Erwinia amylovora phage vB_EamM_Y3 represents another lineage of hairy Myoviridae

Accepted Manuscript Erwinia amylovora phage vB_EamM_Y3 represents another lineage of hairy Myoviridae Colin Buttimer, Yannick Born, Alan Lucid, Martin J. Loessner, Lars Fieseler, Aidan Coffey PII:

S0923-2508(18)30062-7

DOI:

10.1016/j.resmic.2018.04.006

Reference:

RESMIC 3652

To appear in:

Research in Microbiology

Received Date: 20 December 2017 Accepted Date: 23 April 2018

Please cite this article as: C. Buttimer, Y. Born, A. Lucid, M.J. Loessner, L. Fieseler, A. Coffey, Erwinia amylovora phage vB_EamM_Y3 represents another lineage of hairy Myoviridae, Research in Microbiologoy (2018), doi: 10.1016/j.resmic.2018.04.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Erwinia amylovora phage vB_EamM_Y3 represents another lineage

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of hairy Myoviridae

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Colin Buttimera, Yannick Bornb,c‡, Alan Lucida†, Martin J. Loessnerb, Lars Fieselerb‡, Aidan

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Coffeya*

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a

Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland.

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b

Institute of Food, Nutrition, and Health, ETH Zurich, Zürich, Switzerland.

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c

Agroscope, Research Division Plant Protection, Wädenswil, Switzerland

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‡

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Sciences, Wädenswil, Switzerland.

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Present address: Institute of Food and Beverage Innovation, Zurich University of Applied

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Email addresses

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Colin Buttimer - [email protected]

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Yannick Born - [email protected]

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Martin J. Loessner - [email protected]

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Lars Fieseler - [email protected]

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Aidan Coffey - [email protected] *Correspondence and reprints

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ACCEPTED MANUSCRIPT Abstract

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To date, a small number of jumbo myoviruses have been reported to possess atypical

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whisker-like structures along the surface of their contractile tails. Erwinia amylovora phage

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vB_EamM_Y3 is another example. It possesses a genome of 261,365 kbp with 333 predicted

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ORFs. Using a combination of BLASTP, Interproscan and HHpred, about 21% of its putative

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proteins could be assigned functions involved in nucleotide metabolism, DNA replication,

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virion structure and cell wall degradation. The phage was found to have a signal-arrest-

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release (SAR) endolysin (Y3_301) possessing a soluble lytic transglycosylase domain. Like

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other SAR endolysins, inducible expression of Y3_301 caused Escherichia coli lysis, which is

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dependent on the presence of an N-terminal signal sequence. Phylogenetic analysis showed

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that its closest relatives are other jumbo phages including Pseudomonas aeruginosa phage

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PaBG and P. putida phage Lu11, sharing 105 and 87 homologous proteins respectively. Like

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these phages, Y3 also shares a distant relationship to Ralstonia solanacearum phage ΦRSL1

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(sharing 55 homologous proteins). As these phages are unrelated to the Rak2-like group of

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hairy phages, Y3 along with Lu11 represent a second lineage of hairy myoviruses.

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Introduction

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The order of Caudovirales (the tailed phages) consists of three families of bacteriophage

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(phage), with about 25% consisting of members of the family Myoviridae [1]. These

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members possess virions with contractile tails, and include the phages with the largest

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genomes sizes [2]. Those with genomes greater than 200 kbp are typically referred to as

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“jumbo phages”; and currently, there are over ninety in the databases with most having

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Gram negative hosts [3,4]. Those with the largest genomes identified to date are Bacillus

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phage G (498 kbp) and those belonging to the Rak2-like group such Cronobacter sakazakii

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ACCEPTED MANUSCRIPT phage vB_CsaM_GAP32 (358 kbp), Serratia marcescens phage BF (357 kbp) and

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Pectobacterium phage vB_PcaM_CBB (355 kbp) [5–7]. Erwinia phages are of relevance in the

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context of alternative methods for bacterial crop disease control. Indeed, recent years have

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seen intensive investigation into the use of these phages for biocontrol of contagious fire

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blight of apples and pears. Such research is motivated by increasing reports of resistance to

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other antibacterial agents [8–10]. The search for new phages for biocontrol applications has

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resulted in the identification of a number of jumbo phages such as Erwinia amylovora jumbo

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phages Ea35-70 (accession no. NC_023557.1; [11]), φEaH1 [12], φEaH2 [13], including

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another sixteen jumbo phages which have had their genome sequences only recently

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published [14].

