076

                    5.5

                    131

                    AGC35259 hypothetical protein PaP4_028 [Pseudomonas phage PaP4]; proteins motifs: PF06094.7 AIG2, & cd06661 GGCT_like

                    5.55E-27

                    ORF30

                    Conserved hypothetical protein

                    14169..14465

                    +

                    11295

                    5.4

                    98

                    YP_006382488 hypothetical protein tf_026 [Pseudomonas phage tf]

                    6.03E-23

                    ORF31

                    DNA primase/helicase

                    14425..16176

                    +

                    65673

                    5.9

                    583

                    YP_001671897 primase/helicase [Pseudomonas phage LUZ24]; protein motifs: PF13155.1 Toprim_2, & PF03796.10 DnaB_C

                    0

                    ORF32

                    DNA polymerase part I

                    16151..16666

                    +

                    20133

                    4.5

                    171

                    YP_006382490 3′-5′ exonuclease [Pseudomonas phage tf] & YP_001671898 DNA polymerase part I [Pseudomonas phage LUZ24]

                    2.82E-75; 3.72e-68

                    ORF33

                    Conserved hypothetical membrane protein

                    16669..16920

                    +

                    9085

                    10.0

                    83

                    YP_006382491 hypothetical protein tf_29 [Pseudomonas phage tf]; protein motifs: one transmembrane domain discovered using TMHMM and Phobius

                    1.99E-06

                    ORF34

                    Hypothetical protein

                    16931..17068

                    +

                    5096

                    3.4

                    45

                    -

                    -

                    ORF35

                    Hypothetical protein

                    17095..17325

                    +

                    8505

                    9.0

                    76

                    -

                    -

                    ORF36

                    Conserved hypothetical protein

                    17336..17539

                    +

                    7596

                    5.8

                    67

                    YP_001671900 hypothetical protein [Pseudomonas phage LUZ24]

                    2.23E-02

                    ORF37

                    Putative holin

                    17536..17817

                    +

                    10475

                    9.8

                    93

                    YP_006382493 hypothetical protein tf_32 [Pseudomonas phage tf] & YP_001671904 putative holin [Pseudomonas phage LUZ24]; protein motifs: three transmembrane domains discovered using TMHMM and Phobius

                    5.03e-35; 1.92e-18

                    ORF38

                    Hypothetical membrane protein

                    17814..18032

                    +

                    8079

                    9.9

                    72

                    protein motifs: one or two transmembrane domains discovered using TMHMM and Phobius

                     

                    ORF39

                    Conserved hypothetical protein

                    18069..18263

                    +

                    6728

                    3.5

                    64

                    YP_006382494 hypothetical protein tf_34 [Pseudomonas phage tf]

                    6.48E-04

                    ORF40

                    Conserved hypothetical protein

                    18263..18433

                    +

                    5628

                    8.2

                    56

                    YP_007112538 hypothetical protein MAR_61 [Vibrio phage vB_VpaM_MAR]

                    9.20E-05

                    ORF41

                    Conserved hypothetical protein

                    18433..18627

                    +

                    7272

                    10.9

                    64

                    YP_006382495 hypothetical protein tf_35 [Pseudomonas phage tf]

                    1.52E-03

                    ORF42

                    Hypothetical protein

                    18630..18764

                    +

                    4887

                    6.0

                    44

                    -

                    -

                    ORF43

                    DNA polymerase part II

                    18765..20405

                    +

                    60865

                    9.6

                    546

                    YP_007183240 DNA polymerase1 [Pseudomonas phage vB_PaeP_p2-10_Or1] & YP_001671907 DNA polymerase part II [Pseudomonas phage LUZ24]; protein motifs: PF00476.15 DNA_pol_A

                    0; 0

                    ORF44

                    DNA binding protein

                    20472..21068

                    +

                    21853

                    4.8

                    198

                    YP_006382500 DNA binding protein [Pseudomonas phage tf]

                    6.61E-70

                    ORF45

                    Conserved hypothetical protein

                    21139..21495

                    +

                    13200

                    6.3

                    118

                    AGC35272 hypothetical protein PaP4_041 [Pseudomonas phage PaP4]

