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ORIGINAL RESEARCH
published: 17 May 2021
doi: 10.3389/fmicb.2021.628538
Edited by:
Sophia Johler,
University of Zurich, Switzerland
Reviewed by:
Young-Su Seo,
Pusan National University,
South Korea
Laura M. Carroll,
European Molecular Biology
Laboratory (EMBL) Heidelberg,
Germany
*Correspondence:
Jin Xu
xujin@cfsa.net.cn
Fengqin Li
lifengqin@cfsa.net.cn
†These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 12 November 2020
Accepted: 29 March 2021
Published: 17 May 2021
Citation:
Peng Z, Dottorini T, Hu Y, Li M,
Yan S, Fanning S, Baker M, Xu J and
Li F (2021) Comparative Genomic
Analysis of the Foodborne Pathogen
Burkholderia gladioli pv.
cocovenenans Harboring
a Bongkrekic Acid Biosynthesis Gene
Cluster. Front. Microbiol. 12:628538.
doi: 10.3389/fmicb.2021.628538
Comparative Genomic Analysis of
the Foodborne Pathogen
Burkholderia gladioli pv.
cocovenenans Harboring a
Bongkrekic Acid Biosynthesis Gene
Cluster
Zixin Peng1, Tania Dottorini2, Yue Hu2, Menghan Li1, Shaofei Yan1, Séamus Fanning1,3 ,
Michelle Baker2, Jin Xu1*†and Fengqin Li1*†
1NHC Key Laboratory of Food Safety Risk Assessment, Chinese Academy of Medical Sciences Research Unit
(2019RU014), China National Center for Food Safety Risk Assessment, Beijing, China, 2School of Veterinary Medicine
and Science, University of Nottingham, Sutton Bonington Campus, Leicestershire, United Kingdom, 3UCD-Centre for Food
Safety, School of Public Health, Physiotherapy and Sports Science, University College Dublin, Dublin, Ireland
The environmental bacterium Burkholderia gladioli pv. cocovenenans (B. cocovenenans)
has been linked to fatal food poisoning cases in Asia and Africa. Bongkrekic acid (BA),
a mitochondrial toxin produced by B. cocovenenans, is thought to be responsible
for these outbreaks. While there are over 80 species in the Burkholderia genus,
B. cocovenenans is the only pathovar capable of producing BA and causing human
death. However, the genomic features of B. gladioli and the evolution of the BA
biosynthesis gene cluster, bon, in B. cocovenenans remain elusive. In this study,
239 whole genome sequences (WGSs) of B. gladioli, isolated from 12 countries
collected over 100 years, were used to analyze the intra-species genomic diversity
and phylogenetic relationships of B. gladioli and to explore the origin and evolution of
the bon gene cluster. Our results showed that the genome-wide average nucleotide
identity (ANI) values were above 97.29% for pairs of B. gladioli genomes. Thirty-six
of the 239 (15.06%) B. gladioli genomes, isolated from corn, rice, fruits, soil, and
patients from Asia, Europe, North America, and South America, contained the bon gene
cluster and formed three clades within the phylogenetic tree. Pan- and core-genome
analysis suggested that the BA biosynthesis genes were recently acquired. Comparative
genome analysis of the bon gene cluster showed that complex recombination events
contributed to this toxin biosynthesis gene cluster’s evolution and formation. This study
suggests that a better understanding of the genomic diversity and evolution of this
lethal foodborne pathovar will potentially contribute to B. cocovenenans food poisoning
outbreak prevention.