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Another important bacterial pathogen is Ralstonia solanacearum, which colonises the xylem

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and causes wilt in a wide range of economically important crops. Its relevance has resulted

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in the discovery of Ralstonia solanacearum phage ΦRSL1 (231 kbp, accession no.

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NC_010811.2), described in by Yamada et al (2010) and found to possess very little

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homology to known myoviruses. In recent times, a number of other jumbo phages have

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been identified with low but significant homology to phage ΦRSL1 at the protein level.

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These are Pseudomonas phage Lu11 (280 kbp, accession no. NC_017972.1) and PaBG (258

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kbp, accession no. NC_022096.1) as well as phage NTCB (258 kbp, accession no.

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LT598654.1), whose genome sequence was identified in a metagenomic study of a decaying

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Trichodesmium bloom, and whose host range remains unknown [16–18]. As well as the

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recently described Erwinia amylovora phage vB_EamM_Yoloswag (259 kbp, accession no.

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KY448244), which can also be assigned to this group [14].

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Here we report a newly isolated jumbo phage, vB_Eam_Y3 of E. amylovora, which possesses

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atypical whisker-like structures along the surface of its contractile tail; and has been found

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to share homology (at the protein level) to Lu11 and PaBG with distant homology to ΦRSL1.

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Materials and Methods

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Bacterial Strains, Phage, and Cultivation Techniques

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All Erwinia and Pantoea strains were grown in LB medium at 28°C. Phage Y3 was routinely

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cultivated at 28°C with the soft agar overlay method [19] using LB plates and LC soft agar (LB

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supplemented with 10 mM CaCl2 and 2 mM MgSO4; 0.4% agar), and E. amylovora 1/79 as

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host strain. The phage was originally isolated from soil as described previously [20]. Briefly,

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10 g of soil was enriched in 200 mL SM-buffered (50 mM Tris, 100 mM NaCl, 8 mM MgSO4,

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pH 7.4) LB broth, which had been inoculated 1:20 with and overnight culture of E. amylovora

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1/79. After overnight incubation at 28°C, filtered supernatants were spotted onto a freshly

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growing lawn of E. amylovora 1/79 and plaques were picked with a Pasteur pipette. After

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isolation, Y3 was propagated to high titers with the soft agar overlay method. Purification for

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subsequent electron microscopy and DNA isolation was achieved by CsCl density

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centrifugation [21]. Host range analysis involved the use of the soft agar overlay method

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with the use of different bacterial strains and the spotting of phage diluents, a positive result

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was recorded with the detection of plaques after an overnight incubation.

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ACCEPTED MANUSCRIPT Transmission Electron Microscopy

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For transmission electron microscopy, CsCl-purified phages were negatively stained with 2%

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ammonium molybdate and then analysed as previously as described [22], with 10 particles

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being used to determine virion dimensions.

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DNA Isolation and Sequencing

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For the extraction of phage DNA, CsCl-purified phage particles were dialyzed against a 1,000-

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fold excess of SM buffer (6 h, RT). Free nucleic acids were degraded 15 min. at 37°C by

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DNase I (final concentration: 1 U/ml) and RNase A (50 µg/ml). Nucleases were inactivated

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and phage capsids degraded 1 h at 56°C with EDTA (20 mM, pH 8.0), proteinase K (50 μg/ml),

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and SDS (0.5% [wt/vol]). DNA was then purified using a phenol-chloroform extraction

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followed by ethanol-precipitation [23]. DNA sequencing was outsourced to GATC Biotech

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(Konstanz, Germany). To conduct sequencing DNA libraries were first created by DNA

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fragmentation, adapter ligation followed by a size selection and amplification. DNA libraries

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were then measured and quantified on a fragment analyser before sequencing with 2x300

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bp paired end reads using Illumine Hiseq sequencing technology. The de novo assembly was

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performed using default parameters with CLC Genomics Workbench v8.0 (Aarhus,

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Denmark).

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Bioinformatics Analysis

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The RAST server (http://rast.nmpdr.org/; [24]) was employed with GLIMMER [25] to predict

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open reading frames (ORFs) of Y3. With further analysis of ORFs gene product conducted

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with BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins), Pfam

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ACCEPTED MANUSCRIPT (http://pfam.xfam.org/search#tabview=tab1; [24]), Interproscan

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(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3998142/; [25]) and HHpred

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(https://toolkit.tuebingen.mpg.de/#/tools/hhpred; [26]). With the detection of ORFs with

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transmembrane domains, signal peptide sequence and lipoprotein cleavage signal being

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identified with the use of TMHMM v.2 (http://www.cbs.dtu.dk/services/TMHMM/; [27]),

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SignalPv.4.1 (http://www.cbs.dtu.dk/services/SignalP/; [28]) and LipoP v.1

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(http://www.cbs.dtu.dk/services/LipoP/; [29]), respectively. The molecular weights of the

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predicted ORFs were estimated using the batch protein molecular weight determination of

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the sequence manipulation suite (http://www.bioinformatics.org/sms2/protein_mw.html).

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The presence of transfer RNA genes was investigated with the use of tRNAscan-SE

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(http://lowelab.ucsc.edu/tRNAscan-SE/; [30]) and ARAGORN

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(http://130.235.46.10/ARAGORN/; [31]). The circular genomic map of the phage was drawn

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using GCview [34]. Potential Rho-independent terminators in were identified using ARNold

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(http://rna.igmors.u-psud.fr/toolbox/arnold; [33]) with Mfold QuikFold

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(http://unafold.rna.albany.edu/?q=DINAMelt/Quickfold; [34]) using RNA energy rules 3.0 to

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verify predictions. Potential promoters were identified by submitting up streams sequences

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(100bp) of predicted genes to MEME (Multiple Em for Motif Elicitation) (http://meme-

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suite.org/tools/meme; [35]).

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Comparative analysis of phage genomes

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Genome comparisons between phages were performed using TBLASTX and visualised using

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Easyfig [38]. Multiple sequence alignments were created with MEGA7 [39] using MUSCLE.

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Phylograms were constructed using the portal vertex (Y3_003), large terminase (Y3_004),

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DNA polymerase (Y3_173) and ATP dependent DNA helicase (Y3_176) among phages

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possessing homology to ΦRSL1, with the large terminase and portal vertex protein being

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used among jumbo myoviruses of E. amylovora. To estimate the robustness of the

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phylograms, the maximum-likelihood algorithm [40] based on the Whelan and Goldman

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substitution model [41], was conducted with bootstrap support (1000). A total proteome

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comparison between phages was conducted using Coregenes 3.5 with the BLASTP threshold

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set at 75% (http://gateway.binf.gmu.edu:8080/CoreGenes3.5/custdata.html; [39]).

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SAR endolysin expression

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To assess activity of protein Y3_301, its gene (231,516 – 232,190 bp) was amplified using

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forward primer Y3_301-Pci: 5’-CATACatgttagttatcatcaacaatcg -3’ and reverse primer Y3_301-

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Xho1: 5’-AAGCTTctcgagggaagaaggagtatgct-3’. The truncated version of Y3_301, namely rTM-

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Y3_301 without the transmembrane domain (231,614 - 232,190 bp) was amplified using

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forward primer rTM-Y3_301-Pci1: 5’- CATACATgttacgcatttcgccgtc-3’ and the above reverse

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primer Y3_301-Xho1. All amplifications were conducted with High Fidelity PCR Master mix

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(Roche, Basel, Switzerland) using genomic DNA as a template. The resulting PCR product was

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digested and ligated in-frame into plasmid vector pET28a, with the resulting construct

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transformed in E. coli Lemo21 (DE3) [43], following the manufacturer’s standard protocol

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(New England Biolabs, Ipswich, MA, USA). DNA sequencing was performed on purified

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plasmid to ensure integrity of the construct. SAR endolysin activity in E coli was determined

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using 40mL liquid cultures (triplicate) in LB both using selective markers kanamycin (40

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µg/mL) and chloramphenicol (40 μg/mL) with 1% inoculum of an overnight culture. Cultures

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were grown at 30ᵒC with agitation at 100 rpm with the optical density (600nm) being

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measured at 1-hour intervals. At hour 4 (OD600~400), cultures were induced with IPTG (400

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μM) and readings were continued up to hour 8. Control cultures were left un-induced. The

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experiment was conducted in triplicate and averages and standard deviations were

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determined using Microsoft Excel.