                    4.02E-47

                    ORF46

                    Hypothetical protein

                    21525..21755

                    +

                    8335

                    4.7

                    76

                    -

                    -

                    ORF47

                    Hypothetical protein

                    21887..22222

                    +

                    12649

                    9.4

                    111

                    -

                    -

                    ORF48

                    5′-3′ exonuclease

                    22219..23103

                    +

                    33498

                    5.3

                    294

                    NP_775229 exonuclease [Pseudomonas phage PaP3]; protein motifs: PF01367.15 5_3_exonuc, & cd09898 H3TH_53EXO

                    1.47E-149

                    ORF49

                    Conserved hypothetical protein

                    23078..24073

                    +

                    37154

                    4.7

                    331

                    YP_006659984 conserved hypothetical protein [Pseudomonas phage tf]

                    1.15E-39

                    ORF50

                    Endonuclease

                    23874..24341

                    +

                    17655

                    5.4

                    155

                    YP_006382505 endonuclease [Pseudomonas phage tf]

                    1.11E-41

                    ORF51

                    Conserved hypothetical protein

                    24307..25062

                    +

                    28981

                    6.2

                    251

                    YP_001671917 hypothetical protein [Pseudomonas phage LUZ24]

                    2.15E-129

                    ORF52

                    Hypothetical protein

                    25059..25184

                    +

                    4844

                    10.5

                    41

                    -

                    -

                    ORF53

                    Conserved hypothetical protein

                    25255..25458

                    +

                    7717

                    6.5

                    67

                    YP_006659986 hypothetical protein tf_48 [Pseudomonas phage tf]

                    7.14E-14

                    ORF54

                    Hypothetical protein

                    25455..25631

                    +

                    6814

                    4.6

                    58

                    -

                    -

                    ORF55

                    Conserved hypothetical protein

                    25628..25834

                    +

                    8109

                    4.4

                    68

                    YP_001671920 hypothetical protein [Pseudomonas phage LUZ24]

                    1.57E-12

                    ORF56

                    Conserved hypothetical protein

                    25963..26319

                    -

                    13124

                    5.9

                    118

                    AGC35282 hypothetical protein PaP4_051 [Pseudomonas phage PaP4]

                    2.11E-45

                    ORF57

                    Phage structural protein

                    26321..27208

                    -

                    32029

                    5.5

                    295

                    AFD10698 hypothetical protein I7C_020 [Pseudomonas phage MR299-2]

                    3.43E-152

                    ORF58

                    Phage structural protein

                    27219..30383

                    -

                    111420

                    5.3

                    1054

                    YP_001671923 phage particle protein [Pseudomonas phage LUZ24]

                    0

                    ORF59

                    Phage structural protein

                    30389..32101

                    -

                    60235

                    5.5

                    570

                    YP_001671924 phage particle protein [Pseudomonas phage LUZ24]

                    1.35E-86

                    ORF60

                    Phage structural protein

                    32103..32507

                    -

                    13485

                    6.5

                    134

                    YP_001671925 phage particle protein [Pseudomonas phage LUZ24]

                    9.26E-11

                    ORF61

                    Phage particle protein

                    32504..33448

                    -

                    32240

                    4.8

                    314

                    AFD10694 hypothetical protein I7C_016 [Pseudomonas phage MR299-2]

                    3.23E-112

                    ORF62

                    Conserved hypothetical protein

                    33429..33863

                    -

                    16522

                    4.5

                    144

                    AGC35288 hypothetical protein PaP4_057 [Pseudomonas phage PaP4]

                    2.95E-57

                    ORF63

                    Conserved hypothetical protein

                    33860..34570

                    -

                    25585

                    4.8

                    236

                    YP_001671928 hypothetical protein [Pseudomonas phage LUZ24]

                    3.73E-56

                    ORF64

                    Phage particle protein

                    34570..36111

                    -

                    57542

                    5.1

                    513

                    YP_006382515 phage particle protein [Pseudomonas phage tf] & YP_001671929 hypothetical protein [Pseudomonas phage LUZ24]

                    0; 0

                    ORF65

                    Tail fiber protein

                    36119..36754

                    -

                    21879

                    6.7

                    211

                    NP_775246 hypothetical protein PaP3p12 [Pseudomonas phage PaP3]

                    1.68E-62

                    ORF66

                    Conserved hypothetical protein

                    36976..37170

                    -

                    6838

                    6.6

                    64

                    YP_001671932 hypothetical protein LUZ24 [Pseudomonas phage LUZ24]

                    3.02E-28

                    ORF67

                    phage structural protein

                    37175..37804

                    -

                    23887

                    5.0

                    209

                    AFD10687 putative constituent protein [Pseudomonas phage MR299-2]

                    2.54E-82

                    ORF68

                    Conserved hypothetical protein

                    37808..38128

                    -

                    11847

                    5.8

                    106

                    YP_007183215 hypothetical protein BN425_ORF_07 [Pseudomonas phage vB_PaeP_p2-1 0_Or1]

                    1.26E-50

                    ORF69

                    Major capsid protein

                    38180..39133

                    -

                    34740

                    6.0

                    317

                    NP_775251 major head protein [Pseudomonas phage PaP3] & YP_001671935 major head protein [Pseudomonas phage LUZ24]

                    0; 0

                    ORF70

                    Scaffolding protein

                    39152..40138

                    -

                    36190

                    4.3

                    328

                    YP_001671936 scaffolding protein [Pseudomonas phage LUZ24]

                    4.90E-101

                    ORF71

                    Conserved hypothetical protein

                    40128..40379

                    -

                    9559

                    5.2

                    83

                    AGC35298 hypothetical protein PaP4_067 [Pseudomonas phage PaP4] & YP_001671937 hypothetical protein [Pseudomonas phage LUZ24]

                    8.9E-30; 2.00e-29

                    ORF72

                    Portal protein

                    40379..42469

                    -

                    79405

                    5.0

                    696

                    YP_007183212 putative portal protein [Pseudomonas phage vB_PaeP_p2-10_Or1]

                    YP_001671938 portal protein [Pseudomonas phage LUZ24]

                    0; 0

                    ORF73

                    Terminase, large subunit

                    42496..43941

                    -

                    54330

                    6.0

                    481

                    YP_001671939 terminase large subunit [Pseudomonas phage LUZ24]; protein motifs: PF03237.10 Terminase_6

                    0

                    ORF74

                    Lysozyme

                    43945..44451

                    -

                    18961

                    8.6

                    168

                    YP_006382529 lysozyme [Pseudomonas phage tf]; protein motifs: PF00959.14 Phage_lysozyme

                    3.27E-56

                    ORF75

                    Terminase, small subunit

                    44372..44845

                    -

                    17291

                    5.9

                    157

                    YP_007183209 hypothetical protein BN425_ORF_01 [Pseudomonas phage vB_PaeP_p2-1 0_Or1] & YP_006382530 terminase small subunit [Pseudomonas phage tf]

                    5.76E-66; 2.59e-64

                    The searches for consensus sequences of transcriptional promoters revealed the presence of seven promoters, five in the positive strand initiating the transcription of ORFs that encode early proteins, which is a common feature of viral genomes that need bacterial transcription factors to start their infection cycle. The two other promoters are located in late genes modules. These genes are usually transcribed by viral transcription factors.

                    Three rho-independent transcription terminators were predicted using ARNold, one in the positive and two in the negative strand (Figure 1). A bidirectional termination region was found in the region from 25,922 to 25,964. Interestingly, this pattern of termination is also found in the genomes of the phages PaP3 [1] and LUZ24 [2]. The last terminator sequence is located at the terminal end of the gene encoding the major head protein. The low number of sequences of rho-independent terminators compared to the number of predicted ORFs may be due to the existence of other types of terminators or the presence of transcriptional modules and the generation of polycistronic mRNAs, a very common feature of viral genomes.

                    The predicted UFV-P2 genes were functionally classified as its promoters, predicted order of transcription, and its annotated functions.