Keywords: Burkholderia gladioli pv. cocovenenans, bongkrekic acid, food-borne poisoning, bongkrekic acid
biosynthesis gene cluster, recombination, virulence, toxin
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Peng et al. Genomic Analysis of B. cocovenenans
INTRODUCTION
Burkholderia gladioli is ubiquitous in soil and plants (Eberl and
Vandamme, 2016). Some pathogenic strains of this species can
be subdivided into four pathovars: B. gladioli pv. agaricicola,
B. gladioli pv. alliicola,B. gladioli pv. gladioli, and B. gladioli
pv. cocovenenans (B. cocovenenans, also called Pseudomonas
cocovenenans in some previous literature) (Lee et al., 2016). In
contrast to the first three plant pathovars, B. cocovenenans can
cause lethal food poisoning by producing a highly unsaturated
tricarboxylic fatty acid, bongkrekic acid (BA), which is a
mitochondrial toxin that can efficiently block the mitochondrial
adenine nucleotide translocator (ANT) (Moebius et al., 2012;
Anwar et al., 2017). BA is also called a respiratory toxin since this
toxin can prevent the respiratory chain phosphorylation (Rohm
et al., 2010). BA causes food poisoning with an acute toxicity
of 1.41 mg/kg (LD50 by intravenous injection on mice) (Fujita
et al., 2018). In epidemiological investigations, B. cocovenenans
and BA were detected simultaneously in coconut- and corn-based
products responsible for food-borne outbreaks in Indonesia,
China, and Mozambique (Anwar et al., 2017;Gudo et al., 2018;
Li J. et al., 2019). A dose–response relationship has been found
between the amount of BA-contaminated food consumed and
illness severity, with reported manifestations of BA poisoning
including abdominal pain, diarrhea, vomiting, weakness, and
palpitations (Gudo et al., 2018).
As an important fatal toxin, BA is an odorless, tasteless, heat-
stable substance, and contaminated food matrices can have a
normal appearance, smell, and taste. Thus, BA can be difficult
to detect during the food preparation and consumption process
(Gong et al., 2016;Anwar et al., 2017). The compound is not
expected to volatilize or hydrolyze in the environment, but may
be susceptible to direct photolysis by sunlight (Voragen et al.,
1982). It is important to note that cooking foods contaminated
with BA does not render them safe for consumption: although
the bacteria are destroyed, the toxin itself is heat-stable (Falconer
et al., 2017). Previous literature indicated that food poisoning
events involving BA were found only in specific regions of
Indonesia and China, as a result of consuming locally produced
fermented foods, but posed a massive health threat and caused
many lethal intoxications (Matsumoto et al., 2015;Falconer
et al., 2017). Outbreaks of BA food poisoning in these two
regions exhibit high mortality rates, with numbers of 40%
and 60% being reported on China and Indonesia, respectively
(Anwar et al., 2017).
Besides BA, B. cocovenenans also produces another toxin,
toxoflavin (TF), an electron carrier that generates hydrogen
peroxide and subsequent toxicity related to free radical formation
(Meng et al., 1988a,b;Falconer et al., 2017;Li X. et al., 2019).
However, TF toxicity is relatively mild and secondary to that of
BA. BA is well recognized to account for the lethal food poisoning
incidents (Lee et al., 2016;Anwar et al., 2017).
The most recent reported lethal food poisoning case caused
nine people’s deaths in Heilongjiang Province of China in
October 2020, after consuming a homemade sour soup made of
fermented corn, with a case fatality rate of 100% (Yuan et al.,
2020). An earlier BA poisoning outbreak case leading to five
people deaths, was recorded in Guangdong Province of China in
2018, and was caused by consuming a commercially produced
rice noodle product which was not fermented or noticeably
spoiled (Li J. et al., 2019). This clinical case report showed
that non-fermented food could also lead to BA food poisoning.
Notably, the first report of BA food poisoning occurred outside
Asia was severe. In January 2015, 75 people died and 177 were
hospitalized after consuming a traditional beverage made from
corn flour in Mozambique, Africa (Gudo et al., 2018). This tragic
event illustrates that B. cocovenenans and its toxic product BA are
of global concern.
The Burkholderia genus has more than 80 species, including
the notorious human pathogens B. mallei and B. pseudomallei
and some opportunistic human pathogens, but B. cocovenenans
is the only human pathovar found to produce BA and linked
to food poisoning deaths (Depoorter et al., 2016;Anwar et al.,
2017). The genetic basis for BA biosynthesis in B. cocovenenans
depends on harboring the bon gene cluster, which encodes a
trans-AT subgroup of modular type I polyketide synthase (Type I
PKS) and accessory enzymes catalyzing complex polyunsaturated
tricarboxylic acid assembly (Moebius et al., 2012). