ORIGINAL RESEARCH
published: 29 May 2017
doi: 10.3389/fmicb.2017.00852
The Genomic Architecture of Novel
Simulium damnosum Wolbachia
Prophage Sequence Elements and
Implications for Onchocerciasis
Epidemiology
James L. Crainey 1, Jacob Hurst 2, Poppy H. L. Lamberton 3, Robert A. Cheke 4, 5,
Claire E. Griffin 6, Michael D. Wilson 7, Cláudia P. Mendes de Araújo 1,
María-Gloria Basáñez 5*† and Rory J. Post 8, 9 †
1 Laboratório de Ecologia de Doenças Transmissíveis na Amazônia, Fundação Oswaldo Cruz, Instituto Leônidas e Maria
Deane, Manaus, Brazil, 2 Oxford Martin School, Institute for Emerging Infections, University of Oxford, Oxford, UK, 3 Institute
of Biodiversity, Animal Health and Comparative Medicine, Wellcome Centre for Molecular Parasitology, University of Glasgow,
Glasgow, UK, 4 Natural Resources Institute, University of Greenwich at Medway, Chatham, UK, 5 Department of Infectious
Edited by:
Disease Epidemiology, Faculty of Medicine (St Mary’s campus), London Centre for Neglected Tropical Disease Research,
Marina G. Kalyuzhanaya,
School of Public Health, Imperial College London, London, UK, 6 Core Research Laboratories Department, Molecular Biology
San Diego State University, USA
Laboratories Division, Natural History Museum, London, UK, 7 Noguchi Memorial Institute for Medical Research, University of
Reviewed by: Ghana, Accra, Ghana, 8 School of Natural Sciences and Psychology, Liverpool John Moores University, Liverpool, UK,
Marla Trindade, 9 Department of Disease Control, Faculty of Infectious Tropical Diseases, London School of Hygiene and Tropical Medicine,
University of the Western Cape, London, UK
South Africa
Jeremy J. Barr,
Monash University, Australia Research interest in Wolbachia is growing as new discoveries and technical
*Correspondence: advancements reveal the public health importance of both naturally occurring and artificial
María-Gloria Basáñez infections. Improved understanding of the Wolbachia bacteriophages (WOs) WOcauB2
m.basanez@imperial.ac.uk
and WOcauB3 [belonging to a sub-group of four WOs encoding serine recombinases
†
These authors have contributed
equally to this work. group 1 (sr1WOs)], has enhanced the prospect of novel tools for the genetic manipulation
of Wolbachia. The basic biology of sr1WOs, including host range and mode of genomic
Specialty section: integration is, however, still poorly understood. Very few sr1WOs have been described,
This article was submitted to
with two such elements putatively resulting from integrations at the same Wolbachia
Evolutionary and Genomic
Microbiology, genome loci, about 2 kb downstream from the FtsZ cell-division gene. Here, we
a section of the journal characterize the DNA sequence flanking the FtsZ gene ofwDam, a genetically distinct line
Frontiers in Microbiology
ofWolbachia isolated from theWest African onchocerciasis vector Simulium squamosum
Received: 11 October 2016
Accepted: 26 April 2017 E. Using Roche 454 shot-gun and Sanger sequencing, we have resolved >32 kb of WO
Published: 29 May 2017 prophage sequence into three contigs representing three distinct prophage elements.
Citation: Spanning ≥36 distinct WO open reading frame gene sequences, these prophage
Crainey JL, Hurst J, Lamberton PHL,
elements correspond roughly to three different WO modules: a serine recombinase
Cheke RA, Griffin CE, Wilson MD, de
Araújo CPM, Basáñez M-G and Post and replication module (sr1RRM), a head and base-plate module and a tail module.
RJ (2017) The Genomic Architecture The sr1RRM module contains replication genes and a Holliday junction recombinase
of Novel Simulium damnosum
Wolbachia Prophage Sequence and is unique to the sr1 group WOs. In the extreme terminal of the tail module there
Elements and Implications for is a SpvB protein homolog—believed to have insecticidal properties and proposed to
Onchocerciasis Epidemiology.
have a role in how Wolbachia parasitize their insect hosts. We propose that these
Front. Microbiol. 8:852.
doi: 10.3389/fmicb.2017.00852 wDam prophage modules all derive from a single WO genome, which we have named
Frontiers in Microbiology | www.frontiersin.org 1 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
here sr1WOdamA1. The best-match database sequence for all of our
sr1WOdamA1-predicted gene sequences was annotated as of Wolbachia or Wolbachia
phage sourced from an arthropod. Clear evidence of exchange between sr1WOdamA1
and other Wolbachia WO phage sequences was also detected. These findings provide
insights into how Wolbachia could affect a medically important vector of onchocerciasis,
with potential implications for future control methods, as well as supporting the
hypothesis that Wolbachia phages do not follow the standard model of phage evolution.
Keywords: Wolbachia, Wolbachia phages, serine recombinase, SpvB protein homolog, Simulium squamosum E,
onchocerciasis
INTRODUCTION occurringWOs influence vector-borne disease epidemiology and
what risks (if any) they pose to the safety of using artificial
It is estimated that Wolbachia naturally infect about 40% Wolbachia infections for disease control (Bourtzis et al., 2014;
of arthropods, including many important disease vectors Hoffmann et al., 2015; Jeffries and Walker, 2015; Caragata et al.,
(Bourtzis et al., 2014; Zug and Hammerstein, 2015). As these 2016).
infections have an impact on several epidemiologically-relevant In previous studies, we identified a genetically isolated strain
aspects of disease vector biology, such as longevity, insecticide of Wolbachia from the West African onchocerciasis vector
resistance, and refractoriness to infection, it has been argued Simulium squamosum E (a member of the S. damnosum sensu
that Wolbachia are likely to influence disease epidemiology lato [s.l.] species complex [Diptera: Simuliidae]) and identified
(Echaubard et al., 2010; Slatko et al., 2014; Hoffmann et al., bacterial artificial chromosomes (BACs) containing its FtsZ cell-
2015). Much of the present public health interest in arthropod- division gene (Crainey et al., 2010a,b). As shown in Table 1,
infecting Wolbachia focuses on how artificial infections can the FtsZ gene is part of a conserved block (spanning ∼3 kb)
be manipulated as tools for effective disease control (Bourtzis immediately adjacent to where two closely-related prophages
et al., 2014; Hoffmann et al., 2015; Jeffries and Walker, (WOcauB2 and a WOri phage relic) have been identified in
2015). two genetically-distinct Wolbachia genomes: wCau and wRi
Wolbachia bacteriophages (WOs) have received far less genomes (Tanaka et al., 2009; Kent et al., 2011a; Ellegaard et al.,
attention than their bacterial hosts, with some research focusing 2013). This six-gene block begins in both cases with superoxide
on how they could influence disease ecology and epidemiology dismutase and terminates with the magnesium chelatase-related
(Tanaka et al., 2009; Metcalf and Bordenstein, 2012; LePage and protein, which occurs immediately adjacent to the prophages’
Bordenstein, 2013; Wang et al., 2013) and, most commonly, serine recombinase gene. If these WOs belong to a group
how they might be utilized for disease control (Metcalf of site-specific bacteriophages, large cloned fragments of the
and Bordenstein, 2012; LePage and Bordenstein, 2013; Slatko wDam genome, containing FtsZ gene sequences (Crainey et al.,
et al., 2014). Several authors have advocated the possibility of 2010a) could be expected also to contain Wolbachia prophage
developing artificial WO vectors for the genetic modification sequences. Similarly, if, as proposed, certain WOs have a role in
of Wolbachia. Despite the potential of WO-based tools and male-killing (and male-killing is affecting the S. damnosum s.l.
the growing interest in the use of Wolbachia for vector-borne complex), anyWOs might also be expected to harbor SpvB genes
disease control, there are presently no genetic manipulation (Crainey et al., 2010a; Kent et al., 2011a; Metcalf and Bordenstein,
tools available for the genetic engineering of Wolbachia (LePage 2012; LePage and Bordenstein, 2013). In this study, we have
and Bordenstein, 2013; Bourtzis et al., 2014; Slatko et al., characterized the genomic DNA of wDam flanking its FtsZ-
2014; Hoffmann et al., 2015; Jeffries and Walker, 2015). There gene and have recovered three WO phage sequence elements,
is, thus, a growing need for a better understanding of the including one that encodes a SpvB-like gene, that we propose all
basic biology, diversity and distribution of naturally occurring derive from a single WO prophage genome that we have named
WOs to assess the feasibility and potential utility of WO-based sr1WOdamA1.