                    Nucleotide biosynthesis and DNA replication (positive-stranded ORFs)

                    Fifty-five genes (ORFs 01–55) involved in nucleotide biosynthesis and viral replication process were found in the UFV-P2 genome positive strand, named early genes (Figure 1). Among viral replication genes, ORF31 encodes a primase/helicase; ORF44, a DNA-binding protein; ORF48, a 5′-3′ exonuclease; ORF50, a putative endonuclease; and ORFs 32 and 43 encode the two exons of the viral DNA polymerase, between which there is an ORF encoding a putative holin with three transmembrane domains similar to those from the phages tf and LUZ24. Holins are small membrane proteins that accumulate in the membrane until, at a specific time that is “programmed” into the holin, the membrane suddenly becomes permeabilized to the fully folded endolysin [29]. In addition, the UFV-P2 genome contains two endonucleases encoded by ORF24 and ORF50. The first is a HNH endonuclease, a group I homing endonuclease, which may be related to the presence of introns in the UFV-P2 genome [30], like those between the two parts of DNA polymerase. Other enzymes predicted in the positive strand include ORFs 23, 25 and 28, which encode, respectively, an amidoligase, a glutamine amidotransferase and an ATP-grasp enzyme. The other 45 proteins of the early genes module are hypothetical proteins.

                    Virion assembly and host lysis (negative-stranded ORFs)

                    Twenty genes (ORFs 56–75) related to the composition and assembly of the viral particle, DNA packaging, and host lysis were found in the UFV-P2 genome negative strand, named late genes (Figure 1). Two transcriptional modules were found based on predicted terminators. The first is located in the regions comprising the ORFs 75–69, and the second module corresponding to the ORFs 75–56.

                    In the first module, ORF75 and ORF73 encode the small and large terminase subunits, respectively. The terminase is the motor component that assists the translocation of viral genomic DNA to the inner of the capsid during packaging via ATP hydrolysis. There is an ongoing discussion about the role of terminase structure in determining the points for cleavage of the viral DNA, which would influence the entire viral genome organization [31]. Recently, Shen and coworkers [32] functionally identified the two genes encoding PaP3 terminase subunits, located in ORFs 1 and 3, respectively, which have high sequence similarity with ORFs 75 and 73 of the UFV-P2 genome. The PaP3 genome have been annotated as opposing transcriptional gene clusters in relation to the UFV-P2 genome, what explains the difference observed for the numbering of similar ORFs. The same occurred for the earlier annotation of phage UFV-P2 [5], which is revised in this work to correspond to the annotation of phage LUZ24, which represents the genus.

                    ORF72 encodes the portal protein; ORF69 encodes the major head protein; and ORF70 encodes a scaffolding protein, which is a chaperone possibly related to viral particle assembly. In the second module, beyond the ORFs from the first, the ORFs 57–61, 64 and 67 encode particle/structural proteins; ORF65 encodes the tail fiber protein; and the other six ORFs encode hypothetical proteins.

                    ORF74 encodes a lysozyme that is used in the process of host cell breakage through the lysis of the peptidoglycan layer. The occurrence of a lysin, not associated with its cognate holin, is unusual but also found in other members of the Luz24likevirus genus.

                    Structural genomic comparisons and evolutionary clustering

                    Pairwise genomic comparisons has been a useful approach for genotyping and classification of viruses like Circoviridae[33] and Geminiviridae[34]. The alignment of phages genomic sequences and pairwise comparisons revealed that vb_PaeP_p2-10_Or1, vb_PaeP_C1-14_Or, LUZ24, PaP4, PaP3, MR299-2 and tf are the phages most closely related to UFV-P2. Genomic sequences of these phages presented an identity to the UFV-P2 genome ranging from 49.5% to 57.5% (see Table 1).

                    The structural genomic comparisons in Mauve showed that these phages shared a set of conserved locally collinear blocks (LCB) (Figure 2 and Additional file 1: Figure S2). LCBs are conserved segments that appear be free from genome rearrangements, since the orthologous regions of genomes can be reordered or inverted by recombination processes [21]. In addition, a specific comparison between UFV-P2 and LUZ24 showed colinearity across their genomes (Figures 2 and 3).
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-15-7/MediaObjects/12864_2013_5641_Fig2_HTML.jpg
                    Figure 2

                    Phylogenetic clustering and structural genomic comparisons among the UFV-P2 and other phages. Phylogenetic tree of phage genomes (left) was calculated by Bayesian MCMC coalescent analysis. The posterior probability values (PP) (expressed as percentages) calculated using the best trees found by MrBayes are shown beside each node. The outgroup taxon is the Enterobacteria phage T7 (NC_001604). The colored squares in the schematic view of genomes (right) correspond to the conserved locally collinear blocks (LCBs) predicted by Mauve. The numbers and colors indicate the LCBs that are shared between the phages genomes.

                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-15-7/MediaObjects/12864_2013_5641_Fig3_HTML.jpg
                    Figure 3

                    Comparison of the genomes of the phages UFV-P2 and LUZ24. The collinearity between genomes is represented by the conserved locally collinear block (left) and Dot plot alignment (right). Dot plot alignment was calculated using Nucleic Acid Dot Plots (http://​www.​vivo.​colostate.​edu/​molkit/​dnadot/​index.​html), considering a window size of 13 and a mismatch limit of 0.

                    Phages LUZ24, PaP4, and UFV-P2 present a conserved bidirectional genomic organization, which is showed by the shared LCBs (blocks 3–9) (Figure 2). Phage tf also presents this organization, but with some differences in the shared LCBs. On the other hand, phages MR299-2, PaP3, vb_PaeP_p2-10_Or1, and vb_PaeP_C1-14_Or present an inverted set of LCBs (blocks 9–3), representing an opposing arrangement of the gene modules. Proteins of these seven phages were the top hits with the UFV-P2 sequences (Table 2) and can collaborate with each other’s functional annotations. In addition to genomic comparisons, a search for direct terminal repeats (DTRs) indicated the presence of patterns at the ends of the UFV-P2 genome, as described for the phages LUZ24, tf, and vB_PaeP_C1-14_Or1. These repeats are responsible for the recognition and cleavage of the phage genome at the end of the repeat region during packaging. Interestingly, one of the unique features of this group of phages is that PaP3 possesses 20 bp 5′-protuding cohesive ends [1], while LUZ24 has 184 bp DTRs, yet there does not appear to be a significant difference in the amino acid sequence of their terminases.

                    As suggested by the structural genomic comparisons, phylogenetic tree of genomic sequences grouped the phages according the shared LCBs (Figure 2). Phages PaeP_p2-10_Or1, vb_PaeP_C1-14_Or, LUZ24, PaP4, PaP3, MR299-2, tf, and UFV-P2 were included in a distinct monophyletic clade in BI phylogenetic tree, which possibly represents the Luz24likevirus genus. The shared LCBs, blocks 3–9 (Figure 2), may be considered as a genomic signature for this genus. In UFV-P2 genome (Figure 1), as for the other phages, the genes for biosynthesis and DNA replication are included in blocks 5 and 6; genes for virion structure and assembly are in blocks 7 and 8; and genes for host lysis are block 9. In blocks 3 and 4 are included only hypothetical genes. Then, we propose the classification of the phage UFV-P2 in the Luz24likevirus genus. In fact, these analyzes showed that other viruses were also grouped in distinct monophyletic clades or according to specific shared locally collinear blocks (LCB), as those from the T7likevirus (blocks 16 and 17) and Phikmvlikevirus (blocks 22, 24, and 25) genera, beyond a possibly genus including the phages PaP2 and 199X (blocks 4 and 11–15).

                    Conclusions

                    We have presented the functional annotation of UFV-P2, a new Pseudomonas fluorescens phage. Based on structural genomic comparison and phylogenetic clustering, we suggest the classification of UFV-P2 in the Luz24likevirus genus, and present a set of shared locally collinear blocks as the genomic signature for this genus.

                    Declarations

                    Acknowledgments

                    This study was supported by grants from the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The funders had no role in the study design, data collection, analysis, decision to publish, or preparation of this manuscript.

                    Authors’ Affiliations

                    (1)
                    Department of Food Technology, Federal University of Viçosa
                    (2)
                    Bioinformatics Laboratory, Institute of Applied Biotechnology to Agriculture (BIOAGRO), Federal University of Viçosa
                    (3)
                    Department of Biochemistry and Molecular Biology, Federal University of Viçosa
                    (4)
                    Laboratory of Molecular Immunovirology, Federal University of Viçosa
                    (5)
                    Department of General Biology, Federal University of Viçosa
                    (6)
                    Department of Microbiology, Federal University of Viçosa
                    (7)
                    Public Health Agency of Canada, Laboratory for Foodborne Zoonoses
                    (8)
                    Department of Molecular and Cellular Biology, University of Guelph

                    References

                    1. Tan Y, Zhang K, Rao X, Jin X, Huang J, Zhu J, Chen Z, Hu X, Shen X, Wang L, Hu F: Whole genome sequencing of a novel temperate bacteriophage of P. aeruginosa: evidence of tRNA gene mediating integration of the phage genome into the host bacterial chromosome. Cell Microbiol 2007, 9:479–491.PubMedView Article
                    2. Ceyssens P-J, Hertveldt K, Ackermann H-W, Noben J-P, Demeke M, Volckaert G, Lavigne R: The intron-containing genome of the lytic Pseudomonas phage LUZ24 resembles the temperate phage PaP3. Virology 2008, 377:233–238.PubMedView Article
                    3. Kulakov LA, Kochetkov VV, Ksenzenko VN, Krylov VN, Boronin AM: [Physical map of the DNA of bacteriophage tf of Pseudomonas putida ]. Mol Gen Mikrobiol Virusol 1988, (6):12–16.
                    4. Alemayehu D, Casey PG, McAuliffe O, Guinane CM, Martin JG, Shanahan F, Coffey A, Ross RP, Hill C: Bacteriophages φMR299–2 and φNH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells. mBio 2012, 3:e00029–12.PubMed CentralPubMedView Article
                    5. Eller MR, Salgado RL, Vidigal PMP, Alves MP, Dias RS, De Oliveira LL, Da Silva CC, De Carvalho AF, De Paula SO: Complete genome sequence of the Pseudomonas fluorescens bacteriophage UFV-P2. Genome Announcement 2013, 1:1 e00006–12.View Article
                    6. Rasolofo EA, St-Gelais D, LaPointe G, Roy D: Molecular analysis of bacterial population structure and dynamics during cold storage of untreated and treated milk. Int J Food Microbiol 2010, 138:108–118.PubMedView Article
                    7. Munsch-Alatossava P, Alatossava T: Phenotypic characterization of raw milk-associated psychrotrophicbacteria. Microbiol Res 2006, 161:334–346.PubMedView Article
                    8. Baruzzi F, Lagonigro R, Quintieri L, Morea M, Caputo L: Occurrence of non-lactic acid bacteria populations involved in protein hydrolysis of cold-stored high moisture Mozzarella cheese. Food Microbiol 2012, 30:37–44.PubMedView Article
                    9. Arcuri EF, Aparecida M, Paiva V, Lange CC: Contagem, isolamento e caracterização de bactériaspsicrotróficascontaminantes de leite cru refrigerado. Ciência Rural 2008, 38:2250–2255.View Article
                    10. Dogan B, Boor KJ: Genetic diversity and spoilage potentials among Pseudomonas spp. isolated from fluid milk products and dairy processing plants. Appl Environ Microbiol 2003, 69:130–138.PubMed CentralPubMedView Article
                    11. Baum MM, Kainović A, O’Keeffe T, Pandita R, McDonald K, Wu S, Webster P: Characterization of structures in biofilms formed by a Pseudomonas fluorescens isolated from soil. BMC Microbiol 2009, 9:103.PubMed CentralPubMedView Article
                    12. Sillankorva S, Neubauer P, Azeredo J: Isolation and characterization of a T7-like lytic phage for Pseudomonas fluorescens . BMC Biotechnol 2008, 8:80.PubMed CentralPubMedView Article
                    13. Sillankorva S, Neubauer P, Azeredo J: Pseudomonas fluorescens biofilms subjected to phage phiIBB-PF7A. BMC Biotechnol 2008, 08:79.View Article
                    14. Lingohr EJ, Villegas A, She Y-M, Ceyssens P-J, Kropinski AM: The genome and proteome of the Kluyverabacteriophage Kvp1–another member of the T7-like Autographivirinae. Virol J 2008, 5:122.PubMed CentralPubMedView Article
                    15. Sonnhammer EL, Von Heijne G, Krogh A: A hidden Markov model for predicting transmembrane helices in protein sequences.Proceedings/. International Conference on Intelligent Systems for Molecular Biology; ISMB International Conference on Intelligent Systems for. Mol Biol 1998, 6:175–182.
                    16. Käll L, Krogh A, Sonnhammer ELL: A combined transmembrane topology and signal peptide prediction method. J Mol Biol 2004, 338:1027–1036.PubMedView Article
                    17. Derbyshire MK, Lanczycki CJ, Bryant SH, Marchler-Bauer A: Annotation of functional sites with the Conserved Domain Database. Database (Oxford) 2012, 2012:bar058.View Article
                    18. Macke TJ, Ecker DJ, Gutell RR, Gautheret D, Case DA, Sampath R: RNAMotif, an RNA secondary structure definition and search algorithm. Nucleic Acids Res 2001, 29:4724–4735.PubMed CentralPubMedView Article
                    19. Gautheret D, Lambert A: Direct RNA motif definition and identification from multiple sequence alignments using secondary structure profiles. J Mol Biol 2001, 313:1003–1011.PubMedView Article
                    20. Rice P, Longden I, Bleasby A: EMBOSS: the European Molecular Biology Open Software Suite. TIG 2000, 16:276–277.PubMedView Article
                    21. Darling AE, Mau B, Perna NT: progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PloS one 2010, 5:e11147.PubMed CentralPubMedView Article
                    22. Mahadevan P, King JF, Seto D: Data mining pathogen genomes using GeneOrder and CoreGenes and CGUG: gene order, synteny and in silicoproteomes. Int J Comput Biol Drug Des 2009, 2:100–114.PubMedView Article
                    23. Kropinski AM, Borodovsky M, Carver TJ, Cerdeño-Tárraga AM, Darling A, Lomsadze A, Mahadevan P, Stothard P, Seto D, Van Domselaar G, Wishart DS: In silico identification of genes in bacteriophage DNA. Methods Mol Biol 2009, 502:57–89.PubMedView Article
                    24. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP: MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 2012,61(3):539–542.PubMedView Article
                    25. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22:4673–4680.PubMed CentralPubMedView Article
                    26. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011, 28:2731–2739.PubMedView Article
                    27. Darriba D, Taboada GL, Doallo R, Posada D: jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 2012,9(8):772.PubMedView Article
                    28. Glukhov AS, Krutilina AI, Shlyapnikov MG, Severinov K, Lavysh D, Kochetkov VV, McGrath JW, De Leeuwe C, Shaburova OV, Krylov VN, Akulenko NV, L a K: Genomic analysis of Pseudomonas putida phage tf with localized single-strand DNA interruptions. PloS one 2012, 7:e51163.PubMed CentralPubMedView Article
                    29. Wang I, Smith DL, Young R: HOLINS: The Protein Clocks of Bacteriophage Infections. Annu Rev Microbiol 2000, 54:799–825.PubMedView Article
                    30. Hertveldt K, Lavigne R, Pleteneva E, Sernova N, Kurochkina L, Korchevskii R, Robben J, Mesyanzhinov V, Krylov VN, Volckaert G: Genome comparison of Pseudomonas aeruginosa large phages. J Mol Biol 2005, 354:536–545.PubMedView Article
                    31. Feiss M, Rao VB: The bacteriophage DNA packaging machine. Advances in experimental medicine and biology 2012, 726:489–509.PubMedView Article
                    32. Shen X, Li M, Zeng Y, Hu X: Functional identification of the DNA packaging terminase from Pseudomonas aeruginosa phage PaP3. Arch Virol 2012,157(11):2133–2141.PubMed CentralPubMedView Article
                    33. Segalés J, Olvera A, Grau-Roma L, Charreyre C, Nauwynck H, Larsen L, Dupont K, McCullough K, Ellis J, Krakowka S, Mankertz A, Fredholm M, Fossum C, Timmusk S, Stockhofe-Zurwieden N, Beattie V, Armstrong D, Grassland B, Baekbo P, Allan G: PCV-2 genotype definition and nomenclature. Vet Rec 2008, 162:867–868.PubMedView Article
                    34. Fauquet CM, Stanley J: Revising the way we conceive and name viruses below the species level: a review of geminivirus taxonomy calls for new standardized isolate descriptors. Arch Virol 2005, 150:2151–2179.PubMedView Article

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                    © Eller et al.; licensee BioMed Central Ltd. 2014

                    This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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