Comparative whole genome analysis of six diagnostic brucellaphages Jason Farlow, Andrey A. Filippov, Kirill V. Sergueev, Jun Hang, Adam Kotorashvili, Mikeljon P. Nikolich PII: DOI: Reference:
S0378-1119(14)00039-0 doi: 10.1016/j.gene.2014.01.018 GENE 39388
To appear in:
Gene
Received date: Revised date: Accepted date:
15 July 2013 4 November 2013 7 January 2014
Please cite this article as: Farlow, Jason, Filippov, Andrey A., Sergueev, Kirill V., Hang, Jun, Kotorashvili, Adam, Nikolich, Mikeljon P., Comparative whole genome analysis of six diagnostic brucellaphages, Gene (2014), doi: 10.1016/j.gene.2014.01.018
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ACCEPTED MANUSCRIPT Comparative whole genome analysis of six diagnostic brucellaphages Jason Farlow a, Andrey A. Filippov b, Kirill V. Sergueev b, Jun Hang c, Adam Kotorashvili and Mikeljon P. Nikolich b*
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Ilia State University, Tbilisi, Georgia
b
Department of Emerging Bacterial Infections, Bacterial Diseases Branch, Walter Reed
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c
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Army Institute of Research, Silver Spring, Maryland, USA
Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring,
Richard G. Lugar Center for Public Health Research, Tbilisi, Georgia
*corresponding author:
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Maryland, USA
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Department of Emerging Bacterial Infections, Bacterial Diseases Branch Walter Reed Army Institute of Research 503 Robert Grant Avenue
Silver Spring, MD 20910 Tel: +1-301-319-9469
Mobile: +1-301-640-1471 email: [email protected]
[Sequence data for this article have been deposited in GenBank under Accession numbers KC556897, KC556896, KC556898, KC556893, KC556895 and KC556894.]
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ACCEPTED MANUSCRIPT Abstract Whole genome sequencing of six diagnostic brucellaphages, Tbilisi (Tb), Firenze
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(Fz), Weybridge (Wb), S708, Berkeley (Bk) and R/C, was followed with genomic
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comparisons including recently described genomes of the Tb phage from Mexico (TbM) and Pr phage to elucidate genomic diversity and candidate host range determinants.
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Comparative whole genome analysis revealed high sequence homogeneity among these
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brucellaphage genomes and resolved three genetic groups consistent with defined host range phenotypes. Group I was comprised of Tb and Fz phages that are predominantly
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lytic for Brucella abortus and Brucella neotomae; Group II included Bk, R/C, and Pr phages that are lytic mainly for B. abortus, Brucella melitensis and Brucella suis; Group
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III was comprised of Wb and S708 phage that are lytic for B. suis, B. abortus and B. neotomae. We found that the putative phage collar protein is a variable locus with
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features that may be contributing to the host specificities exhibited by different brucellaphage groups. The presence of several candidate host range determinants is illustrated herein for future dissection of the differential host specificity observed among these phages.
1. Introduction Brucellosis is a common global zoonotic disease caused by members of the Brucella genus with severe animal and human health impacts worldwide [1]. The genus is composed of multiple species based on specificity to a primary mammalian host, including the six classical species Brucella melitensis (sheep and goats), Brucella suis
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ACCEPTED MANUSCRIPT (hogs), Brucella abortus (cattle), Brucella ovis (sheep), Brucella canis (dogs), Brucella neotomae (wood rats) [2], as well as marine mammalian species Brucella pinnipedialis
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and Brucella ceti, and more recently identified atypical clades. Traditional typing of
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Brucella is based on colony morphology, CO2 requirement, H2S production, growth on serum dextrose agar (SDA) dye plates, serum agglutination and brucellaphage lysis at
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routine test dilution (RTD). Lytic bacteriophages infecting Brucella have been described
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for over half a century and are routinely used in diagnostic applications to confirm the identity of Brucella species [2, 3]. Brucellaphages are linear nonenveloped double-
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stranded DNA viruses currently classified as Podoviridae members (C1 Group morphology) with a 50-80 nm diameter icosahedral head and a 10-15 × 7-9 nm long
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noncontractile tail [4, 5]. To date all reported brucellaphages display a host range restricted to the genus Brucella. Most brucellaphages were isolated from various
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Brucella strains possessing smooth lipopolysaccharide (LPS), including strains of B. abortus, B. suis and B. melitensis [6-11]. The brucellaphages that are active on rough LPS host strains were derived in the laboratory from phage lines lytic for smooth LPS Brucella hosts [12, 13]. The classical typing scheme used globally includes six groups of lytic brucellaphages represented by Tb, Fz, Wb, Bk, rough-specific phages R/O and R/C, and Iz. This brucellaphage typing set allows the identification of smooth and rough strains belonging to six species: B. abortus, B. melitensis, B. suis, B. canis, B. ovis and B. neotomae [3]. The biology of brucellaphage-host interaction is rarely reported in the current literature, and the genetic basis of and molecular determinants for observed host range patterns have not yet been characterized. In B. abortus, phage resistance has previously
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ACCEPTED MANUSCRIPT been suggested to involve enzyme-resistant cell wall modifications preventing phage penetration [14] though this observation was not experimentally validated. Historically,
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brucellaphages have been noted to cause significant phenotypic changes in the colony
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morphology of Brucella cultures [7,15,16], and though phage exposure can lead to alterations in biochemical and metabolic phenotypes [17], specific genes and mechanisms
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involved in these processes remain unknown. Brucella spp. are genetically homogenous
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[2] and phage resistance systems such as CRISPRs are notably absent in Brucella genomes with the exception of minor eroded genetic structures in select B. melitensis and
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B. pinnipedialis strains [http://crispr.u-psud.fr/crispr/]. Recently the whole genomic sequences of a Tb phage utilized in Mexico (TbM)
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and an additional brucellaphage (Pr) were reported [18], providing the first whole genome data for this bacteriophage group. These authors observed high sequence
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similarity between these brucellaphage genomes, Tb and Pr, and found two prominent major InDel differences between the two phages. They also noted a coding sequence (ORF) for a putative DnaA-like protein and an oriC-like origin of replication in both genomes, a finding previously unreported for bacteriophage genomes. Over 40 brucellaphages have been isolated over the past 60 years [19]. However, the population structure and fine scale molecular diversity of different brucellaphages with differential host specificity remains unknown. Thus, a comparative whole genome analysis of an expanded collection of commonly used typing brucellaphages is needed for better understanding of the phylogenetic structure of this phage group and to identify candidate loci involved in differential host range phenotypes. In this study we sequenced the complete genomes of six brucellaphages (see Table 1), including a reference Tb phage
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ACCEPTED MANUSCRIPT along with Fz, Wb, S708, Bk and R/C. All these brucellaphages were acquired from the Félix d'Hérelle Reference Center for bacterial viruses (Université Laval, Canada) and
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propagated and analyzed at Walter Reed Army Institute of Research (herein the WRAIR-
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propagated Tb phage is designated TbW). These phage genomes were compared to the only two available sequences, those reported by Flores et al [18]: a Tb phage from
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Mexico (designated TbM herein) and the Pr phage. The bacteriophages sequenced and
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analyzed in this study are important diagnostic brucellaphages representing five out of the six typing groups [3]. Our data confirmed that the two major genome insertions
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or deletions (+ InDels and – InDels) that were observed previously are conserved among all the brucellaphage genomes represented in the present study. Comparative whole
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genome analysis in our expanded survey revealed extensive sequence homogeneity among these brucellaphage genomes and resolved three phylogenetic groups that were
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consistent with defined host range phenotypes. We also noted a small but significant level of sequence divergence of the TbM phage from TbW and the other brucellaphages that were analyzed.
2. Results
2.1. Assessment of the phage lytic activity on different Brucella species
We acquired six important brucellaphages that are used globally for Brucella species determination from the Felix d’Herelle collection at Université Laval. These belong to five out of the six known typing groups, as all except the Iz group were
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ACCEPTED MANUSCRIPT represented: Tb phage (Tb group), Fz (Fz group), Wb and S708 (Wb group), Bk (Bk group) and R/C (rough-specific phage group) [3]. The Izatnagar phage (Iz) was not
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available for this study. This set of typing brucellaphages allows the definitive
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identification of smooth and rough strains of B. abortus, B. suis, B. melitensis, B. neotomae, B. canis and B. ovis even without use of a representative of the Iz phage group
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(Table 1). In order to evaluate specific lytic activities, the ability of these phages to form
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plaques and provide confluent lysis was tested on different Brucella indicator strains commonly employed as controls in confirmatory testing regimes. Attenuated animal
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vaccine strain B. abortus S19 and three virulent reference Brucella strains were used: B. melitensis strain 16M, B. abortus strain 2308 and B. suis strain 23444 (also known as
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strain 3130). Both routine test dilutions (RTD, defined as the minimal bacteriophage titer
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able to produce a confluent spot of lysis) and 10,000 routine test dilutions (104 × RTD)
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were used in assays (Table 2). All the brucellaphages tested produced clear and large plaques on attenuated B. abortus S19 but formed smaller and turbid plaques on virulent B. abortus 2308. We also observed confluent lysis of B. suis 23444 by bacteriophages S708, Wb, Bk and R/C. The plaque morphology of the brucellaphages grown on B. suis 23444 differed: S708 and Wb phages produced turbid plaques, while Bk and R/C formed clear plaques. Additionally, none of the bacteriophages tested except for Bk phage were able to form clear plaques on B. melitensis 16M. Bk phage produced very small clear plaques at 10,000 RTD. These results were in agreement with the host range and specificity data published previously (see Table 1) and confirmed the authenticity of these brucellaphages acquired from Université Laval.
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ACCEPTED MANUSCRIPT 2.2. Genomic comparison and phylogenetic structure
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Genomic sequences of each phage were determined and deposited in GenBank
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with Nucleotide Sequence Accession Numbers as follows: TbW (KC556897), S708 (KC556896), Wb (KC556898), Bk (KC556893), R/C (KC556895) and Fz (KC556894).
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We performed whole genome de novo assembly as well as assembly mapped to the
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previously reported TbM and Pr genomic sequences [18]. Similar genome sizes and GC content were observed across the phages in this study: TbW (41.142 Kb, GC content =
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48.2%), Fz (41.142 Kb, GC content = 48.2%), Wb (38.253 Kb, GC content = 48.2%), S708 (38.253 Kb, GC content = 48.2%), Bk (38.253 Kb, GC content = 48.2%), and R/C
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(38.253 Kb, GC content = 48.2%). Genome alignment comparison revealed pairwise nucleotide sequence identity of 96.1% or greater with strong synteny across all genomes,
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consistent gene number (N=57 ORFs) and complete conservation of gene order. Despite the high sequence similarity observed across the phage genomes in this expanded collection of geographically and temporally diverse strains, three major evolutionary groups were evident based on our whole genome phylogenies. We noted the consistent separation of two of the major groups defined by single nucleotide polymorphisms (SNPs) and InDels (Figure 1A, 1B). Unrooted phylogenetic reconstructions using both whole genome alignments containing the two major deletions (Figure 2B) and the SNP data alone (Figure 2A) allowed us to detect this phylogenetic structure with significant bootstrap confidence. The brucellaphage genomes were consequently separated into three groups which well correlated to the distinctive lytic phenotypes: Group I was comprised of Tb and Fz phages that are predominantly lytic for
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ACCEPTED MANUSCRIPT B. abortus and B. neotomae; Group II included Bk, R/C, and Pr phages that are lytic mainly for B. abortus, B. melitensis and B. suis; Group III was comprised of Wb and
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S708 phage that both originated from B. suis hosts and are lytic for B. suis, B. abortus
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and B. neotomae. Significant bootstrap confidence (> 0.50) support was observed for each of these major genetic groups: Group I phages TbM-TbW-Fz phages (95%), the TbW-
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Wb (99.4%) and the R/C-Pr subgroup (58%).
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Fz subgroup (85.4%), the Group II phages Bk-R/C-Pr (66.4%), Group III phages S708-
Within Group I phages (TbM, TbW and Fz), the TbW and Fz sequences differed by
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only a single nucleotide (SNP 22,365; TbW=T, Fz=G). In contrast, the TbM sequence had an overall nucleotide identity of 99.93% with TbW and Fz but was divergent from both in
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several intergenic (IG) regions and an insertion in ORF 21 (Figure 2A, 2B, Table 3). Only two IG loci discriminated other phage strains in this comparative study: SNP 40,644
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shared by only Pr and TbM and SNP 40,845 that distinguished the Group I strains. In addition, genetic relationships observed from the SNP data (Figure 1A) are consistent with the distinction of the Group I clade based on whole genome comparisons (Figure 1B).
2.3. Candidate loci for host specificity
To guide future experimental identification of phage loci responsible for host ranges, we performed a comparative bioinformatic analysis of putative brucellaphage genes that might mediate host binding and specificity. In our analysis, the three major evolutionary groups identified here exhibited diversity within several putative genes with
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ACCEPTED MANUSCRIPT sequence similarity to genes involved in host attachment and adhesion (Table 3). In Tb phage, the first major InDel region extends ORF 21 that encodes a structural protein that
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exhibits significant orthology to hypothetical proteins in other diverse phage and in some
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cases limited orthology to the neck proteins of Prochlorococcus phages. The second major InDel region both extends ORF 28 that encodes the tail fiber protein and contains
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an additional ORF (ORF29) that exhibits significant similarity to a pectin lyase-like
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domain containing carbohydrate binding proteins. It is possible the altered length of the tail fibers distinguishing Group I and Group II/III phages may reflect adaptations to
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distinct requisites in the receptor structure of their original bacterial hosts. ORF 14 in the TbM phage genome encodes a hypothetical protein of 137 residues
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(AEY69683) with similarity to phage head fiber proteins at C-terminal residues 86-136. An insertion at nucleotide position G96 in this gene (genomic position 9,189) is present
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in all Group II and III members (Table 3) and results in the truncation of 17 C-terminal amino acid residues and the generation of a unique 13 residue N-terminus. ORF 29 and ORF 30 were previously shown to encode putative peptidoglycan binding and DUF847 domains that normally function in lytic activity and peptidoglycan degradation [18]. ORFs 29 and 30 showed no sequence diversity across the phage genomes analyzed in the present study. Our analysis revealed that TbM has an insertion in ORF 21 relative to all other brucellaphages. Residues G726C and A697V in the protein encoded by ORF 21 distinguished Group I members, including TbW and Fz. In addition, all Group I members including TbM were distinguished by residue E196 of ORF 43, which encodes a putative DNA methyltransferase. A substantial amount of diversity was observed within ORF 27 that encodes a putative tail collar protein (Pfam acc. no. PF07484). Seven amino acid
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ACCEPTED MANUSCRIPT substitutions were observed in this gene, with residue 239 (TbM numbering) exhibiting extensive polymorphism across individual brucellaphage groups (Table 3). We tested for
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the presence of positive (diversifying) selection acting on the collar protein gene using
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the dN/dS ratio. Results were significant for positive selection with an overall dN/dS=2.4 (P = 0.009). ORF 27 terminates 106 bp upstream of the second deletion region that
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truncates ORF 28 and deletes ORF 29 in Group II brucellaphages. ORF 28 shows
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homology to a putative enzymatic tail fiber protein that encodes a putative GDSL/SGNH hydrolase. Enzymatic tail fiber proteins often function in the degradation of the host cell
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membrane. Group I brucellaphages contain the full amino acid complement of ORF 28 while Group II and III members encode a C-terminal truncation of the tail fiber gene
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leaving only an 36 N-terminal domain (Figure 3) that terminates at Pro36. ORF 29 is present only in the Group I brucellaphages and encodes a putative carbohydrate binding
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tail spike protein containing pectin-lyase domains similar to phage P22. The P22 phage tail spike protein functions as an enzymatic viral adhesion protein that mediates binding to the bacterial host cell surface via pectin-lyase-like structural motifs (http://www.ebi.ac.uk/interpro/IEntry?ac =IPR012332). Overall, ORF 28 and 29 both appear to be large enzymatic tail fiber proteins that could be important in host cell adhesion and penetration. ORF 23 encodes a putative structural protein with a C-terminal peptidase S74 motif as well as a SNP at position 16,678 (K7E) and the InDel at positions 17,184-17,189 (A175/V176) relative to the published TbW and Pr genome comparisons (18). In our study, an additional SNP was identified at position 16,952 in ORF 23 resulting in a R98Q mutation in genomes of Group I members TbW and Fz. BLAST analysis using default search settings detected moderate similarity to the N-terminal
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ACCEPTED MANUSCRIPT peptidase domains of the bacterial phage-related tail fiber protein of Pedobacter spp. (ZP_01885447) and the phage tail fiber protein of Burkholderia phage DC-1
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(YP_006589987). The Burkholderia phage DC1 tail fiber protein peptidase domain
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exhibited similarity to ORF 23 both in positional topology at the N-terminus and amino
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acid sequence identity (38%) suggesting this gene may encode a tail fiber protein.
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2.4. Discussion
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In an effort to characterize the genetic structure of bacteriophage specific for Brucella more broadly, and to identify the potential genetic requisites for differential host
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specificity, we sequenced the genomes of six diagnostic brucellaphage strains that belong to five of the six known phage typing groups and compared their whole genomic
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sequences. The strong sequence homogeneity that was observed across these brucellaphages of differing origin suggested a lack of diversifying selection within the genetically homogenous Brucella host species. This high synteny and complete colinearity indicates that the brucellaphage group evolved in a close and perhaps clonal evolutionary history. Brucellaphages originally isolated in Georgia (Tb), England (Wb), Mexico (Pr) and Italy (Fz) were highly homogenous, consistent with the homogenous restriction profiles observed previously [22, 24] and recent genome comparisons of the TbM and Pr phages [18]. The presence of the ORF encoding a DnaA-like protein and the putative oriC-like origin of replication was conserved in the genomes of all the brucellaphages analyzed (Table 3). The oriC-like region was conserved in all the phage strains that were analyzed. Some strains have highly similar sequence and genomic
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ACCEPTED MANUSCRIPT structures therefore are very likely to have originated from the same lineage. For example, phage Bk was isolated after incubation of a mixture of B. melitensis culture and
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phage Wb [23] and thus appears to be a host range mutant of Wb. R/C was derived from
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the R/O phage, whose ancestor was isolated from a mixture of three brucellaphages including Wb [12]. The highly homogenous genetic structure of brucellaphages observed
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even in our present expanded survey may be the result of hyperadaptation to the bacterial
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host, as suggested by Flores et al [18]. Alternatively, this feature may be the result of a genetic bottleneck in the brucellaphage population. Lack of genetic change through
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recombination or the absence of selective pressure for such recombination to occur could also account for this great genetic homogeneity. This may be influenced by host factors
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such as niche isolation and the observed lack of natural conjugative genetic elements in the host genus. It has also been suggested that lytic phages may exhibit more pronounced
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genomic homogeneity and lower phylogenetic congruence with their bacterial hosts due to a lack of dependency on the host replication systems and lower frequencies of lateral genetic exchange [25]. In any case, this pronounced genetic similarity across the sequenced brucellaphages warrants formal designation as a single taxonomic species and suggests that host range selection does not impart a significant lasting structure on the population, at least among the brucellaphages that have been sequenced to date with this study. The recent discovery of atypical Brucella species that are divergent from the six classical species may provide new sources of bacteriophages specific for the genus with greater potential genetic diversity.
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ACCEPTED MANUSCRIPT Future sequencing efforts could include other diverse brucellaphage such as Np and Iz as well as other novel strains isolated from the environment and not subject to
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extensive laboratory passage. Such efforts may confirm whether the genetic structure
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currently observed is a characteristic feature of genomes within the brucellaphage family. Genome-to-phenotype connections can also be further refined by including wider range
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of definitive assay results in the analysis. For instance, Fz and TbW show a high genetic
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similarity consistent with their shared host range; however previous studies suggest that Fz exhibits a distinct plaque morphology which enables the differentiation of B. suis
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biotype 4, as well as differences in physical and chemical stability [10, 26]. Similarly, the phylogenetic placement of the Wb and S708 phages had a close association (Figure
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2A, 2B) consistent with their shared origin from B. suis hosts.
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The significance of the differences observed between the different Tb phage strains that have been sequenced is still speculative at this stage. Despite being derived from the same Georgian phage isolate, the TbM and TbW phage strains were more recently acquired from different sources, the Gamaleya Scientific Research Institute of Epidemiology and Microbiology, Moscow, Russian Federation [18] and the Félix d'Hérelle Reference Center for Bacterial Viruses of the Université Laval, Québec, Canada, respectively. The greater genetic distance observed between the TbM phage strain and other within-group brucellaphages TbW and Fz suggests that TbM may have been subjected to some diversifying selection and/or genetic drift in the laboratory, perhaps through the use of non-standard propagation host strains. In our study, lytic specificities were determined for all six brucellaphages prior to purification and genome
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ACCEPTED MANUSCRIPT sequencing (Table 3) and all were propagated on attenuated vaccine strain B. abortus S19. In any case, confirmation of the phenotypic properties of TbM is warranted to
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compare its combined lytic profile with other Tb reference strains including TbW.
The phenotypic characteristics of the Tb phage have been studied extensively [21,
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4]. Tb shows the highest efficiencies of adsorption and infection on smooth LPS B.
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abortus cultures and moderate and mild lytic activity towards B. neotomae and B. suis, respectively [27]. B. melitensis shows low susceptibility to the Tb phage, while cultures
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of naturally rough LPS B. ovis and B. canis are resistant to Tb [27]. In general, the phenotypes exhibited by Tb phage are also shared by Fz phage [28]. Wb phage is highly
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lytic for B. suis while Bk phage is primarily lytic for B. abortus and mildly lytic for B. suis and B. melitensis [28]. To date, the host receptor(s) utilized by brucellaphages
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across their bacterial hosts have not been identified. Bacteriophage receptor ligands are specified by the distinct structure and composition of the bacterial cell wall architecture [29]. In Brucella, the structure of the outer envelope that distinguishes individual phage strain host range is characterized by the rough or smooth phenotype determined by the presence of the LPS O-polysaccharide, deeper LPS structure and the exposure of outer membrane proteins (OMPs). It has been noted that phage adsorption on smooth LPS Brucella occurs regardless of host strain-specific variations in O-polysaccharide structure, suggesting that fine scale differences in LPS profiles alone may not determine phage host specificity [2]. Indeed, previous studies of B. neotomae showed an association of the phage receptor with a protein-phospholipid-LPS complex [30]. Previously authors have postulated that because of their highly similar restriction profiles,
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ACCEPTED MANUSCRIPT differences in brucellaphage host ranges are caused primarily by mutations in phage tail fiber receptors, or by host receptor accessibility since higher multiplicities of infection
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can induce lysis of phage resistant hosts [22].
Our comparative analysis of multiple closely related brucellaphages led to the
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identification of an array of genetic differences among the brucellaphages analyzed,
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revealing SNPs and InDels in genes encoding enzymes and structural proteins, including in putative tail fiber proteins, DNA polymerase, the major head protein and a highly
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variable locus within the putative phage collar protein. Based on the differences observed across these genomes, the putative tail collar protein encoded by ORF 27 could
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be the phage receptor-binding protein, as it appears to be under some diversifying selective pressure. Changes in this gene and corresponding conformational alterations of
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the protein could be responsible in part for the variety of brucellaphage host range phenotypes that have been described, but this will need to be tested. In structurally similar phages such as T7, conformational changes in tail collar proteins have been proposed to mediate complex interactions for penetration of the bacterial outer membrane and structural transitions in the channel for DNA injection [31, 32]. As with other Podoviridae members, brucellaphages may encode multiple tail fiber proteins, so it is possible that in addition to ORF 28 and 29, ORF 23 may also function in mediating host recognition. The truncation or absence of these proteins in the Group II brucellaphages may impart selective differences in host range phenotype.
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ACCEPTED MANUSCRIPT Taken together, these observed genetic differences may indicate loci that are under adaptive selection which may contribute to the differential host specificity
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observed among these bacteriophage strains. Differences in bacterial cell surface
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receptors across Brucella species may be driving this observed variation in putative brucellaphage receptor-binding proteins. These relationships deserve further exploration.
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The molecular diversity and speculation of attributes for these putative host attachment
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and adhesion genes is provided here to inform future genetic interrogation and
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3. Materials and Methods
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experimental dissection of brucellaphage host range determinants.
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3.1. Bacterial strains, bacteriophages and media used in this work
The characteristics of the brucellaphages used in this work are presented in Table 1. The phages were purchased from the bacteriophage collection of the Félix d'Hérelle Reference Center for Bacterial Viruses of the Université Laval (Laval, Québec, Canada). Attenuated vaccine strain B. abortus S19 and virulent strain B. abortus 2308 were both used for bacteriophage propagation. Virulent strain B. melitensis 16M and strain B. suis 23444 (also known as strain 3130) were used for phage characterization. BBL ™ Brucella Broth and BBL ™ Brucella Agar (BD Biosciences, San Jose, California USA) were used for all Brucella and brucellaphage growth procedures.
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3.2. Bacteriophage propagation and lysis assays
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Bacteriophages were propagated on attenuated vaccine strain B. abortus S19 as described in [27] with a few modifications. Bacteriophage stock lysates were added to
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150 ml of early logarithmic phase broth bacterial cultures at a multiplicity of infection
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(MOI) of 0.01 and incubated at 37°C for 48 h in 1-L plastic erlenmeyer flasks with moderate shaking. Phage lysates were allowed to stand undisturbed at 4°C for another 24
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h and were treated with chloroform added to final concentration of 10% in order to destroy all remaining live bacteria. Bacterial debris was removed by 15 min of
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centrifugation at 5,000 g. The resulting lysates were filtered through sterile 0.22-µm membranes. Final titers of bacteriophage lysates were determined by the mixing of
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phage lysate dilutions with a semi-solid (0.7%) agar overlay containing a B. abortus S19 suspension. Results were scored after 48-72 hours of growth at 37°C in the presence of 5% CO2.
3.3. Bacteriophage DNA isolation
Brucellaphage lysates were centrifuged at 13,000 g for 4 h and resuspended in SM buffer [33] in 1/100 of the original volume. Concentrated lysates were treated for 1 h at 37°C with RNaseA (60 µg/ml) and DNaseI (20 µg/ml) to remove residual Brucella host nucleic acids. After nuclease treatment, concentrated bacteriophage particles were incubated with proteinase K (final concentration 50 µg/ml) and SDS (0.5%) at 56°C for 1
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ACCEPTED MANUSCRIPT h. DNA was purified by phenol:chloroform extraction as described [32] followed by overnight DNA precipitation with 70% ethanol at -20°C. The resulting DNA was re-
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suspended in nuclease-free water. The purity of the bacteriophage genomic DNA
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preparations was confirmed by restriction analysis with BamHI, EcoRI and XbaI endonucleases (New England BioLabs Inc, Ipswich, Massachusetts, USA) using standard
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agarose gel electrophoresis.
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3.4. Whole genome pyrosequencing and genome assembly
Whole genome sequencing of the six brucellaphages was performed by using
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Roche/454 GS FLX Titanium next-generation high throughput pyrosequencing system
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with Roche’s rapid shotgun genomic DNA library preparation method (Roche 454 Life
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Sciences, Branford, CT, USA). Sequencing data were subjected to de novo assembly using Roche GSAssembler software (Newbler) version 2.5.3 and reference mapping assembly against the TbM (JN939331.1) and the Pr (JN939332.1) genomic sequences using the Geneious software package version 6.0.5 [34] with medium sensitivity and fine tuning at 5X iterations using trimmed sequences (yielded >100-fold coverage of each genome). Annotations were performed using Geneious and Sequin with the TbM and Pr strain genomes as references. Geneious software was used for whole genome alignments and to analyze the physical organization of reference genomic sequences including ORF content and synteny.