Wolbachia manipulation tools (Tanaka et al., 2009; LePage and
Bordenstein, 2013; Wang et al., 2013). Similarly, there is also a
pressing need to improve our understanding about how naturally MATERIALS AND METHODS
Shotgun Sequencing of the Genomic DNA
Regions Flanking the wDam Cell-Division
Abbreviations: BAC, bacterial artificial chromosome; BLAST, Basic Local
Alignment Search Tool; bp, base pair; FtsZ, Filamenting temperature-sensitive Protein FtsZ
mutant Z; gp, gene product; ITR, inverted terminal repeat; kb, kilo base; LargeWolbachia-DNA-containing BAC clone mini cultures from
MITE, Miniature Inverted-repeat Transposable Element; nts, nucleotides; PCR, seven FtsZ-positive BACs (identified previously) were grown
polymerase chain reaction; pgp, paralogous gene product; SpvB-like protein,
shaking over-night in BAC library growth media (Crainey
Salmonella virulence plasmid B protein homolog; sr1RRM, group 1 serine
recombinase and replicationmodule; TcdB toxin,Clostridium difficile toxin B;WO, et al., 2010b). Thick mini-culture preparations from each BAC
Wolbachia bacteriophage. colony were pooled and their BAC DNA was isolated in
Frontiers in Microbiology | www.frontiersin.org 2 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
TABLE 1 | wDam shot-gun sequence contigs from matching known and predicted serine recombinase WO upstream integration sites.
Gene name Distance (in nucleotides, nts) Gene length wDam sequence match wDam wDam contig
from WO phage terminal end contig(s) length
Mg chelatase-related protein wCau 158 nts from 5′ terminal end of wCau: 1,238 wCau: co-ordinates: 261–891 KY695242 632
protein ID: BAH22205 wRi protein ID: WOcauB2 wRi: 1,464 Identity: 527/632 (83%)
ACN95503 0 nts from 5′ terminal end of BLAST score: 587 bits (738)
WOriRelic1 wRi: co-ordinates: 259–888
Identity: 523/631(83%)
BLAST score: 648 bits (718)
Cell-division protein FtsZ wCau 2,263 nts from 5′ terminal end of wCau: 1,145 wCau: co-ordinates: 18–1,145 KY695243 1,943
protein ID: BAH22203 wRi protein ID: WOcauB2 wRi: 1,197 Identity: 959/1,166 (82%)
ACN95501 1,477 nts from 5′ terminal end of BLAST score: 1,166 bits (1,292)
WOriRelic1 wRi: co-ordinates: 1–1,193
Identity: 1,004/1,222 (82%)
BLAST score: 1,048 bits (1,318)
Hypothetical protein GF1gp18 wCau 3,390 nts from 5′ terminal end of wCau: 399 wCau: co-ordinates: 1–161 KY695244 729
protein ID: BAH22202 wRi protein ID: WOcauB2 wRi: 399 Identity: 132/161 (82%)
ACN95500 3,371 nts from 5′ terminal end of BLAST score:159 bits (176)
WOriRelic1 wCau: co-ordinates: 202–399 KY695243 1,943
Identity: 162/202 (80%)
BLAST score: 157 bits (196)
wRi: coordinates: 11–161 KY695244 729
Identity: 124/151 (82%)
BLAST score: 133 bits (166)
wRi: coordinates: 200–399 KY695243 1,943
Identity: 164/200 (82%)
BLAST score: 199 bits (220)
Hypothetical protein GF1gp17 wCau 3,782 nts from 5′ terminal end of wCau: 459 wCau: co-ordinates: 40–459 KY695244 729
protein ID: BAH22201 wRi protein ID: WOcauB2 wRi: 453 Identity: 337/423 (80%)
ACN95499 4,151 nts from 5′ terminal end of BLAST score: 327 bits (410)
WOriRelic1 wRi: co-ordinates: 14–453
Identity: 365/442 (83%)
BLAST score: 394 bits (494)
Peptidase, M16 family wCau protein 16,926 nts from 5′ terminal end wCau: 1,275 wCau: co-ordinates: 1–1,262 KY695245 1,970
ID: BAH22189 wRi protein ID: of WOcauB2 wRi: 1,275 Identity: 1,022/1,262 (81%)
ACN95488 17,238 nts from 5′ terminal end BLAST score: 1,195 bits (1,324)
of WOriRelic1 wRi: coordinates: 16–1,268
Identity: 1,004/1,255 (80%)
BLAST score: 992 bits (1,248)
Superoxide dismutase wCau protein 15,656 nts from 5′ terminal end wCau: 609 wCau: co-ordinates: 1–596 KY695245 1,970
ID: BAH22188 wRi protein ID: of WOcauB2 wRi: 618 Identity: 486/614 (79%)
ACN95487 18,514 nts from 5′ terminal end BLAST score: 529 bits (586)
of WOriRelic1 wRi: co-ordinates: 1–606
Identity: 480/616 (78%)
BLAST score: 425 bits (534)
Quoted sequence matches are based on BLASTn sequence comparisons implementing the “align two or more sequences” function. The BLAST scores were calculated after the default
search comparison parameters were modified to a word size of 7 and gap opening penalty system of 0 for existence and 4 for extension. Only significant matches with BLAST scores
over 130 bits are shown.
single preparations using a QIAGEN large-construct kit and contigs using Phrap (Ewing and Green, 1998; Ewing et al.,
protocol (https://www.qiagen.com/kr/resources/resourcedetail? 1998). Shot-gun sequence contigs were screened for the
id=8f67b644-6d21-4ef3-b33e-a60f32623785&lang=en). A 10- presence of WOcauB2 and WOcauB2-flanking sequences using
mg sample of purified BAC DNA was shot-gun sequenced BLAST (Basic Local Alignment Search Tool) homology searches
commercially using a Roche 454 FLX system sequencer at (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the NCBI sequence
the Cambridge University Biochemistry Department. Sequence deposits AB478515 and AB478516 as well as a library of
reads were quality-checked using Phred Software (http://www. previously proposedWolbachia phage sequences (Supplementary
phrap.org/phredphrapconsed.html) and assembled into 8,238 File 1).
Frontiers in Microbiology | www.frontiersin.org 3 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
wDam Wolbachia Prophage Sequence The resulting alignments were used to construct
Assembly maximum likelihood trees using the software from the
Contigs showing significant matches were classified as being of PHYLIP package (Felsenstein, 2002). The robustness of
bacteriophage origin if two of their three best matches in the the constructed trees’ topologies was tested with 1000
NCBI’s non-redundant sequence data bank were annotated as pseudoreplicates (http://evolution.genetics.washington.edu/
a Wolbachia phage sequence. Contigs identified as containing phylip.html). Final alignment files used in the tree construction
possible phage sequences were aligned to the WOcauB2 are provided in Supplementary Files 3, 4.
reference genome to identify putative gap sequences. Primers
were designed to amplify predicted phage genome “gap” RESULTS
DNA sequences. All “gap-closing” PCRs that produced PCR
products of the expected size had their PCR fragments Sanger Identification and Structural Resolution of
sequenced in the forward and reverse directions (http://www. Three wDam Prophage Sequence
lifesciences.sourcebioscience.com/genomic-services/sanger- Elements and the Proposed Genomic
sequencing-service/). A full list of the primers used for this step
is provided in Supplementary File 2. The primer design and Architecture of the sr1WOdamA1
PCR conditions used to amplify these “gap regions” followed an Prophage Genome
approach described previously (Post et al., 2009). “Gap-closing” In total, 22 contigs were identified as containing putative
Sanger-sequence reads were aligned to those generated from 454 bacteriophage sequences and showing homology with 33
sequence runs and used to extend the original contigs into a WOcauB2 genes. In each case, only one allele for each phage gene
total of three large non-contiguous sequences, spanning what is was identified, which led to the hypothesis that the sequences
proposed here to be a complete WO genome sequence (i.e., from recovered from the shot-gun sequence analysis had originated
its first gene sequence to its last). from just one phage genome sequence that might be resolvable by
gap-closing PCR amplification and Sanger sequencing. Following
Confirmation of WO Prophage Sequence gap-closing PCRs, the 22 original phage contigs were extended
Proximity to the wDam FtsZ Gene and assembled into three large contigs totalling 32.439 kb
of unique sequence, which we propose here represents the
Sequence
near-complete genome of sr1WOdamA1 and which has been
A BLAST search using the S. squamosum E FtsZ sequence
deposited at the NCBI with accession numbers KY695239–
(FN563974) confirmed that the Roche 454 sequence reads were
KY695241.
from the targeted S. squamosum E Wolbachia described in
Crainey et al. (2010a). To confirm that the WO prophage
sequences occur adjacent to the wDam FtsZ gene, all seven FtsZ- The Characterization of a wDamWO Serine
positive BAC colonies used in the shotgun sequence run were Recombinase Replication and Repair
individually PCR-screened for the presence of WO genes using Module (sr1RRM) Prophage Sequence
four primer sets (Supplementary File 2). Two of the primer sets Element (sr1WOdamA1 Contig Number 1)
targeted the serine recombinase gene (i.e., the phage’s WOcauB2 wDamWO prophage contig number 1 (NCBI accession number
gp1 paralog), which was predicted to occur at the 5′ end of the KY695239) is 11.689 kb in length and is predicted to contain
bacteriophage (as inWOcauB2 andWOcauB3) and the other two a block of 12 genes that show high levels of sequence identity
primer sets targeted the gene sequences from the tail end of the with the first 12 predicated genes in WOcauB2 [WOcauB2
phage (corresponding to WOcauB2 gp32 and gp33 paralogous gp1–gp12 (Table 2)]. TheWOcauB2 gp1 paralog (sr1WOdamA1
sequences). gp1p) occurs at the extreme 5’ terminal end of this contig
and corresponds to the sr1WOdamA1 recombinase gene,
Phylogenetic Classification of the wDam whose phylogenetic analysis robustly groups with the four
Wolbachia Prophage previously described WO group serine recombinases (Figure 1).