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Accession Number

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The genome sequence of phage Y3 was submitted to Genbank under accession number

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KY984068

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Results

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Growth parameters, host range and morphology

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Erwinia amylovora phage vB_EamM_Y3 was isolated from soil, which had been collected at

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an apple orchard with trees displaying fire blight symptoms in Suree (canton of Lucerne,

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Switzerland) in 2008. The bacterial host strain used for its isolation was Erwinia amylovora

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stain 1/79, which had originally been isolated from an apple tree in Germany in 1979 [44];

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and its use as a host strain resulted in tiny clear pin-sized plaques. The phage was found to

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infect a number of strains of E. amylovora as well as strains of Pantoea agglomerans and

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Pantoea vagans (Table 1). Essentially, its host range was similar to other previously

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identified E. amylovora phages [20].

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Examination of the morphology of Y3 by transmission electron microscopy showed the

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phage belonged to the family of Myoviridae possessing an A1 morphotype [45], with an

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icosahedral head of 129 nm (±4 nm) in diameter and with a contractile tail with a length of

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192 nm (±12 nm) (Fig 1). The phage was named in accordance with the bacterial virus

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nomenclature set out by Kropinski et al (2009). The most unusual feature of Y3 was the hair-

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like structures along the surface of its contractile tail, a characteristic that has only been

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observed on a small number of phages to date: these being the jumbo Pseudomonas phage

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Lu11 [16] which shares protein homology with Y3, and those belonging to jumbo phage

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Rak2-like group such as Pectobacterium phage vB_PccM_CBB [7] and Escherichia phage

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121Q (accession no. NC_025447.1; [47]), and phages of unknown genome sizes and lineages

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such as X particle and Escherichia phage PhAPEC6 [48,49].

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General genome features

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The genome size of Y3 (261,365 bp) places it among the double stranded DNA jumbo

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phages. The assembly pattern of the reads obtained from sequencing indicated that its

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genome is likely to be circularly permuted with terminal repeats like that of coliphage T4

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[50]. Due to this observation, the start position of its Genbank file was set to match that of

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Erwinia phage Yoloswag, its closest relative available on public databases. The average G+C

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content of Y3 was found to be 47%, which is lower than that which would typically be found

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for its host chromosomal DNA at c.53% [51,52].

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A total of 333 ORFs were identified with putative gene products ranging in size of 39 to 1951

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amino acids. These ORFs were largely divided into two major gene clusters (Fig 2). The

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largest region 1 was situated between Y3_75 to 221, with ORFs mostly in the anticlockwise

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direction and region 2 situated from Y3_001 to 74 and Y3_222 to 333 with ORFs mostly in

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the clockwise direction. The GC skew correlates well with the ORF orientation of the

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proposed clusters [53]. The ORFs were found to be tightly organised having little intergenic

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space, with a number of examples of neighbouring ORFs’ start and end points overlapping.

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Most ORFs were found to start with AUG, while twenty and three ORFs were predicted to

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start with GUG and UUG, respectively. Of the gene products of the identified ORFs, seventy

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could be allocated a possible function (supplementary information 1, Table S1) with others

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identified as putative transmembrane proteins (fourteen) putative lipoproteins (two) and

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hypothetical proteins (247). No tRNA genes were identified on the genome.

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In terms of lysogeny, no integrase, excisionase or repressor genes were identified. However,

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one gene product (Y3_031) was found to possess the AntA/AntB antirepressor domain

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(IPR013557), which has been associated with anti-repressor proteins such as that encoded

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by LO142 of temperate phage 993W [54]. These proteins are believed to be involved in the

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lysis/lysogenic decision making process of the phage, possibly suggesting a temperate

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ancestry [55], with a homolog of this protein also evident in Yoloswag.

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A potential promoter with a consensus sequence of CTGTAAATA was identified at thirty two

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locations in the genome of Y3 (supplementary information 2, Fig S1, Table S2), with a highly

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similar promoter (ACTGTAAATA[N7]A) also having been predicted in the genome of

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Pseudomonas phage Lu11 [16]; and like that of Lu11, this promotor is mostly situated before

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ORFs with products predicted to play roles in nucleotide metabolism and DNA replication. In

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addition, forty-five potential rho factor independent terminators were located throughout

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the genome of Y3 (supplementary information 2 Table S3).

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Phylogenetic position of phage Y3 within the family Myoviridae

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At the DNA level, Y3 was found to have little or no homology to phage genomes currently

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available on public databases, with its closest match being Yoloswag (coverage 6%, identity

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75%) using BLASTN analysis. However, during BLASTP analysis, a small number of other

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jumbo shared a number of homologs (large terminase, major capsid protein, etc). These

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were phage NTCB, Pseudomonas phages Lu11 and PaBG and Ralstonia phage ΦRSL1, which

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hinted at a possible evolutionary relationship (Fig 3). Like Y3 at the DNA level, these five

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phages (Yoloswag, NCTB, Lu11, PaBG and ΦRSL1) also exhibited no homology to each other

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indicating that they are at most only distantly related.

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To determine the relatedness to each other among above six phages, a phylogenetic analysis

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was conducted using four shared proteins (portal vertex, DNA polymerase, large terminase

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and ATP dependent DNA helicase) to generate phylogenetic trees with confident bootstrap

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values (Fig 4). Similar phylogenetic markers have been used in studies of other jumbo phages

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[4,7,56]. These trees showed that among the six, two separate clades were formed: one

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included Y3 and Yoloswag; and the second was comprised of those phages infecting the

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genus Pseudomonas (Lu11 and PaBG). Phages NCTB and ΦRSL1 were outliers. The NCTB

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position on the tree is somewhere between the Erwinia and Pseudomonas phages. Phage

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ΦRSL1 appeared on a branch that sits outside of all the other five phages implying a far

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more distant relatedness. This interpretation was further supported by the analysis of the

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phages proteomes using Coregene (Table 2). The E. amylovora phages were found to share

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225 proteins with each other; the Pseudomonas phages shared 136; NTCB shared between

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88 and 112 proteins with both the Erwinia clade and the Pseudomonas clade respectively.

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ΦRSL1 was still the most distantly related, sharing only 55 to 65 homologues with the other

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five phages. This distant evolutionary relationship could possibly be explained in the context

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of host range with the genera Erwinia and Pseudomonas belonging to the class of

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Gammaproteobacteria while Ralstonia belongs to the class Betaproteobacteria [57].

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Finally, phylogenetic analysis was preformed using the large terminase and portal protein of

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all jumbo myoviruses of E. amylovora that have had their genome sequences submitted to

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Genbank to date. Both these proteins have been used as phylogenetic markers in such

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studies of distantly related phages of Enterobacteriaceae [58]. The resulting phylogram

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shows that there is a minimum of four clades of these phages infecting this bacterium. With

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Y3, along with phage Yoloswag, forming a novel clade that is highly distinct from the other

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three clades identified, as the branch that these phages reside on in phylograms sits outside

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of those representing the other clades (supplementary information 2, Table S4, Fig 5).

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Morphogenesis proteins of phage Y3

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The majority of the putative structural proteins of Y3 were situated in a cluster with a length

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of 65 kbp (Y3_104 -158) within region 1, with majority of these shared with phages

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Yolosway, NTCB, PaBG and Lu11. These putative proteins were the tail tube (Y3_132, 134),

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the tail sheath (Y3_135, 158), the baseplate (113, 114, 124), the tail fibres (Y3_104, 108, 109,

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110), the tail-collar fibre protein (Y3_110) and the major capsid protein (Y3_139). A putative

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portal protein (Y3_006) was located outside of this region, being adjacent to the large

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terminase (Y3_004). Interestingly, the homology of the portal protein to that of Bacillus

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phage G (gp14, 5E-8, 23%) was found to be high when using BLASTP. Proteins Y3_106 and

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Y3_143 also shared strong homology with gp431 (2e-9, 28%) and gp19 (3e-09, 33%)

260

respectively of Bacillus phage G. Y3_106 is situated between ORFs predicted to form the tail

261

fibres that possess collagen domains [59]. This gene product was predicted to be a tail fibre

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hinge protein due a presence of an ILEI domain (PF15711.4, 1.7E-7). This domain belongs to

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the FAM3 superfamily to which the T4-like phage tail hinge gp35 protein is distantly related

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[60]. Three of the structural proteins that have been identified in phage ΦRSL1 (ΦRSL1_135,

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136, 140) as confirmed by N-terminal sequencing [15], were found to have homologues in

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phage Y3 (namely Y3_135, 139, 140, 158) providing further evidence that they play a role in

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the virion structure of Y3.

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Nucleotide metabolism, DNA replication and DNA modification

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Typical of large myoviruses, Y3 has a number of proteins with functions predicted to

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influence the nucleotide pool within its host [3]. These include a CMP deaminase (Y3_035)

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allowing the hydrolysis of dCMP to dUMP and a putative dUTPase (Y3_300) allowing the

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conversion of dUTP to dUMP. The dUMP can then be acted upon by a thymidylate synthase

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(Y3_045), which can catalyse the methylation of dUMP to dTMP, which a dTMP kinase

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(Y3_118) can in turn convert to dTDP. Another feature typical of large phages is the

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possession of a number of proteins involved in DNA replication. It is worth mentioning that

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smaller phages typically use a lesser number of proteins to recruit the host replication

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machinery to carry out this role [61]. Phage Y3 was identified to possess helicases (Y3_001,

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168, 176), primases (Y3_024, 227), a DNA ligase (Y3_148), topoisomerase subunits (Y3_327,

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329), as well as class 1 polymerases (Y3_011, 173) and the delta component (PF13177.5,

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5.7E-26) of a class III polymerase (Y3_209). Y3 also has proteins involved in DNA repair and

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maintenance including RecA (Y3_243), UV damage endonuclease UvsE (Y3_287) and MmcB-

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like DNA repair protein (Y3_119) as well as those involved in DNA recombination (Y3_186)

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and DNA degradation (Y3_229). DNA methylation is a well-known strategy used by some

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phages to protect against host restriction endonucleases [62] and accordingly, Y3 appears to

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be able to methylate both cytosine and adenine (Y3_29, 50).

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While the genome of phage Y3 does not possess tRNA genes, it does possess proteins

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potentially involved in their maturation such as RNA 2'-phosphotransferase (Y3_272) and a

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carboxy-S-adenosyl-L-methionine synthase (Y3_017). Curiously, the phage was found to

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possess an asparagine synthase (Y3_020), which would allow the conversion of aspartate to

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asparagine. Such auxiliary phage genes that produce proteins that compliment a host’s

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metabolism have been identified in a number phages of marine environments; and the role

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of their encoded proteins is understood to be the removal of potential bottle-necks in their

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host metabolism that could potentially hinder the lytic cycle [63].

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Selfish genetic elements

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Inteins are selfish genetic elements that self-cleave and remove themselves from a

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translated protein [64]. Proteins identified with these elements in phage Y3 are the large

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terminase (Y3_004), C-5 cytosine methyltransferase (Y3_050) and one of the four predicted

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tail fibre proteins (Y3_110). Also identified was a homing endonuclease of the HNH family

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(Y3_179), situated between two hypothetical proteins. These homing endonuclease are

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mobile genetic elements with endonuclease activity that only promote the spread of their

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own encoding gene [65].

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Cell wall degrading enzymes

306

Analysis of the protein Y3_301 showed that it possesses an N-terminal transmembrane

307

domain (12-34 aa) with a high alanine content (23%), with one basic residue (arginine), and a

308

C-terminal soluble lytic transglycosylase (SLT) domain (IPR008258; 108-218 aa). The latter

309

domain degrades peptidoglycan by cleavage of the β-1,4 glysosidic bond that results in the

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formation of a 1,6-anhydrobond in the muramic acid residue. A homolog of this protein was

311

also found in phage Yoloswag (YOLOSWAG_297). Due the composition of the domains in this

312

protein, it was suspected to be an endolysin that followed the signal arrest (SAR) system like

313

gp45 of φKMV and lyz of phage P1 [66,67]. Analysis of the protein using SignalP 3.0, showed

314

that it possessed an N-terminal signal sequence with a probability of 0.729. However, the

315

more recent version of SignalP did not identify this domain suggesting that the N-terminal

316

secretory signal is not removed after export, but rather remains tethered to the inner

317

membrane after translocation by the host sec translocon system.

318

For number of these types of endolysin, an inducible system with E. coli permits

319

demonstration of SAR endolysin activity using a detectable reduction in culture turbidity

320

upon induction [67,68]. A similar experiment was conducted here for protein Y3_301.

321

Accordingly, gene Y3_301 was cloned into pET28a and transformed into E coli Lemo21 (DE3).

322

Expression of the cloned gene was lethal to cells, as addition of IPTG after 4 hours of growth

323

caused the OD600 to drop from ~0.480 to ~0.320 at 8 hours (Fig 6). No lytic activity was

324

detected after the removal of the signal sequence as seen with the induction of the

325

truncated transmembrane domain Y3_301 (rTM-Y3_301). As the toxic activity of this protein

326

was found to be dependent on the presence of its transmembrane domain, it could be

327

concluded that this protein behaves as a typical SAR endolysin, requiring the

328

transmembrane domain to act as a signal sequence for its translocation enabling the

329

catalytic domain of the protein to reach the periplasm where it acts on the peptidoglycan of

330

the bacterial cell resulting in lysis.

331

An important feature of SAR endolysins is the regulation of their enzymatic activity while in

332

their membrane-tethered form. The two main mechanisms that have been identified for

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those with the lysozyme domain are (a) those which utilise steric hindrance of catalytic

334

residues due to the proximity of the bilayer to which the enzyme is embedded (as seen with

335

R21 of lambdoid phage 21) and (b) those which regulate activity by the formation of

336

disulphide bridges as seen with LyzP1 of coliphage P1 [69]. Little is understood of how SAR

337

endolysins with transglycosylase domains regulate their activity. However, the formation of

338

disulphide bridges is a possible explanation of how Y3_301 might regulate itself. Five

339

cysteine resides were identified in its amino acid sequence, one situated within the

340

transmembrane domain (Cys23) and the four-remaining located within its catalytic domain

341

(Cys127, 147, 182 & 188).

342

A second protein, namely Y3_197 was found to possess a soluble lytic transglycosylase (SLT)

343

domain (PF05838), with a peptidoglycan binding domain (IPR018537). A homolog of this

344

protein was also found in phage Yoloswag (YOLOSWAG_189). To date, we have not

345

succeeded in demonstrating its predicted enzymatic activity after cloning in E. coli (data not

346

shown).

347

Conclusion

348

As mentioned above, a number of hairy Myoviridae jumbo phages to date have been placed

349

in the Rak2-like group. The possession of whisker-like structures is shared with the unrelated

350

E. amylovora phage Y3, suggesting that these may be more widespread in the Myoviridae

351

family than previously thought. For the Rak2-like Escherichia phage 121Q, this feature has

352

been linked to a protein which resembles a phage tail fibre [47]. The exact gene products

353

responsible for the atypical whisker-like structures of Y3 is yet unknown. However, a few

354

genes have been predicted to encode tail fibre structures (Y3_104, 106, 108, 109, 110), and

355

any of these could potentially be responsible. The role of these atypical hairs of these phages

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is still unknown, but is has been speculated to play a role in enhancing phage ability to

357

detect and infect its host by facilitating the phage adsorption processes [7,47].

358

Phylogenetic studies show that phages Y3 and Yoloswag establish a novel clade of jumbo

359

phages infecting E. amylovora (Fig 5). In the context of shared homologous proteins, Lavigne

360

et al (2009) defined phage subfamily and genus boundaries at ≥20% and ≥40%, respectively.

361

Based on these cut-off values, it would place phages Y3 and Yoloswag within a genus within

362

a subfamily to which NTCB, PaBG and Lu11 are also members and which is peripherally

363

related to ΦRSL1 (Table 2).

364

Little has been described about SAR endolysins in jumbo phages to date. However, a small

365

number of jumbo phages have been predicted to possess them. These include members of

366

the Rak2-like group such as Klebsiella phage vB_KleM-RaK2 (gp506), Pectobacterium phage

367

CBB (CBB_187), Cronobacter phage GAP32 (gp180) and coliphage Q121 (gp532) [5,70]; and

368

upon examination within our study, Erwinia phages Ea35-70 (Ea357_029), Deimos-Minion

369

(accession no. ANH52127.1; DM_29) and Simmy50 (accession no. KU886223.1; gp_30).

370

Phages Y3 and Yoloswag can be included in this group. These endolysins are known to be

371

highly prevalent in phages that infect Enterobacteriaceae [71]; and this may also be the case

372

for their jumbo phages, as a number of such endolysins have already been identified among

373

them.

374

Conflict of interest

375

There is no conflict of interest.

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ACCEPTED MANUSCRIPT Acknowledgements

379

†Alan Lucid passed away on 31 August 2017 during the preparation of the manuscript and

380

this paper is dedicated to his memory.

381

TEM imagines of phage Y3 were prepared by Rudi Lurz at the Max Planck Institue for

382

Molecular Genetic, Berlin.

383

This work was supported by Science Foundation Ireland Project Ref:12/R1/2335,

384

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Table 1. Host range of bacteriophage vB_EamM_Y3 tested on strains of Erwinia amylovora

590

and closely related genera and species, as determined by spot testing with dilution.