3.5. Comparative genomics, phylogenetic structure and selection
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Phylogenies were reconstructed using whole genome alignment data of TbW, TbM,
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Fz, Wb, S708, Bk and Pr genomic sequences using Geneious software. Unrooted
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Neighbor-Joining phylogenies were performed based on Tamura-Nei model with bootstrap performed with 1000 replicates. MEGA 5 software [35] was used to determine
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phylogenetic relationships among brucellaphage strains using unrooted Maximum-
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Likelihood parameters on SNP variants from Table 3 based on a Tamura-Nei model (Figure 2A). Branch lengths were measured in number of substitutions per site with
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bootstrap (BS) performed using 1000 replicates. The significance of dN/dS ratios in testing for the presence of positive/adaptive selection was determined by the two tailed Z-
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Acknowledgements
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test (P
S0378-1119(14)00039-0 doi: 10.1016/j.gene.2014.01.018 GENE 39388
To appear in:
Gene
Received date: Revised date: Accepted date:
15 July 2013 4 November 2013 7 January 2014
Please cite this article as: Farlow, Jason, Filippov, Andrey A., Sergueev, Kirill V., Hang, Jun, Kotorashvili, Adam, Nikolich, Mikeljon P., Comparative whole genome analysis of six diagnostic brucellaphages, Gene (2014), doi: 10.1016/j.gene.2014.01.018
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ACCEPTED MANUSCRIPT Comparative whole genome analysis of six diagnostic brucellaphages Jason Farlow a, Andrey A. Filippov b, Kirill V. Sergueev b, Jun Hang c, Adam Kotorashvili and Mikeljon P. Nikolich b*
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Ilia State University, Tbilisi, Georgia
b
Department of Emerging Bacterial Infections, Bacterial Diseases Branch, Walter Reed
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c
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Army Institute of Research, Silver Spring, Maryland, USA
Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring,
Richard G. Lugar Center for Public Health Research, Tbilisi, Georgia
*corresponding author:
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Maryland, USA
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Department of Emerging Bacterial Infections, Bacterial Diseases Branch Walter Reed Army Institute of Research 503 Robert Grant Avenue
Silver Spring, MD 20910 Tel: +1-301-319-9469
Mobile: +1-301-640-1471 email: [email protected]
[Sequence data for this article have been deposited in GenBank under Accession numbers KC556897, KC556896, KC556898, KC556893, KC556895 and KC556894.]
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ACCEPTED MANUSCRIPT Abstract Whole genome sequencing of six diagnostic brucellaphages, Tbilisi (Tb), Firenze
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(Fz), Weybridge (Wb), S708, Berkeley (Bk) and R/C, was followed with genomic
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comparisons including recently described genomes of the Tb phage from Mexico (TbM) and Pr phage to elucidate genomic diversity and candidate host range determinants.
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Comparative whole genome analysis revealed high sequence homogeneity among these
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brucellaphage genomes and resolved three genetic groups consistent with defined host range phenotypes. Group I was comprised of Tb and Fz phages that are predominantly
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lytic for Brucella abortus and Brucella neotomae; Group II included Bk, R/C, and Pr phages that are lytic mainly for B. abortus, Brucella melitensis and Brucella suis; Group
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III was comprised of Wb and S708 phage that are lytic for B. suis, B. abortus and B. neotomae. We found that the putative phage collar protein is a variable locus with
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features that may be contributing to the host specificities exhibited by different brucellaphage groups. The presence of several candidate host range determinants is illustrated herein for future dissection of the differential host specificity observed among these phages.
1. Introduction Brucellosis is a common global zoonotic disease caused by members of the Brucella genus with severe animal and human health impacts worldwide [1]. The genus is composed of multiple species based on specificity to a primary mammalian host, including the six classical species Brucella melitensis (sheep and goats), Brucella suis
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ACCEPTED MANUSCRIPT (hogs), Brucella abortus (cattle), Brucella ovis (sheep), Brucella canis (dogs), Brucella neotomae (wood rats) [2], as well as marine mammalian species Brucella pinnipedialis
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and Brucella ceti, and more recently identified atypical clades. Traditional typing of
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Brucella is based on colony morphology, CO2 requirement, H2S production, growth on serum dextrose agar (SDA) dye plates, serum agglutination and brucellaphage lysis at
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routine test dilution (RTD). Lytic bacteriophages infecting Brucella have been described
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for over half a century and are routinely used in diagnostic applications to confirm the identity of Brucella species [2, 3]. Brucellaphages are linear nonenveloped double-
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stranded DNA viruses currently classified as Podoviridae members (C1 Group morphology) with a 50-80 nm diameter icosahedral head and a 10-15 × 7-9 nm long
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noncontractile tail [4, 5]. To date all reported brucellaphages display a host range restricted to the genus Brucella. Most brucellaphages were isolated from various
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Brucella strains possessing smooth lipopolysaccharide (LPS), including strains of B. abortus, B. suis and B. melitensis [6-11]. The brucellaphages that are active on rough LPS host strains were derived in the laboratory from phage lines lytic for smooth LPS Brucella hosts [12, 13]. The classical typing scheme used globally includes six groups of lytic brucellaphages represented by Tb, Fz, Wb, Bk, rough-specific phages R/O and R/C, and Iz. This brucellaphage typing set allows the identification of smooth and rough strains belonging to six species: B. abortus, B. melitensis, B. suis, B. canis, B. ovis and B. neotomae [3]. The biology of brucellaphage-host interaction is rarely reported in the current literature, and the genetic basis of and molecular determinants for observed host range patterns have not yet been characterized. In B. abortus, phage resistance has previously
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ACCEPTED MANUSCRIPT been suggested to involve enzyme-resistant cell wall modifications preventing phage penetration [14] though this observation was not experimentally validated. Historically,
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brucellaphages have been noted to cause significant phenotypic changes in the colony
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morphology of Brucella cultures [7,15,16], and though phage exposure can lead to alterations in biochemical and metabolic phenotypes [17], specific genes and mechanisms
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involved in these processes remain unknown. Brucella spp. are genetically homogenous
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[2] and phage resistance systems such as CRISPRs are notably absent in Brucella genomes with the exception of minor eroded genetic structures in select B. melitensis and
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B. pinnipedialis strains [http://crispr.u-psud.fr/crispr/]. Recently the whole genomic sequences of a Tb phage utilized in Mexico (TbM)
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and an additional brucellaphage (Pr) were reported [18], providing the first whole genome data for this bacteriophage group. These authors observed high sequence
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similarity between these brucellaphage genomes, Tb and Pr, and found two prominent major InDel differences between the two phages. They also noted a coding sequence (ORF) for a putative DnaA-like protein and an oriC-like origin of replication in both genomes, a finding previously unreported for bacteriophage genomes. Over 40 brucellaphages have been isolated over the past 60 years [19]. However, the population structure and fine scale molecular diversity of different brucellaphages with differential host specificity remains unknown. Thus, a comparative whole genome analysis of an expanded collection of commonly used typing brucellaphages is needed for better understanding of the phylogenetic structure of this phage group and to identify candidate loci involved in differential host range phenotypes. In this study we sequenced the complete genomes of six brucellaphages (see Table 1), including a reference Tb phage
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ACCEPTED MANUSCRIPT along with Fz, Wb, S708, Bk and R/C. All these brucellaphages were acquired from the Félix d'Hérelle Reference Center for bacterial viruses (Université Laval, Canada) and
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propagated and analyzed at Walter Reed Army Institute of Research (herein the WRAIR-
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propagated Tb phage is designated TbW). These phage genomes were compared to the only two available sequences, those reported by Flores et al [18]: a Tb phage from
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Mexico (designated TbM herein) and the Pr phage. The bacteriophages sequenced and
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analyzed in this study are important diagnostic brucellaphages representing five out of the six typing groups [3]. Our data confirmed that the two major genome insertions
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or deletions (+ InDels and – InDels) that were observed previously are conserved among all the brucellaphage genomes represented in the present study. Comparative whole
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genome analysis in our expanded survey revealed extensive sequence homogeneity among these brucellaphage genomes and resolved three phylogenetic groups that were
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consistent with defined host range phenotypes. We also noted a small but significant level of sequence divergence of the TbM phage from TbW and the other brucellaphages that were analyzed.
2. Results
2.1. Assessment of the phage lytic activity on different Brucella species
We acquired six important brucellaphages that are used globally for Brucella species determination from the Felix d’Herelle collection at Université Laval. These belong to five out of the six known typing groups, as all except the Iz group were
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ACCEPTED MANUSCRIPT represented: Tb phage (Tb group), Fz (Fz group), Wb and S708 (Wb group), Bk (Bk group) and R/C (rough-specific phage group) [3]. The Izatnagar phage (Iz) was not
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available for this study. This set of typing brucellaphages allows the definitive
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identification of smooth and rough strains of B. abortus, B. suis, B. melitensis, B. neotomae, B. canis and B. ovis even without use of a representative of the Iz phage group
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(Table 1). In order to evaluate specific lytic activities, the ability of these phages to form
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plaques and provide confluent lysis was tested on different Brucella indicator strains commonly employed as controls in confirmatory testing regimes. Attenuated animal
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vaccine strain B. abortus S19 and three virulent reference Brucella strains were used: B. melitensis strain 16M, B. abortus strain 2308 and B. suis strain 23444 (also known as
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strain 3130). Both routine test dilutions (RTD, defined as the minimal bacteriophage titer
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able to produce a confluent spot of lysis) and 10,000 routine test dilutions (104 × RTD)
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were used in assays (Table 2). All the brucellaphages tested produced clear and large plaques on attenuated B. abortus S19 but formed smaller and turbid plaques on virulent B. abortus 2308. We also observed confluent lysis of B. suis 23444 by bacteriophages S708, Wb, Bk and R/C. The plaque morphology of the brucellaphages grown on B. suis 23444 differed: S708 and Wb phages produced turbid plaques, while Bk and R/C formed clear plaques. Additionally, none of the bacteriophages tested except for Bk phage were able to form clear plaques on B. melitensis 16M. Bk phage produced very small clear plaques at 10,000 RTD. These results were in agreement with the host range and specificity data published previously (see Table 1) and confirmed the authenticity of these brucellaphages acquired from Université Laval.
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ACCEPTED MANUSCRIPT 2.2. Genomic comparison and phylogenetic structure
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Genomic sequences of each phage were determined and deposited in GenBank
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with Nucleotide Sequence Accession Numbers as follows: TbW (KC556897), S708 (KC556896), Wb (KC556898), Bk (KC556893), R/C (KC556895) and Fz (KC556894).
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We performed whole genome de novo assembly as well as assembly mapped to the
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previously reported TbM and Pr genomic sequences [18]. Similar genome sizes and GC content were observed across the phages in this study: TbW (41.142 Kb, GC content =
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48.2%), Fz (41.142 Kb, GC content = 48.2%), Wb (38.253 Kb, GC content = 48.2%), S708 (38.253 Kb, GC content = 48.2%), Bk (38.253 Kb, GC content = 48.2%), and R/C
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(38.253 Kb, GC content = 48.2%). Genome alignment comparison revealed pairwise nucleotide sequence identity of 96.1% or greater with strong synteny across all genomes,
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consistent gene number (N=57 ORFs) and complete conservation of gene order. Despite the high sequence similarity observed across the phage genomes in this expanded collection of geographically and temporally diverse strains, three major evolutionary groups were evident based on our whole genome phylogenies. We noted the consistent separation of two of the major groups defined by single nucleotide polymorphisms (SNPs) and InDels (Figure 1A, 1B). Unrooted phylogenetic reconstructions using both whole genome alignments containing the two major deletions (Figure 2B) and the SNP data alone (Figure 2A) allowed us to detect this phylogenetic structure with significant bootstrap confidence. The brucellaphage genomes were consequently separated into three groups which well correlated to the distinctive lytic phenotypes: Group I was comprised of Tb and Fz phages that are predominantly lytic for
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ACCEPTED MANUSCRIPT B. abortus and B. neotomae; Group II included Bk, R/C, and Pr phages that are lytic mainly for B. abortus, B. melitensis and B. suis; Group III was comprised of Wb and
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S708 phage that both originated from B. suis hosts and are lytic for B. suis, B. abortus
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and B. neotomae. Significant bootstrap confidence (> 0.50) support was observed for each of these major genetic groups: Group I phages TbM-TbW-Fz phages (95%), the TbW-
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Wb (99.4%) and the R/C-Pr subgroup (58%).
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Fz subgroup (85.4%), the Group II phages Bk-R/C-Pr (66.4%), Group III phages S708-
Within Group I phages (TbM, TbW and Fz), the TbW and Fz sequences differed by
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only a single nucleotide (SNP 22,365; TbW=T, Fz=G). In contrast, the TbM sequence had an overall nucleotide identity of 99.93% with TbW and Fz but was divergent from both in
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several intergenic (IG) regions and an insertion in ORF 21 (Figure 2A, 2B, Table 3). Only two IG loci discriminated other phage strains in this comparative study: SNP 40,644
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shared by only Pr and TbM and SNP 40,845 that distinguished the Group I strains. In addition, genetic relationships observed from the SNP data (Figure 1A) are consistent with the distinction of the Group I clade based on whole genome comparisons (Figure 1B).