The phylogenetic classification of the WO prophage sequences The next 11 gene sequences occur in the same order and
was performed using the minor capsid (sometimes referred to orientation as in WOcauB2, representing the conserved group
as the WO orf7 gene) and recombinase genes corresponding 1 serine recombinase and replication module (sr1RRM) which
to WOcauB2 gp17 and gp1 paralogs, respectively. Clustal X is unique to and highly conserved among, the sr1WO group
(Thompson et al., 1997) was used to align the serine recombinase bacteriophages (Figure 2 and below). The extreme 3′ terminal
amino acid sequence of our WO gp1 paralog to the serine end of contig 1 shows very high levels of sequence identity with
recombinases amino acid sequences of WO phage used in the WOcauB2 predicated gene protein 13 (WOcauB2 gp13). The
the recombinase analysis of Kent et al. (2011a). Clustal X first 545 base pairs of WOcauB2 gp13 thus correspond with
was also used to align the nucleotide sequence of the minor the last 469 nucleotides of contig 1 (Table 2). As the first 354
capsid gene of our WO gp17 paralog to the minor capsid nucleotides of contig 2 correspond to the last 369 nucleotides
genes of the same WO phage, as well as the genes of three of the same gene (WOcauB2 gp13), we assumed that the two
other Wolbachia prophages that lack integrase/recombinase contigs would be easily joined by PCR (Table 2). Despite repeated
genes. efforts (using eight different primer sets), we were unable to
Frontiers in Microbiology | www.frontiersin.org 4 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
TABLE 2 | Conservation of gene content, synteny and sequence similarity in the sr1WO group Wolbachia phage, and gene-content inventory for the three
sr1WOdamA1 Wolbachia phage contigs generated and characterized in this study.
Gene name† sr1WOdamA1 sr1WOdamA1 sr1WOdamA1 WOcauB2 WOcauB3 WOvitA WOsimwRi
contig #1 contig #2 contig #3 AB478515‡ AB478516‡ HQ906663‡ CP001391‡
KY695239§ KY695240§ KY695241§
Coordinates Coordinates Coordinates Coordinates
Similarity (percent) Similarity (percent) Similarity (percent) Similarity (percent)
Divergence (percent) Divergence (percent) Divergence (percent) Divergence (percent)
BLAST score BLAST score BLAST score BLAST score
WOB2pgp1 34–1,565 – – 20,032–21,575 16,816–18,347 89–1,632 81,7,261–81,8794
Recombinase 1,346/1,550 (87%) 1,318/1,538 (86%) 1,350/1,550 (87%)* 1,31,4/1,540 (85%)
24/1,550 (1%) 24/1,538 (1%) 24/1,550 (1%) 24/1,540 (1%)
1,633 bits (2,056) 1,551 bits (1,952) 1,649 bits (2,076)* 1,530 bits (1,926)
WOB2pgp2 1,906–3,104 – – 21,893–23,090 18,700–19,897 1,950–3,1,47 819,117–820,31,3
1,112/1,201 (93%) 1,108/1,201 (92%) 1,118/1,201 (93%) 1,132/1,200 (94%)*
5/1,201 (0.4%) 5/1,201 (0.4%) 5/1,201 (0.4%) 5/1,200 (0.4%)
1,551 bits (1,952) 1,535 bits (1,932) 1,574 bits (1,982) 1,632 bits (2,054)*
WOB2pgp3 3,070–4,179 – – 23,056–24,1,65 19,863–20,972 3,1,13–4,222 820,279–821,388
1,055/1,110 (95%) 1,058/1,110 (95%) 1,087/1,110 (98%)* 1,034/1,110 (93%)
0/1,110 (0%) 0/1,110 (0%) 0/1,110 (0%) 0/1,110 (0%)
1,544 bits (1,944) 1,557 bits (1,960) 1,671 bits (2,104)* 1,462bits (1,840)
WOB2pgp4 4,185–6,205 – – 24,191–26,187 20,998–22,994 4,228–6,238 821,402–823,408
1,882/1,998 (94%) 1,899/1,998 (95%) 1,903/2,012 (95%) 1,867/2,011 (93%)
4/1,998 (0.2%) 4/1,998 (0.2%) 4/2,012 (0.2%) 11/2,011 (0.5%)
2,709 bits (3,412) 2,776 bits (3,496) 2,758 bits (3,474) 2,612 bits (3,290)
WOB2pgp5 6,211–7,441 – – 26,222–27,448 23,029–24,255 6,273–7,499 823,443–824,669
1,190/1,233 (97%) 1,166/1,233 (95%) 1,191/1,233 (97%)* 1,180/1,233 (96%)
8/1,233 (0.6%) 8/1,233 (0.6%) 8/1,233 (0.6%) 8/1,233 (0.6%)
1,781 bits (2242) 1,686 bits (21,22) 1,785 bits (2248)* 1,741 bits (2,192)
WOB2pgp6 7,438–7,926 – – 27,445–27,933 24,252–24,731 7,496–7,984 824,705–825,166
459/489 (94%) 463/489 (95%) 461/489 (94%) 364/462 (79%)
0/489 (0%) 9/489 (2%) 0/489 (0%) 1,2/462 (3%)
659 bits (828) 667 bits (838) 667 bits (838) 337 bits (422)
WOB2pgp7 7,950–8,745 – – 27,957–28,754 25,283–26,023 8,008–8,805 825,190–825,987
771/798 (97%) 625/744 (84%) 771/798 (97%)* 753/799 (94%)
2/798 (0.3%) 11/744 (1,.5%) 2/798 (0.3%) 4/799 (0.5%)
1,159 bits (1,458) 702 bits (882) 1,159 bits (1,458)* 1,084 bits (1,364)
WOB2pgp8 8,797–9,288 – – 28,806–29,297 25,537–26,021 8,857–9,348 826,039–826,530
460/493 (93%) 448/486 (92%) 465/493 (94%) 475/492 (97%)*
2/493 (0.4%) 2/486 (0.4%) 2/493 (0.4%) 0/492 (0%)
651 bits (818) 621 bits (780) 671 bits (844) 714 bits (898)*
WOB2pgp9 9,336–9,493 – – 29,343–29,501 26,071–26,220 9,394–9,552 826,578–826,727
150/159 (94%) 144/150 (96%) 151/159 (95%) 148/150 (99%)*
1/159 (0.6%) 1/150 (0.7%) 1/159 (0.6%) 1/150 (0.7%)
218 bits (272) 214 bits (268) 221 bits (276) 230 bits (288)*
(Continued)
Frontiers in Microbiology | www.frontiersin.org 5 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
TABLE 2 | Continued
Gene name† sr1WOdamA1 sr1WOdamA1 sr1WOdamA1 WOcauB2 WOcauB3 WOvitA WOsimwRi
contig #1 contig #2 contig #3 AB478515‡ AB478516‡ HQ906663‡ CP001391‡
KY695239§ KY695240§ KY695241§
Coordinates Coordinates Coordinates Coordinates
Similarity (percent) Similarity (percent) Similarity (percent) Similarity (percent)
Divergence (percent) Divergence (percent) Divergence (percent) Divergence (percent)
BLAST score BLAST score BLAST score BLAST score
WOB2pgp10 9,642–9,799 – – 29,650–29,713 26,370–26,430 9,701–9,764 826,877–826,937
63/64 (98%) 61/61 (100%) 63/64 (98%) 60/61 (98%)
0/64 (0%) 0/61 (0%) 0/64 (0%) 0/61 (0%)
99.1 bits (122) 99.1 bits (122) 99.1 bits (122) 94.3 bits (116)
29,647–29,814 26,367–26,534 9,698–9,865 826,874–827,041
155/168 (92%) 155/168 (92%) 154/168 (92%) 154/168 (92%)
7/168 (4%) 7/168 (4%) 7/168 (4%) 7/168 (4%)
211 bits (264) 211 bits (264) 206 bits (258) 206 bits (258)
WOB2pgp11 10,128–10,594 – – 30,170–30,639 26,891–27,351 10,222–10,691 827,414–827,883
439/470 (93%) 431/470 (92%) 438/470 (93%) 444/471 (94%)
4/470 (0.9%) 1,3/470 (3%) 4/470 (0.9%) 6/471 (1%)
621 bits (780) 583 bits (732) 617 bits (776) 637 bits (800)
WOB2pgp12 10,727–11,203 – – 30,774–31,244 27,485–27,942 10,825–11,295 828,029–828,499
439/477 (92%) 423/464 (91%) 439/477 (92%)* 436/477 (91%)
6/477 (1%) 6/464 (1%) 6/477 (1%) 6/477 (1%)
603 bits (758) 570 bits (716) 603 bits (758)* 591 bits (742)
WOB2pgp13 11,220–11,689 – – 31,261–31,457 27,966–28,160 11,312–11,508 828,51,6–828,712
192/197 (97%) 181/195 (93%) 197/197 (100%) 188/197 (95%)
0/197 (0%) 0/195 (0%) 0/197 (0%) 0/197 (0%)
294 bits (368) 256 bits (320) 31,4 bits (394) 278 bits (348)
31,523–31,808 28,238–28,509 11,574–11,859 828,790–828,858
267/288 (93%) 243/273 (89%) 246/287 (86%) 66/69 (96%)
6/288 (2%) 3/273 (1%) 4/287 (1%) 0/69 (0%)
370 bits (464) 313 bits (392) 299 bits (374) 99.1 bits (122)
1–354 32,392–32760 29,086–29,454 13,027–13,386 –
341/369 (92%) 339/369 (92%) 334/360 (93%)
15/369 (4%) 15/369 (4%) 6/360 (2%)
464 bits (582) 456 bits (572) 465 bits (584)
WOB2pgp14 – 348–21,78 – 32,754–34,575 29,448–31,278 13,380–15,200 –
1,653/1,832 (90%) 1,664/1,840 (90%) 1,679/1,831 (92%)
19/1,832 (1%) 18/1,840 (1%) 22/1,831 (1%)
21,84 bits (2,750) 2,209 bits (2,782) 2,287 bits (2,880)
WOB2pgp15 – 2,176–2,400 – 34,576–34,800 31,276–31,500 15,212–15,430 –
172/225 (76%) 224/225 (99%)* 194/219 (89%)
0/225 (0%) 0/225 (0%) 0/219 (0%)
167 bits (184) 401 bits (444)* 282 bits (312)
(Continued)
Frontiers in Microbiology | www.frontiersin.org 6 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
TABLE 2 | Continued
Gene name† sr1WOdamA1 sr1WOdamA1 sr1WOdamA1 WOcauB2 WOcauB3 WOvitA WOsimwRi
contig #1 contig #2 contig #3 AB478515‡ AB478516‡ HQ906663‡ CP001391‡
KY695239§ KY695240§ KY695241§
Coordinates Coordinates Coordinates Coordinates
Similarity (percent) Similarity (percent) Similarity (percent) Similarity (percent)
Divergence (percent) Divergence (percent) Divergence (percent) Divergence (percent)
BLAST score BLAST score BLAST score BLAST score
WOB2pgp16 – 2,401–3,785 – 34,801–36,228 31,501–32,928 16,129–16,829 –
Phage portal 1,215/1,439 (84%) 1,228/1,440 (85%)* 584/713 (82%)
protein 65/1,439 (5%) 67/1,440 (5%) 15/713 (2%)
1,344 bits (1,692) 1,392 bits (1,752)* 610 bits (766)
– – 16,880–17,508
540/650 (83%)
25/650 (4%)
576 bits (724)
WOB2pgp17 – 3,818–4,888 – 36,255–37,331 32,955–33,967 17,551–18,566 –
Putative minor 985/1,077 (91%)* 907/1,024 (89%) 809/1,030 (79%)
capsid protein 6/1,077 (1%) 12/1,024 (1%) 21/1,030 (2%)
1,528 bits (1,694)* 1,312 bits (1,454) 830 bits (920)
WOB2pgp18 – 4,863–5,231 – 37,306–37,667 34,030–34,391 18,629–18,996 –
331/362 (91%)* 299/367 (81%) 323/368 (88%)
3/362 (1%) 13/367 (4%) 0/368 (0%)
451 bits (566)* 303 bits (380) 406 bits (51,0)
WOB2pgp19 – 5,273–6,280 – 37,715–38,715 34,430–35,429 19,031–20,015 –
928/1,002 (93%)* 918/1,001 (92%) 910/986 (92%)
1/1,002 (0.1%) 1/1,001 (0.1%) 1/986 (0.1%)
1,297 bits (1632)* 1,260 bits (1586) 1,263 bits (1590)
WOB2pgp20 – 7,485–7,805 – 38,807–39,1,26 35,51,4–35,833 20,1,29–20,193 –
298/320 (93%) 295/320 (92%) 53/65 (82%)
0/320 (0%) 0/320 (0%) 0/65 (0%)
422 bits (530) 410 bits (51,4) 57.8 bits (70)
WOB2pgp21 – 7,798–8,285 – 39,1,20–39,608 35,827–36,306 20,496–20,915 –
467/489 (96%) 458/489 (94%) 300/422 (71%)
1/489 (0.2%) 1,0/489 (2%) 37/422 (9%)
689 bits (866) 646 bits (812) 157 bits (196)
WOB2pgp22 – 8,266–8,698 – 39,589–40,021 36,287–36,761 20,952–21,359 –
430/433 (99%) 419/478 (88%) 351/409 (86%)
0/433 (0%) 6/478 (1%) 2/409 (0.5%)
676 bits (850) 521 bits (654) 419 bits (526)
WOB2pgp23 – 8,726–9,054 – – – – –
WOB2pgp25 – – 316–651 40,773–41,08 37,871–38,206 – –
326/336 (97%) 328/336 (98%)
0/336 (0%) 0/336 (0%)
562 bits (622) 571 bits (632)
WOB2pgp26 – – 661–1,470 4,118–41,894 38,21,6–39,001 22,471–23,245 –
739/789 (94%) 747/798 (94%) 535/799 (67%)
1,2/789 (2%) 12/798 (2%) 24/799 (3%)
1,204 bits (1,334) 1,216 bits (1,348) 250 bits (276)
(Continued)
Frontiers in Microbiology | www.frontiersin.org 7 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
TABLE 2 | Continued
Gene name† sr1WOdamA1 sr1WOdamA1 sr1WOdamA1 WOcauB2 WOcauB3 WOvitA WOsimwRi
contig #1 contig #2 contig #3 AB478515‡ AB478516‡ HQ906663‡ CP001391‡
KY695239§ KY695240§ KY695241§
Coordinates Coordinates Coordinates Coordinates
Similarity (percent) Similarity (percent) Similarity (percent) Similarity (percent)
Divergence (percent) Divergence (percent) Divergence (percent) Divergence (percent)
BLAST score BLAST score BLAST score BLAST score
WOB2pgp27 – – – 41,922–42,083 39,020–39,181 23,439–24,361 –
136/162 (84%) 136/162 (84%) 672/933 (72%)
0/162 (0%) 0/162 (0%) 20/933 (2%)
176 bits (194) 176 bits (194) 484 bits (536)
42,132–43,139 39,230–40,237 –
980/1,008 (97%) 978/1,008 (97%)
0/1,008 (0%) 0/1,008 (0%)
1,692 bits (1876) 1,683 bits (1866)
WOB2pgp28 – – 2,794–3,476 43,160–43,844 40,258–40,939 – –
61,7/685 (90%) 543/688 (79%)
4/685 (1%) 13/688 (2%)
816 bits (1,026) 508 bits (638)
WOB2pgp29 – – 3,492–5,414 43,863–45,796 40,958–42,910 – –
1,769/1,935 (91%) 1,742/1,953 (89%)
14/1,935 (1%) 30/1.953 (2%)
2,403 bits (3,026) 2,239 bits (2,820)
WOB2pgp30 – – 5,427–6,610 45,822–46,996 42,922–44,108 – –
1,076/1,175 (92%) 1,082/1,187 (91%)
1/1,175 (0.1%) 4/1,187 (0.3%)
1,473 bits (1,854) 1,465 bits (1,844)
WOB2pgp31 – – 6,615–6,811 47,001–47,195 44,124–44,310 – –
157/197 (80%) 145/191 (76%)
2/197 (1%) 6/191 (3%)
154 bits (192) 118 bits (146)
WOB2pgp32 – – 6,808–8,298 47,192–47,518 44,330–44,651 – –
302/327 (92%) 279/322 (87%)
0/327 (0%) 0/322 (0%)
477 bits (528) 387 bits (428)
47,607–48,612 44,740–45,719
926/1,018 (91%) 909/992 (92%)
12/1,018 (1%) 12/992 (1%)
1,424 bits (1578) 1,416 bits (1,570)
WOB2pgp33 – – 8,304–8,594 48,626–48,910 45,760–46,051 – –
225/286 (79%) 239/292 (82%)
3/286 (1%) 1/292 (0.3%)
211 bits (264) 254 bits (318)
WOB2pgp42 – – 8,593–9,193 55,791–56,398 51,536–52,143 – –
581/608 (96%) 581/608 (96%)
7/608 (1%) 7/608 (1%)
976 bits (1082) 976 bits (1,082)
(Continued)
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Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
TABLE 2 | Continued
Gene name† sr1WOdamA1 sr1WOdamA1 sr1WOdamA1 WOcauB2 WOcauB3 WOvitA WOsimwRi
contig #1 contig #2 contig #3 AB478515‡ AB478516‡ HQ906663‡ CP001391‡
KY695239§ KY695240§ KY695241§
Coordinates Coordinates Coordinates Coordinates
Similarity (percent) Similarity (percent) Similarity (percent) Similarity (percent)
Divergence (percent) Divergence (percent) Divergence (percent) Divergence (percent)
BLAST score BLAST score BLAST score BLAST score
WOB2pgp43 – – 9,121–9,692 56,319–56,900 52,064–52,645 – –
488/590 (83%) 488/590 (83%)
26/590 (4%) 26/590 (4%)
513 bits (644) 513 bits (644)
WOB2pgp44 – – 9,831–10,049 57,025–57,243 52,770–52,988 – –
216/219 (99%) 21,0/219 (96%)
0/219 (0%) 0/219 (0%)
337 bits (422) 313 bits (392)
WOB2pgp45 – – 10,023–10,922 57,217–58,116 52,962–53,866 – –
858/902 (95%) 848/905 (94%)
4/902 (0.4%) 5/905 (0.6%)
1,255 bits (1,580) 1,208 bits (1,520)
WOB3pgp45 – – 11,124–11,696 – 54,016–54,588 – –
SpvB insect toxin 475/573 (83%)
0/573 (0%)
522 bits (656)
†
Predicted gene products are named and numbered in the “gene name” column; homology-based predicted functional information is also provided. Gene names are based on their
homology to gene products reported for the WO reference genome WOcauB2 (Kent et al., 2011a). WOcauB2 paralogs gene product is abbreviated as “WOB2pgp” followed by an
identifying number; WOB2pgp13 is thus a paralogs sequence of WOcauB2 gene product 13.
§NCBI accession numbers of the three contigs generated in this study are provided directly below their names: sr1WOdamA1 contig 1 to 3.
‡
GenBank accession numbers, gene co-ordinates and similarity values for all four of the other sr1WO phage genomes (for a schematic overview of shared sr1WO group genomic
architecture see Figure 2). Divergence measurements from WOdamA1 predicted products are displayed for all the paralogs gene sequences that occur in these other four sr1WO
genomes. Quoted sequence co-ordinates and similarity values were obtained from individual gene product BLASTn sequence searches of GenBank’s non-redundant nucleotide
sequence deposits using sr1WOdamA1 predicted gene products as queries and implementing the following search parameters: word size 7; gap opening penalty 0; extension penalty
4. Similarity values are displayed only if they were recovered from the top 10 most significant sequence matches (based on bit scores) found from GenBank’s entire non-redundant
sequence repository.
*Bold type face highlights BLASTn sequence similarity matches that were the most significant sequence matches found in all of GenBank’s entire non-redundant sequence repository.
bridge what we predicted would correspond to a 584 base-pair for the minor capsid (orf7) protein, which is a B2gp17 paralog
(bp) gap of WOcauB2 gp13 paralog gene sequence, which we and has been used to construct the phylogenetic tree shown
expected to occur between contigs 1 and 2 (Table 2). It is, thus, in Figure 3. The 10 whole gene sequences that occur in contig
most probable that the sr1WOdamA1 genome is not orientated 2 appear in the same order and orientation as their paralogs
as the WOcauB2 genome (Figure 2 shows its similarity to the in the WOcauB2 genome. The synteny between the WOcauB2
other serine recombinase WOs). and sr1WOdamA1 genomes is only interrupted by the existence
of a transposable element-like sequence occurring between the
The Characterization of a wDam WO Head sr1WOdamA1 WOcauB2 gene protein paralogs B2gp19 and
and Base-Plate Module Prophage B2gp20.
Sequence Element (sr1WOdamA1 Contig
Number 2) The Characterization of a wDam WO Tail
wDam WO prophage contig 2 (NCBI accession number Module Prophage Sequence Element
KY695240) is 9.054 kb and spans from the 3′ end of our (sr1WOdamA1 Contig Number 3)
WOcauB2 gp13 paralog to the middle of our WOcauB2 wDam WO prophage contig 3 (NCBI accession number
gp23 paralog (Figure 2). It contains a block of 10 WOcauB2 KY695241) is 11.696 kb in length and contains 14 predicted
gene sequences which, as shown in Figure 2, code for genes gene sequences, spanning from a WOcauB2 gene protein 25
corresponding to what Kent et al. (2011a) defined as WO head paralog at one end through to a WOcauB3 gp45 gene protein
and base-plate modules. It also includes gene sequence coding paralog at the other end (Table 2 and Figure 2). As can be
Frontiers in Microbiology | www.frontiersin.org 9 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
FIGURE 1 | Maximum likelihood consensus tree constructed from an alignment of Wolbachia phage recombinase amino acid sequences. Three
bootstrap-supported sequence clusters (labeled sr1WO-sr3WO) recovered in the analysis of Kent et al. (2011a) and Wang et al. (2013) were recovered in this analysis
and are indicated. All the WOs known to have the structural group 1 serine recombinase replication module (sr1RRM) can be seen to occur in the sr1WO group.
Sr1WO group recombinases are marked with a circle. When these circles are colored in yellow, the phage is known to occur adjacent to the FtsZ cell-division gene,
white coloring indicates that the phage’s genomic location is unknown and black is used to indicate that the phage does not appear to be located close to the FtsZ
gene. The recombinase amino acid sequences are provided in Supplementary File 3.
seen in Figure 2, this contig contains the 3′ prime end of the Preliminary Characterization of the wDam
phage base-plate gene modules as well as its virulence and Genomic DNA Flanking the FtsZ
tail regions (Kent et al., 2011a). The first predicted nine gene Cell-Division Gene: a Possible Wolbachia
sequences in this contig correspond to paralogs of WOcauB2
gene protein sequences spanning from 25 to 33 (Table 2 and Genome Target Site for sr1 Group WO
Figure 2). This wDam WO prophage sequence element then Integration
appears to have a deletion. Thus, after this nine-gene block, BLASTn and tBLASTx homology searches of the shot-gun
there is a 3′-truncated gene protein sequence (a paralog of sequence data using the 16,703 nucleotide sequence reported
the WOcauB2 gene protein 33), which is immediately followed to be upstream of WOcauB3 did not identify any significant
by a block of five gene sequences corresponding to paralogs sequence matches. However, four shot-gun sequence contigs
of WOcauB2 gene proteins B2gp42 to B2gp45 (Table 2 and (representing 5.265 kb of unique DNA) showed significant levels
Figure 2). At the extreme 3′ end of this prophage sequence of homology with the upstream sequence ofWOcauB2. As shown
element and contig 3 (directly after the WOcauB2 gp45 paralog), in Table 1, these four contigs show high levels of sequence
there is a gene sequence matching the B3gp45 gene protein identity with six of the 16 genes found immediately upstream
(see below) which has no paralog in the WOcauB2 genome. of the WOcauB2 and WOri relic serine recombinase prophage
Repeated efforts to close a predicted gap between contigs 2 and sequences. Importantly, the gene sequences in these contigs
3 failed; it is, thus, unclear if there is a WOcauB2 gene protein 24 correspond to four of the five gene sequences that are closest to
paralog within the genome of sr1WOdamA1 or not. The extreme the WOcauB2 and WOri relic integration sites. Hence, our 454
5′ end of contig 3 contains what we propose here is a 217- sequence run recovered DNA sequence matching the 4,239 bp
nucleotide transposable element sequence with all the features immediately upstream of the WOcauB2 prophage and the 3,769
of a Miniature Inverted-repeat Transposable Element (MITE), bp sequence immediately upstream of the WOri relic prophage
including 24-nucleotide inverted terminal repeats (ITRs), as well (Table 1). In contrast with the other four genes immediately
as a 9-nucleotide target-site duplication (Delihas, 2011). This upstream of the WOcauB2 and WOri relic prophages, BLAST
MITE—named here as wDam-MITE-1—could be the beginning searches revealed that the gene for which we did not recover a
of a stretch of repetitive DNA lying between the head and base- paralog (corresponding to ACN95502 in wRi and BAH22204 in
plate and tail modules (i.e., contigs 2 and 3) of the sr1WOdamA1 wCau) is incompletely conserved among the Wolbachia strains.
genome that was too long for our PCR bridging efforts to gap- The absence of this gene from the shot-gun sequence reads
close. could be a consequence of its absence from the wDam genome.
Frontiers in Microbiology | www.frontiersin.org 10 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
FIGURE 2 | Schematic representation of the predicted structural organization of sr1WOdamA1 in relation to all four other members of the sr1WO
group bacteriophage group (sr1WO). Arrows are used to indicate the direction in which predicted gene sequences are encoded and shading is used to indicate
functional homology. The gene sequences of the WOcauB2 reference genome (used in this paper and in Kent et al., 2011a) are annotated with numbers and the gene
product (gp) abbreviation. A conserved block of genes spanning from WOcauB2 gp1 to WOcauB2 gp12 with functions involved in recombination and replication
functions, and referred to in the main text as “sr1RMM,” is highlighted with a red box. See also Figure 1. Nine perfectly conserved genes found within this box are
indicated with pink highlighting. Predicted gene protein functional groups have been colored following the classification of Kent et al. (2011a). Blue is used to show
“head” proteins; purple is used for “base-plate” proteins; orange is used for virulence proteins, and black is used for “tail” proteins. Red coloring is used to highlight a
block of three predicted gene proteins, which appears only to occur in sr1WOdamA1, WOcauB2, and WOcauB3, suggesting a special relationship between these
phages. The inset with WOcauB2 and WOcauB3 genes that have no WOdamA1 paralogs represents that there is no non-coding DNA separating the 3′-truncated
gp33 paralog and the gp42 paralog in sr1WOdamA1 contig 3. The two proposed transposable elements referred to in the main text are indicated with pink arrows;
the wDam-MITE-1 element is also labeled with its name.
Sequence comparisons made between the wDam FtsZ gene just the tail-end of sr1WOdamA1 (i.e., the gp31 and gp32
contig recovered from our shot-gun sequence reads and the genes).
previous report confirmed that the FtsZ-positive BACs used
in this study were of the same origin as the FtsZ gene first
reported in 2010 (Crainey et al., 2010a). The 454 sequence contig All Three wDam Prophage Sequence
showed >99% sequence identity with the previously published Elements Likely Derive from an Inactive
FtsZ (FN563974) sequence. sr1WOdamA1 Prophage Relic
PCR screening for sr1WOdamA1 gene sequences within the The three contigs recovered in this work can be seen to
seven FtsZ-positive BACs used in the original shot-gun sequence correspond, roughly, to three distinct (and non-overlapping)
run, identified three colonies as containing sr1WOdamA1 WO modules (1–3), that can be considered, when compared
phage sequences. Consistent with the notion that a full- to the WOcauB2 reference genome, collectively to make-up a
length sr1WOdamA1 prophage element occurs immediately near complete sr1WO genome. wDam WO contig 1, contains a
adjacent to the FtsZ gene of wDam, one FtsZ-positive BAC was replication and repair module thus far only associated with WOs
confirmed (by two independent PCR reactions) as containing that contain sr1 recombinases (sr1RRM); the wDam WO contig
the sr1WOdamA1 serine recombinase gene (i.e., a WOcauB2 2 contains a head and base-plate module and some “virulence”
gp1 paralog) and also B2gp33 and B2gp34 paralogs (by two genes, and wDam WO contig 3 is a tail module (Figure 2).
independent PCR reactions). As previous analysis of BAC clones While the exact relationship between these wDamWO prophage
from the S. squamosum E BAC library used in this study sequences is presently unclear, the gene sequence order and
suggested that the average BAC contains around 128 kb of orientation within these contigs can be seen to be almost identical
cloned DNA and the sr1WOdamA1 genomic sequence is over 32 to those reported for the WOcauB2 and WOcauB3 phage
kb, this strongly suggests that the sr1WOdamA1 prophage has genomes. Althoughwe acknowledge that alternative explanations
integrated within 100 kb of the wDam FtsZ gene. Unexpectedly, for our results may exist (see Section Discussion), we believe that
two BACs tested positive (in two independent PCR tests) for the most parsimonious explanation is that all three of the wDam
Frontiers in Microbiology | www.frontiersin.org 11 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
the extreme 5′-end of contig 3, immediately downstream of
sr1WOdamA1’s WOcauB2 gp25 gene protein paralog (where the
WOcauB2 gp24 gene should occur). Because of this and because
there was no trace of aWOcauB2 gp24 paralog detected from our
initial shot-gun sequence run, we believe that the sr1WOdamA1
genome reported here is probably a dysfunctional prophage relic.
wDam WO Prophage Sequences Are
Isolated from Both
Non-wolbachia-infecting Bacteriophage
and Other WOs
As shown in Table 2, most BLAST searches with the wDam WO
prophage sequences recovered in this work, returned best match
paralogous sequences from the genomes of other sr1WO group
FIGURE 3 | Representative maximum likelihood tree constructed from prophages. In every search performed with our 36 predicted
a 1031 nucleotide position alignment of 17 WO minor capsid protein
sr1WOdamA1 gene sequences, the paralogous sequence matches
gene sequence. The tree (historically used for WO classification) includes
sequences from all the WOs used in Figure 1, as well as two additional WO listed in Table 2 were among the top ten closest matches in
sequences (from WOPip1 and WOPip5), the genomes of which lack the non-redundant NCBI sequence repository. In addition to
integrase/recombinase genes (Kent et al., 2011a). The gene sequences of this, every search returned a best match sequence annotated as
WOPip1 and WOPip5 are highlighted with blue boxing. Minor capsid gene deriving from a Wolbachia genome or WO. Moreover, when
sequences originating from WOs with the group 1 serine replication
the search results returned five or more significant matches, the
recombinase module (sr1RRM) referred to in the main text are indicated, as are
their integration sites (labeled as in Figure 1). The serine recombinase-based top five hits were always annotated as not just deriving from
phylogenetic classification of these WOs is also highlighted, using branch tip a Wolbachia or WO genome but to be also sourced from an
suffixes written in red (sr1–sr3). The bootstrap support for two monophyletic arthropod.
groups containing WO sequence representatives with varying Figure 1 presents a phylogenetic tree constructed using WO
integrase/recombinase gene sequences is also shown. The minor capsid
protein sequences alignment is provided in Supplementary File 4. recombinase genes and shows that the wDam WO phage
sequence element recovered in contig 1 belongs to the same
group of serine recombinase WOs to which WOcauB2 and
WOcauB3 belong, and that was previously identified in the
WO contigs reported here derive from the same sr1WO genome, analysis of both Kent et al. (2011a) and Wang et al. (2013).
which we are proposing be named as sr1WOdamA1. In relation As mentioned above, in addition to sharing closely-related
to the reference genome WOcauB2, the sr1WOdamA1 appears recombinase genes, this group of five phages (WOcauB2,
to be missing nine phage gene sequences corresponding to WOcauB3, WOvitA2, WOri relic, and now sr1WOdamA1)
WOcauB2 gp24 and gp34–41 (Figure 2), but to contain paralogs share an sr1RRM (spanning around 11 kb), which is not
for all other WOcauB2 genome sequences. found in other (unrelated) WOs and corresponds almost
In WOcauB2, and the other serine recombinase phage exactly with contig 1 (Figure 2). Gene order and orientation is
genomes that have paralogs, the genes gp34–41 (and their near perfectly conserved in the sr1RRM, with nine conserved
paralogs) have been ascribed tail functions (Figure 2 and WOcauB2 spanning gp1–6 paralog sequences recognizable in
Table 2). As this deletion begins following a 3′-truncated gp33 sr1WOdamA1 and all other serine recombinase WOs (Table 2
paralog, our findings indicate that sr1WOdamA1 lacks paralogs and Figure 2). Our first six predicted gene sequences have clear
for these sequences because of a recent deletion and, thus, that paralogs (appearing in the same order and orientation) and the
sr1WOdamA1 is missing genes that its progenitor contained. As, last two gene sequences of the module (including a Holliday
however, this part of the tail region is highly variable among junction recombinase) have clear paralogs in all five phage
WOs and some, including other sr1WO group bacteriophages genomes (Table 1 and Figure 2).
like WOvitA2 (Figure 2), lack recognizable tail modules, it As shown in Table 2, BLAST searches (against the NCBI’s
is possible that sr1WOdamA1 and/or its progenitor retained entire non-redundant sequence database) with the 12wDamWO
active functions in the absence of a gp34–41 section. On the contig gene sequences from this module returned best match
other hand, the absence of a WOcauB2 gp24 paralog from the sequences deriving from another serine recombinase WO eight
sr1WOdamA1 genome, a well conserved gene component of times. In most cases the wDam WO-predicted gene sequences
the highly preserved phage base-plate region, is likely to render share similar levels of identity (>90%) with the other predicted
sr1WOdamA1 immobile. Although our failure to detect this gene phage gene sequences. For five out of eight of these genes, all
could be an artifact of its expected location occurring in the break differences between the sequences and their closest ones in the
between two of our three sr1WOdamA1 contigs (Figure 2 and database are attributable to nucleotide substitutions, suggesting
above), we believe that it is more likely that the gene has been that these genes have been the subject of point mutation-based
disrupted by a transposable element integration. Our analysis evolution. Indications of recombination, however, can be seen
identified a wDam-MITE-1 transposable element sequence at in Table 2. For example, most WOvitA2, WOri relic, WOcauB2,
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Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
and WOcauB3 genes show similar levels of divergence from Alternative explanations may include, for instance, the
their sr1WOdamA1 paralogs, but the WOvitA2 B2gp3 paralog is generation of chimeric BAC clones, created during the cloning
markedly closer than the others. Similar signs of point-mutation- process by ligating WO and wDam genomic fragments (with
based WO evolution and of between-WO gene recombination different origins and which do not occur close together in
are also evident from the BLAST search returns of wDam WO the wDam genome in nature). This would have required these
prophage head base-plate gene sequences (contig 2) and tail sequences co-incidentally being cloned into the same BAC vector.
module gene sequences (contig 3) (Table 2). However, there are good reasons to doubt such an explanation.
Firstly, BAC libraries have been widely used in genome research
for over 30 years and reports of such chimeric ligation being
The wDam WO Prophage Tail-Module generated by the cloning process are extremely rare. Second, our
Sequence Element Harbors an SpvB former characterization of the BAC library used for this work
Protein Homolog at its Terminal End suggests that wDam DNA represents only about 1% of the total
In addition to the 36 wDam prophage genes with paralogs cloned DNA (Crainey et al., 2010a). Hence, one would expect
in the WOcauB2 genome that were identified from the three that there is about a 99% chance that any randomly created
prophage sequence elements recovered in this work, an SpvB- BAC chimera including a wDam genomic fragment would be
like protein was observed to occur at the terminal end of the composed of wDam and non-wDam DNA (i.e., most likely S.
wDam WO prophage tail module element (contig 3). BLASTn squamosum genomic DNA). The PCRs we did on our BAC
searches with the last 377 nucleotides of contig 3, best match clones showed that three of our FtsZ-positive BACs contain both
the first 378 nucleotides of the WOcauB3 gp45 protein which wDam genomic DNA andWO phage sequences; thus, this would
is annotated as coding for an SpvB-motif protein (BAH22314). require three such events to have occurred (each with a 1%
The two sequences share 83% identity (315/378). The second- chance) ignoring that the cloning process only very rarely creates
best (and only other significant) match is with the first 378 chimeric BAC clones. Therefore, the possibility that our results
nucleotides of the Wolbachia phage wNo_WO4 “SpvB and are explained by such a phenomenon is in the order of one in
TcdB toxin domain protein” (AGJ99401), which shares 79% a million. We are, therefore, reasonably confident that all the
identity (299/378). BLASTx searches also provided best matches wDam WO sequences recovered are from the wDam genome
with these gene sequences (83 and 85% identity across 110 and thus of prophage origin. As a result, we are tentatively
residues, respectively), as well as support for this gene having proposing that they are all from the sameWO prophage genome,
an insecticidal toxin function. Thus, while there are presently no named here sr1WOdamA1. This sr1WOdamA1 is most similar
other close relatives to these proteins in the NCBI database, the to the Wolbachia phage from the almond moth Cadra cautella,
next 17 best matches are all with bacterial proteins, which share WOcauB2 (Tanaka et al., 2009). Several similarities exist between
between 50 and 60% amino acid level identity and have similar our sequences and otherWOs, as well as unique aspects which we
properties to those predicted for the BAH22314 and AGJ99401 discuss in relation to phage evolution, Wolbachia-based disease
proteins. All 10 of these hits that have functional annotation, are control programmes and the relevance of sr1WOdamA1 to
described as SpvB proteins and/or toxins or “insecticidal toxins.” onchocerciasis epidemiology below.
As in the WOcauB3 genome, the SpvB-like protein appears to
occur at the terminal tail end of the wDam WO tail module The Wolbachia Prophages of wDam Show
contig.
Indications of an Integration-Site
Preference
DISCUSSION Traditional phylogenetic classification of Wolbachia phages has
focused on the minor capsid or “orf7” gene (Bordenstein and
In previous work we reported a novel Wolbachia FtsZ cell- Wernegreen, 2004; Gavotte et al., 2007; Chafee et al., 2010).
division gene sequence and showed the genome of thisWolbachia More recently, however, WO researchers have begun performing
to be well represented in a BAC library prepared from S. phylogenetic analysis on the integrase/recombinase genes of
squamosum E blackfly larvae (Crainey et al., 2010a,b). In this WOs (Kent et al., 2011a; Wang et al., 2013). To classify our
work we have taken the first step toward characterizing this novel wDamWO prophage sequence elements, we used both
bacterium’s genome and have provided evidence that it harbors approaches (Figures 1, 3). Using the serine recombinase gene
Wolbachia prophage sequence elements close to its FtsZ cell- of the wDam WO sr1RRM prophage sequence module, our
division gene. Following gap-closing PCRs, we have resolved>32 analysis resulted in the same four serine phylogenetic groupings
kb of WO prophage sequence elements, corresponding to three generated by Kent et al. (2011a) and Wang et al. (2013), and
distinctWO functional modules, namely, an sr1RRM, a head and showed that thiswDamWO prophage sequence element, at least,
base-plate and a tail module. Although alternative explanations belongs to a cluster of four other serine recombinase phages
may exist (see below) as to why we recovered these three WO (sr1WOs) that share several structural features (Figures 1, 2).
sequences from the pool of FtsZ-gene positive BACs sequenced, Phylogenetic analysis with the minor capsid gene from the
the most parsimonious explanation is that they all derive from wDam WO head and base-plate module prophage sequence
a single WO prophage genome that occurs close to the FtsZ element (Figure 3), by contrast, did not share the same degree
cell-division gene. of congruence with WO structural features or agree well with
Frontiers in Microbiology | www.frontiersin.org 13 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
the phylogeny constructed using the serine recombinase genes. phages, suggest that the sr1RRM prophage may be involved in a
Supporting the notion that our wDam WO sr1RMM prophage common and targeted genomic integration method. However, it
element and our wDamWO head and base-plate modules derive should be noted that while the WOri relic prophage, WOcauB2
from the same (sr1WOdamA1) genome, the minor capsid gene and sr1WOdamA1 all appear to have integrated close to the
phylogeny shown in Figure 3, clustered the wDam prophage Wolbachia FtsZ gene, the WOcauB3 appears to have integrated
minor capsid gene in a bootstrap-supported monophyletic group at a different genomic location (Klasson et al., 2008; Tanaka et al.,
with the genes of three other sr1 group WOs. This group, 2009). As more serine recombinaseWolbachia phages have their
however, also contained two non-sr1 group WOs and excluded genome sequences and integration sites resolved, the nature of
the sr1group WOvitA2 bacteriophage, supporting previous this apparent genomic targeting will become better understood.
reports that this gene has been exchanged between WO families Our observation that the tail end of sr1WOdamA1 appears to
via recombination and suggesting that this is not a reliable be closer to the FtsZ gene than the recombinase gene, suggests
gene for WO classification. As the conservation of the sr1RRM that the integration process may not require the bacteriophage
probably reflects a fundamental difference in phage life-cycle and to be integrated in a fixed orientation and highlights how
serine recombinase-based phylogeny grouped all of theWOs that little is presently known about the process by which Wolbachia
share this feature together, we think that classifying and naming phages integrate into their host Wolbachia genomes, with the
our phage based on this feature (rather than by its minor capsid data presented here contributing substantially to the current
protein) has more biological meaning. knowledge base.
With the inclusion of sr1WOdamA1 in the sr1WO group,
the latter can be considered as, currently, having five members,
sr1WOdamA1: Evolution and Relevance to
namely sr1WOdamA1, sr1WOvitA2, WOcauB2, WOcauB3, and
WOri relic (Figures 1, 2). While we have been unable to resolve Wolbachia-Based Disease Control
completely the modular organization of sr1WOdamA1 recorded Strategies
here, we have been able to resolve most of its within-modular Modular theory predicts that phages can exchange gene
structure, and from this it is apparent that gene sequence identity, sequences freely across a broad range of ecological niches (Kent
gene sequence order and orientation are all well preserved et al., 2011b; Metcalf and Bordenstein, 2012). It has been
among this group (Figure 2 and Table 2). Our results do, proposed, however, that the normal rules of modular evolution
however, suggest that modular architecture of sr1WO group do not apply to WOs and that, while WOs can exchange gene
bacteriophages may vary as for many otherWO families (Klasson sequences among themselves, they do not appear commonly to
et al., 2008; Kent et al., 2011a). Thus, while the occurrence of exchange genes with non-Wolbachia phages (Kent et al., 2011b;
the B2gp13 homolog gene sequence—corresponding to the first Metcalf and Bordenstein, 2012). The BLAST searches performed
portion of the gene—at the end of contig 1, and the occurrence with each of the sr1WOdamA1 36 predicted gene sequences,
of the B2gp13 homolog gene sequence—corresponding to the returned a best match sequence deriving from a previously
end of the gene—in contig 2, strongly suggest that they are characterized WO sequence. In most cases the best match
from the same WO genome, the fact that we were unable to sequence was from another serine recombinase WO, suggesting
bridge the gap by PCR suggests that they may not be orientated that there may $$even be restriction of gene flow between WO
in the same way as the WOcauB2, WOcauB3, and WOvitA2 subgroups (Kent et al., 2011b; Metcalf and Bordenstein, 2012). In
genomes (Figure 2). Consistent with the idea of variant modular line with previous analysis, however, these searches did provide
architectures occurring within the sr1WO group, the “terminal” clear evidence of genetic exchange between sr1WOdamA1 and
end of the WOri relic (like the end of the sr1WOdamA1 contig other WO sequences that infect arthropod-infecting Wolbachia
1) corresponds to the 5′-end of a B2gp13 paralog (Klasson et al., (Klasson et al., 2008; Kent et al., 2011b; Wang et al., 2013).
2009). The sr1RRM of the sr1WO phage group may have become Although evidence of frequentWolbachia phages horizontally
separated from the head and base-plate modules (and therefore transferring between strains has recently emerged, the evidence
not occur in sr1WOdamA1 as they do in WOcauB2, WOcauB3, for this has been entirely based on the minor capsid protein and,
and WOvitA2 genomes) in the progenitors of this relic and thus, the structure and biology of the bacteriophages involved in
the sr1WOdamA1 prophage. A variant modular organization these transfers have hitherto been completely unknown (Wang
of sr1WOdamA1 may also help to explain the non-joining of G. H. et al., 2016; Wang N. et al., 2016). In this study, we have
contigs 2 and 3 (and thus the sr1WOdamA1 head and tail isolated three novel WO prophage sequence elements (which
modules). probably all derive from the same WO genome) from the wDam
In addition to shared sequence and structural features, some Wolbachia genome. The wDamWolbachia strain is the first from
of the sr1WO bacteriophages also share a common integration outside the A and B super clades to be shown to be infected
preference. The occurrence of the sr1WOdamA1 genome within with an sr1WO group bacteriophage (Crainey et al., 2010a; Kent
BACs that contain four of the five genes immediately upstream et al., 2011a; Ellegaard et al., 2013). This has two important
of two other sr1 group prophages (WOcauB2 and a WOri implications for Wolbachia-based disease control strategies.
relic) suggest that the sr1WOdamA1 prophage belongs to a Firstly, it suggests that artificially-introduced Wolbachia, like
group of Wolbachia prophages with a target site preference. those being used to control Aedes aegypti-transmitted dengue
This observation and the fact that most of the sr1RRM genes in Australia, Brazil and elsewhere, could themselves be infected
do not have clear paralogs in the genomes of other Wolbachia by naturally occurring phages (Hoffmann et al., 2011, 2015;
Frontiers in Microbiology | www.frontiersin.org 14 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
Caragata et al., 2016). Given the present plans to expand the use male-killing, this could be expected to increase the proportion
ofWolbachia-based disease control techniques and the possibility of female flies in the onchocerciasis foci where this species
that phage integrations into artificialWolbachia infections could occurs, and this could, in turn, be expected to increase disease
impact on vector characters of epidemiological importance, this transmission in areas where such infections occur. Similarly, it
is not a trivial observation but one that may have wide-reaching would suggest that antibiotic treatment might aid getting this
consequences (Woolfit et al., 2013; Sutton et al., 2014; Jeffries notoriously difficult species into laboratory colonies.
and Walker, 2015). For example, although variant strains of
wMelWolbachia bacteria currently used for control appear to be Conclusions
near-identical in gene-coding regions, very minor differences in In this study we have shown that the genome of a genetically-
repeat-region sequences have a major impact on the longevity distinct Wolbachia named here as wDam harbors at least one
(and thus epidemiological importance) of Ae. aegypti (Woolfit serine recombinaseWolbachia prophage relic. Although the three
et al., 2013). On the other hand, this observation suggests that if prophage sequence elements we have characterized correspond
aWO can be manipulated to modify geneticallyWolbachia (such to three distinct non-overlapping WO functional modules (and
as lambda, which is routinely used to infect E. coli), one phage could in theory havemultiple origins), we believe that they almost
could potentially be used to modify a broad range of Wolbachia certainly all derive from a singleWO genome that we have named
stains (Tanaka et al., 2009; Kent and Bordenstein, 2010; Wang sr1WOdamA1. Although this WO is unlikely to be active, its
et al., 2013). In this context, our discovery that sr1WOdamA1 existence in the wDam genome implies that active, naturally
and WOcauB2 belong to a group of phages that may integrate occurring bacteriophages can infect a broad range of genetically
into a single Wolbachia genomic site is particularly interesting diverse Wolbachia strains and that naturally occurring WOs
as it suggests that they may be adapted to provide a genetic could pose a greater risk to the artificial Wolbachia infections
modification system forWolbachia. currently used for disease control than previously thought. The
occurrence of an sr1RRM prophage sequence in the same BAC
clones in which the FtsZ gene is found is consistent with the
The Relevance of wDam WOs to notion that at least some of the sr1WO group WOs, notably
Onchocerciasis Epidemiology the WOcauB2 phage, may have a target site preference and
Because some Wolbachia strains seem to be able to change could be used for targeted introduction of recombinant genes
radically and spontaneously the way in which they infect into Wolbachia genomes. The occurrence of an SpvB gene in
their insect hosts (for example, from exerting a cytoplasmic the genome of the wDam WO prophage sequences suggests
incompatibility to male-killing), WOs have long been suspected that these genes may be a more common feature of Wolbachia
as having an important influence on these characteristics (Kent bacteriophages than hitherto realized, an observation consistent
and Bordenstein, 2010; Metcalf and Bordenstein, 2012). Thus far, with previous proposals that WOs could be important drivers
however, precious little evidence has been uncovered to support of Wolbachia reproductive parasitism and thus could be causing
this hypothesis. The existence of an SpvB-like protein at the male-killing in the onchocerciasis S. damnosum s.l. species
extreme terminal end of the WOcauB3 phage is regarded as the complex with implications for the laboratory colonization of
best evidence yet that these bacteriophages could influence the vector species and the epidemiology of onchocerciasis.
insect host as this gene is believed to have insecticidal properties
(Kent and Bordenstein, 2010; Metcalf and Bordenstein, 2012). AUTHOR CONTRIBUTIONS
The impact that wDam and sr1WOdamA1 might have on S.
squamosum E is presently unknown (Crainey et al., 2010b), but RJP, MGB, and RAC secured financing for this work. JLC, RJP,
the occurrence of an SpvB-like gene at the terminal end of the MGB, and MDW contributed to the planning of the work.
tail module of the WO prophage sequence elements isolated in JLC performed the laboratory work, except for the Sanger
this study suggests that the WOs of wDam could be influencing sequencing which was done by CEG, JLC, JH, and CPMA
S. squamosum E biology. performed the DNA sequence analysis. RJP, RAC, and PHLL
Male-killing is a common form of reproductive parasitism collected S. squamosum E. MGB, RJP, RAC, and PHLL made
induced by Wolbachia (Zug and Hammerstein, 2015). Selective substantial contributions to the interpretation and editing of the
expression of insecticidal proteins such as SpvB in a male manuscript. All authors read and approved the final version of
insect environment could provide a molecular mechanism for the manuscript.
such a phenomenon (Kent and Bordenstein, 2010; LePage and
Bordenstein, 2013; Metcalf and Bordenstein, 2012). Although FUNDING
there are presently no reports of male-killing Wolbachia
infections in the S. damnosum s.l. complex, which contains PHLL, RAC, MDW, MGB, and RJP acknowledge financial
the most important vectors of human onchocerciasis in Africa support from The Wellcome Trust (grant 085133/Z/08/Z
(including S. squamosum E), efforts to get the species into to MGB); PHLL received funding from a Junior Research
laboratory colonies have repeatedly failed because the entire Fellowship at Imperial College London. Presently PHLL is
population has become female over time (Simmons and Edman, funded by a European Research Council Starting Grant
1982; Raybould and Boakye, 1986; Crainey et al., 2017). If the [680088 SCHISTO_PERSIST], a Wellcome Trust ISSF Grant
Wolbachia infecting S. squamosum E is promoting its spread by [105614/Z/14/Z] and a Lord Kelvin Adam Smith Leadership
Frontiers in Microbiology | www.frontiersin.org 15 May 2017 | Volume 8 | Article 852
Crainey et al. sr1WOdamA1: A Novel Wolbachia Prophage
Fellowship. RJP acknowledges funding from the Medical We are also grateful to the Natural History Museum’s DNA
Research Council (grant 77615). The funders had no role in the sequencing facility, and especially to Julia Llewellyn-Hughes and
study design, data collection and analysis, decision to publish, or Lisa Smith, for assisting with the Sanger sequencing used in this
preparation of the manuscript. study.
ACKNOWLEDGMENTS SUPPLEMENTARY MATERIAL
We are grateful for the support provided in Ghana by The Supplementary Material for this article can be found
Daniel A. Boakye, Mike Y. Osei-Atweneboana, and Anthony online at: http://journal.frontiersin.org/article/10.3389/fmicb.
Tetteh-Kumah for the collection of the simuliid larval samples. 2017.00852/full#supplementary-material
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determination of a highly degenerated prophage WO genome in a Wolbachia Conflict of Interest Statement: The authors declare that the research was
strain infecting a fig wasp species. Appl. Environ. Microbiol. 79, 7476–7478. conducted in the absence of any commercial or financial relationships that could
doi: 10.1128/AEM.02261-13 be construed as a potential conflict of interest.
Wang, N., Jia, S., Xu, H., Liu, Y., and Huang, D. (2016). Multiple horizontal
transfers of bacteriophage WO and host Wolbachia in fig wasps in Copyright © 2017 Crainey, Hurst, Lamberton, Cheke, Griffin, Wilson, de Araújo,
a closed community. Front. Microbiol. 7:136. doi: 10.3389/fmicb.2016. Basáñez and Post. This is an open-access article distributed under the terms of
00136 the Creative Commons Attribution License (CC BY). The use, distribution or
Woolfit, M., Iturbe-Ormaetxe, I., Brownlie, J. C., Walker, T., Riegler, M., reproduction in other forums is permitted, provided the original author(s) or licensor
Seleznev, A., et al. (2013). Genomic evolution of the pathogenic Wolbachia are credited and that the original publication in this journal is cited, in accordance
strain, wMelPop. Genome Biol. Evol. 5, 2189–2204. doi: 10.1093/gbe/ with accepted academic practice. No use, distribution or reproduction is permitted
evt169 which does not comply with these terms.
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