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Sensitivity + + + + + + + + + + + + + + + + + -

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Strain CFBP 1430 Erwinia amylovora CFBP 1232 Ea153 1/74 1/79 ACW 38899 ACW 56400 ACW 44274 Ea Rac 3075 ACW 55500 ACW 55835 ACW 55955 IPV 1077/7 01SFR-BO LA 469 LA 071 LA 411 LA 468 LA 477 ACW 40943 Erwinia persicina ACW 40560x ACW 41072 Erwinia billingiae Pagg B90 Eh 42 Pantoea agglomerans Em 406 Em 283 Pantoea vagans C9-1 Pantoea ananatis 351 Lys Results recorded as +, sensitive; −, no infecqon.

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Table 2. Homologous proteins shared between the phages related to Erwinia amylovora

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phage Y3 (Yoloswag, NTCB, Pseudomonas phages PaBG and Lu11 and Ralstonia phage

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ΦRSL1).

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Yoloswag NCTB PaBG Lu11 ΦRSL1 225 112 105 87 55 Y3 (333) (67.57%) (33.63%) (31.53%) (26.13%) (16.52%) Yoloswag 225 113 102 87 53 (334) (67.37%) (26.05%) (15.87%) (33.83%) (30.54%) 112 113 95 88 53 NCTB (301) (37.21%) (37.54%) (31.56%) (29.24%) (17.61) 105 102 95 136 65 PaBG (308) (34.09%) (33.12%) (30.84%) (44.16%) (21.1%) 87 87 88 136 62 Lu11 (391) (22.25%) (22.25%) (22.51) (34.78%) (15.86%) 55 53 53 65 62 ΦRSL1 (343) (16.03%) (15.45%) (15.45) (18.95%) (18.08%) Number in bracket next phage name is total predict proteins with percentages representing

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the portion of shared proteins within the proteome of the phage in question. Analyses were

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conducted using Coregenes with the BLASTP threshold set to 75%.

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Fig 1. Electron micrograph of Erwinia amylovora phage Y3. Phage particles were negatively

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stained with 2% ammonium molybdate. Hair-like structures are evident around the tails.

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Scale bar represents 100 nm

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Fig 2. Summary of the genomic organisation of the 261,365 bp genome of Erwinia amylovora

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phage Y3. On the outer ring, putative ORFs are represented by purple arrowheads labelled

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with predicted function where possible. On the middle ring is shown GC content relative to

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the mean GC content of the genome. On the inner ring, GC skew is illustrated where green

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represents positive skew and purple a negative skew.

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Fig 3. Comparison of the genomes of phage Y3 with jumbo phages Erwinia amylovora phage

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Yoloswag, phage NCTB, Pseudomonas phages Lu11 and PaBG and Ralstonia phage ΦRSL1

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using currently available annotations employing TBLASTX and visualised with Easyfig. The

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blue/red bar chart (top) shows the G+C skew of the Y3 genome. The genome maps comprise

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of arrows indicating locations and orientation of ORFs. Lines between genome maps indicate

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level of homology. To assist in the comparison, the largest gene of Y3 was set to the first

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position of its map with the corresponding homologous gene being set to the first position

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for the other phage genomes. Level of amino acid identity is shown via the gradient scale.

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Fig 4. Phylogenetic analysis using the portal vertex protein (phylogram A), DNA polymerase

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(B), large terminase (C) and ATP dependent DNA helicase (D) using the six phages Y3,

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Yoloswag, NCTB, Lu11, PaBG and ΦRSL1. The amino acid sequences were compared using

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MUSCLE. The tree was constructed using the maximum likelihood algorithm. The

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percentages of replicate trees were assessed with the bootstrap test (1000).

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Fig 5. Phylogenetic analysis using the large terminase (phylogram A) and portal protein (B)

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of jumbo Erwinia amylovora myoviruses with genome sequences submitted to Genbank to

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date. The amino acid sequences were compared using MUSCLE. The tree was constructed

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using the maximum likelihood algorithm. The percentages of replicate trees were assessed

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with the bootstrap test (1000)

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Fig 6. Growth curve of E. coli Lemo21 (DE3) carrying pET28a with the SAR endolysin Y3_301

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and its truncated derivative without the transmembrane domain (rTM-Y3_301). The culture

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was induced at OD600 ~0.450 with 400 µM IPTG (indicated with arrow). All OD measurements

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were taken in triplicate and the averages are represented.

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Supplementary material

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Table S1, Table S2 and Table S3, Table S4

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