2.3. Candidate loci for host specificity
To guide future experimental identification of phage loci responsible for host ranges, we performed a comparative bioinformatic analysis of putative brucellaphage genes that might mediate host binding and specificity. In our analysis, the three major evolutionary groups identified here exhibited diversity within several putative genes with
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ACCEPTED MANUSCRIPT sequence similarity to genes involved in host attachment and adhesion (Table 3). In Tb phage, the first major InDel region extends ORF 21 that encodes a structural protein that
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exhibits significant orthology to hypothetical proteins in other diverse phage and in some
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cases limited orthology to the neck proteins of Prochlorococcus phages. The second major InDel region both extends ORF 28 that encodes the tail fiber protein and contains
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an additional ORF (ORF29) that exhibits significant similarity to a pectin lyase-like
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domain containing carbohydrate binding proteins. It is possible the altered length of the tail fibers distinguishing Group I and Group II/III phages may reflect adaptations to
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distinct requisites in the receptor structure of their original bacterial hosts. ORF 14 in the TbM phage genome encodes a hypothetical protein of 137 residues
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(AEY69683) with similarity to phage head fiber proteins at C-terminal residues 86-136. An insertion at nucleotide position G96 in this gene (genomic position 9,189) is present
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in all Group II and III members (Table 3) and results in the truncation of 17 C-terminal amino acid residues and the generation of a unique 13 residue N-terminus. ORF 29 and ORF 30 were previously shown to encode putative peptidoglycan binding and DUF847 domains that normally function in lytic activity and peptidoglycan degradation [18]. ORFs 29 and 30 showed no sequence diversity across the phage genomes analyzed in the present study. Our analysis revealed that TbM has an insertion in ORF 21 relative to all other brucellaphages. Residues G726C and A697V in the protein encoded by ORF 21 distinguished Group I members, including TbW and Fz. In addition, all Group I members including TbM were distinguished by residue E196 of ORF 43, which encodes a putative DNA methyltransferase. A substantial amount of diversity was observed within ORF 27 that encodes a putative tail collar protein (Pfam acc. no. PF07484). Seven amino acid
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ACCEPTED MANUSCRIPT substitutions were observed in this gene, with residue 239 (TbM numbering) exhibiting extensive polymorphism across individual brucellaphage groups (Table 3). We tested for
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the presence of positive (diversifying) selection acting on the collar protein gene using
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the dN/dS ratio. Results were significant for positive selection with an overall dN/dS=2.4 (P = 0.009). ORF 27 terminates 106 bp upstream of the second deletion region that
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truncates ORF 28 and deletes ORF 29 in Group II brucellaphages. ORF 28 shows
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homology to a putative enzymatic tail fiber protein that encodes a putative GDSL/SGNH hydrolase. Enzymatic tail fiber proteins often function in the degradation of the host cell
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membrane. Group I brucellaphages contain the full amino acid complement of ORF 28 while Group II and III members encode a C-terminal truncation of the tail fiber gene
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leaving only an 36 N-terminal domain (Figure 3) that terminates at Pro36. ORF 29 is present only in the Group I brucellaphages and encodes a putative carbohydrate binding
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tail spike protein containing pectin-lyase domains similar to phage P22. The P22 phage tail spike protein functions as an enzymatic viral adhesion protein that mediates binding to the bacterial host cell surface via pectin-lyase-like structural motifs (http://www.ebi.ac.uk/interpro/IEntry?ac =IPR012332). Overall, ORF 28 and 29 both appear to be large enzymatic tail fiber proteins that could be important in host cell adhesion and penetration. ORF 23 encodes a putative structural protein with a C-terminal peptidase S74 motif as well as a SNP at position 16,678 (K7E) and the InDel at positions 17,184-17,189 (A175/V176) relative to the published TbW and Pr genome comparisons (18). In our study, an additional SNP was identified at position 16,952 in ORF 23 resulting in a R98Q mutation in genomes of Group I members TbW and Fz. BLAST analysis using default search settings detected moderate similarity to the N-terminal
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ACCEPTED MANUSCRIPT peptidase domains of the bacterial phage-related tail fiber protein of Pedobacter spp. (ZP_01885447) and the phage tail fiber protein of Burkholderia phage DC-1
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(YP_006589987). The Burkholderia phage DC1 tail fiber protein peptidase domain
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exhibited similarity to ORF 23 both in positional topology at the N-terminus and amino
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acid sequence identity (38%) suggesting this gene may encode a tail fiber protein.
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2.4. Discussion
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In an effort to characterize the genetic structure of bacteriophage specific for Brucella more broadly, and to identify the potential genetic requisites for differential host
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specificity, we sequenced the genomes of six diagnostic brucellaphage strains that belong to five of the six known phage typing groups and compared their whole genomic
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sequences. The strong sequence homogeneity that was observed across these brucellaphages of differing origin suggested a lack of diversifying selection within the genetically homogenous Brucella host species. This high synteny and complete colinearity indicates that the brucellaphage group evolved in a close and perhaps clonal evolutionary history. Brucellaphages originally isolated in Georgia (Tb), England (Wb), Mexico (Pr) and Italy (Fz) were highly homogenous, consistent with the homogenous restriction profiles observed previously [22, 24] and recent genome comparisons of the TbM and Pr phages [18]. The presence of the ORF encoding a DnaA-like protein and the putative oriC-like origin of replication was conserved in the genomes of all the brucellaphages analyzed (Table 3). The oriC-like region was conserved in all the phage strains that were analyzed. Some strains have highly similar sequence and genomic
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ACCEPTED MANUSCRIPT structures therefore are very likely to have originated from the same lineage. For example, phage Bk was isolated after incubation of a mixture of B. melitensis culture and
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phage Wb [23] and thus appears to be a host range mutant of Wb. R/C was derived from
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the R/O phage, whose ancestor was isolated from a mixture of three brucellaphages including Wb [12]. The highly homogenous genetic structure of brucellaphages observed
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even in our present expanded survey may be the result of hyperadaptation to the bacterial
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host, as suggested by Flores et al [18]. Alternatively, this feature may be the result of a genetic bottleneck in the brucellaphage population. Lack of genetic change through
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recombination or the absence of selective pressure for such recombination to occur could also account for this great genetic homogeneity. This may be influenced by host factors
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such as niche isolation and the observed lack of natural conjugative genetic elements in the host genus. It has also been suggested that lytic phages may exhibit more pronounced
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genomic homogeneity and lower phylogenetic congruence with their bacterial hosts due to a lack of dependency on the host replication systems and lower frequencies of lateral genetic exchange [25]. In any case, this pronounced genetic similarity across the sequenced brucellaphages warrants formal designation as a single taxonomic species and suggests that host range selection does not impart a significant lasting structure on the population, at least among the brucellaphages that have been sequenced to date with this study. The recent discovery of atypical Brucella species that are divergent from the six classical species may provide new sources of bacteriophages specific for the genus with greater potential genetic diversity.
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ACCEPTED MANUSCRIPT Future sequencing efforts could include other diverse brucellaphage such as Np and Iz as well as other novel strains isolated from the environment and not subject to
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extensive laboratory passage. Such efforts may confirm whether the genetic structure
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currently observed is a characteristic feature of genomes within the brucellaphage family. Genome-to-phenotype connections can also be further refined by including wider range
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of definitive assay results in the analysis. For instance, Fz and TbW show a high genetic
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similarity consistent with their shared host range; however previous studies suggest that Fz exhibits a distinct plaque morphology which enables the differentiation of B. suis
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biotype 4, as well as differences in physical and chemical stability [10, 26]. Similarly, the phylogenetic placement of the Wb and S708 phages had a close association (Figure
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2A, 2B) consistent with their shared origin from B. suis hosts.
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The significance of the differences observed between the different Tb phage strains that have been sequenced is still speculative at this stage. Despite being derived from the same Georgian phage isolate, the TbM and TbW phage strains were more recently acquired from different sources, the Gamaleya Scientific Research Institute of Epidemiology and Microbiology, Moscow, Russian Federation [18] and the Félix d'Hérelle Reference Center for Bacterial Viruses of the Université Laval, Québec, Canada, respectively. The greater genetic distance observed between the TbM phage strain and other within-group brucellaphages TbW and Fz suggests that TbM may have been subjected to some diversifying selection and/or genetic drift in the laboratory, perhaps through the use of non-standard propagation host strains. In our study, lytic specificities were determined for all six brucellaphages prior to purification and genome
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ACCEPTED MANUSCRIPT sequencing (Table 3) and all were propagated on attenuated vaccine strain B. abortus S19. In any case, confirmation of the phenotypic properties of TbM is warranted to
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compare its combined lytic profile with other Tb reference strains including TbW.
The phenotypic characteristics of the Tb phage have been studied extensively [21,
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4]. Tb shows the highest efficiencies of adsorption and infection on smooth LPS B.
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abortus cultures and moderate and mild lytic activity towards B. neotomae and B. suis, respectively [27]. B. melitensis shows low susceptibility to the Tb phage, while cultures
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of naturally rough LPS B. ovis and B. canis are resistant to Tb [27]. In general, the phenotypes exhibited by Tb phage are also shared by Fz phage [28]. Wb phage is highly
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lytic for B. suis while Bk phage is primarily lytic for B. abortus and mildly lytic for B. suis and B. melitensis [28]. To date, the host receptor(s) utilized by brucellaphages
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across their bacterial hosts have not been identified. Bacteriophage receptor ligands are specified by the distinct structure and composition of the bacterial cell wall architecture [29]. In Brucella, the structure of the outer envelope that distinguishes individual phage strain host range is characterized by the rough or smooth phenotype determined by the presence of the LPS O-polysaccharide, deeper LPS structure and the exposure of outer membrane proteins (OMPs). It has been noted that phage adsorption on smooth LPS Brucella occurs regardless of host strain-specific variations in O-polysaccharide structure, suggesting that fine scale differences in LPS profiles alone may not determine phage host specificity [2]. Indeed, previous studies of B. neotomae showed an association of the phage receptor with a protein-phospholipid-LPS complex [30]. Previously authors have postulated that because of their highly similar restriction profiles,
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ACCEPTED MANUSCRIPT differences in brucellaphage host ranges are caused primarily by mutations in phage tail fiber receptors, or by host receptor accessibility since higher multiplicities of infection
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can induce lysis of phage resistant hosts [22].
Our comparative analysis of multiple closely related brucellaphages led to the
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identification of an array of genetic differences among the brucellaphages analyzed,
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revealing SNPs and InDels in genes encoding enzymes and structural proteins, including in putative tail fiber proteins, DNA polymerase, the major head protein and a highly
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variable locus within the putative phage collar protein. Based on the differences observed across these genomes, the putative tail collar protein encoded by ORF 27 could
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be the phage receptor-binding protein, as it appears to be under some diversifying selective pressure. Changes in this gene and corresponding conformational alterations of
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the protein could be responsible in part for the variety of brucellaphage host range phenotypes that have been described, but this will need to be tested. In structurally similar phages such as T7, conformational changes in tail collar proteins have been proposed to mediate complex interactions for penetration of the bacterial outer membrane and structural transitions in the channel for DNA injection [31, 32]. As with other Podoviridae members, brucellaphages may encode multiple tail fiber proteins, so it is possible that in addition to ORF 28 and 29, ORF 23 may also function in mediating host recognition. The truncation or absence of these proteins in the Group II brucellaphages may impart selective differences in host range phenotype.
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ACCEPTED MANUSCRIPT Taken together, these observed genetic differences may indicate loci that are under adaptive selection which may contribute to the differential host specificity
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observed among these bacteriophage strains. Differences in bacterial cell surface
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receptors across Brucella species may be driving this observed variation in putative brucellaphage receptor-binding proteins. These relationships deserve further exploration.
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The molecular diversity and speculation of attributes for these putative host attachment
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and adhesion genes is provided here to inform future genetic interrogation and
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3. Materials and Methods
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experimental dissection of brucellaphage host range determinants.
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3.1. Bacterial strains, bacteriophages and media used in this work
The characteristics of the brucellaphages used in this work are presented in Table 1. The phages were purchased from the bacteriophage collection of the Félix d'Hérelle Reference Center for Bacterial Viruses of the Université Laval (Laval, Québec, Canada). Attenuated vaccine strain B. abortus S19 and virulent strain B. abortus 2308 were both used for bacteriophage propagation. Virulent strain B. melitensis 16M and strain B. suis 23444 (also known as strain 3130) were used for phage characterization. BBL ™ Brucella Broth and BBL ™ Brucella Agar (BD Biosciences, San Jose, California USA) were used for all Brucella and brucellaphage growth procedures.
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3.2. Bacteriophage propagation and lysis assays
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Bacteriophages were propagated on attenuated vaccine strain B. abortus S19 as described in [27] with a few modifications. Bacteriophage stock lysates were added to
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150 ml of early logarithmic phase broth bacterial cultures at a multiplicity of infection
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(MOI) of 0.01 and incubated at 37°C for 48 h in 1-L plastic erlenmeyer flasks with moderate shaking. Phage lysates were allowed to stand undisturbed at 4°C for another 24
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h and were treated with chloroform added to final concentration of 10% in order to destroy all remaining live bacteria. Bacterial debris was removed by 15 min of
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centrifugation at 5,000 g. The resulting lysates were filtered through sterile 0.22-µm membranes. Final titers of bacteriophage lysates were determined by the mixing of
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phage lysate dilutions with a semi-solid (0.7%) agar overlay containing a B. abortus S19 suspension. Results were scored after 48-72 hours of growth at 37°C in the presence of 5% CO2.
3.3. Bacteriophage DNA isolation
Brucellaphage lysates were centrifuged at 13,000 g for 4 h and resuspended in SM buffer [33] in 1/100 of the original volume. Concentrated lysates were treated for 1 h at 37°C with RNaseA (60 µg/ml) and DNaseI (20 µg/ml) to remove residual Brucella host nucleic acids. After nuclease treatment, concentrated bacteriophage particles were incubated with proteinase K (final concentration 50 µg/ml) and SDS (0.5%) at 56°C for 1
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ACCEPTED MANUSCRIPT h. DNA was purified by phenol:chloroform extraction as described [32] followed by overnight DNA precipitation with 70% ethanol at -20°C. The resulting DNA was re-
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suspended in nuclease-free water. The purity of the bacteriophage genomic DNA
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preparations was confirmed by restriction analysis with BamHI, EcoRI and XbaI endonucleases (New England BioLabs Inc, Ipswich, Massachusetts, USA) using standard
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agarose gel electrophoresis.
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3.4. Whole genome pyrosequencing and genome assembly
Whole genome sequencing of the six brucellaphages was performed by using
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Roche/454 GS FLX Titanium next-generation high throughput pyrosequencing system
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with Roche’s rapid shotgun genomic DNA library preparation method (Roche 454 Life
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Sciences, Branford, CT, USA). Sequencing data were subjected to de novo assembly using Roche GSAssembler software (Newbler) version 2.5.3 and reference mapping assembly against the TbM (JN939331.1) and the Pr (JN939332.1) genomic sequences using the Geneious software package version 6.0.5 [34] with medium sensitivity and fine tuning at 5X iterations using trimmed sequences (yielded >100-fold coverage of each genome). Annotations were performed using Geneious and Sequin with the TbM and Pr strain genomes as references. Geneious software was used for whole genome alignments and to analyze the physical organization of reference genomic sequences including ORF content and synteny.
3.5. Comparative genomics, phylogenetic structure and selection
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Phylogenies were reconstructed using whole genome alignment data of TbW, TbM,
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Fz, Wb, S708, Bk and Pr genomic sequences using Geneious software. Unrooted
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Neighbor-Joining phylogenies were performed based on Tamura-Nei model with bootstrap performed with 1000 replicates. MEGA 5 software [35] was used to determine
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phylogenetic relationships among brucellaphage strains using unrooted Maximum-
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Likelihood parameters on SNP variants from Table 3 based on a Tamura-Nei model (Figure 2A). Branch lengths were measured in number of substitutions per site with
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bootstrap (BS) performed using 1000 replicates. The significance of dN/dS ratios in testing for the presence of positive/adaptive selection was determined by the two tailed Z-
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Acknowledgements
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test (P
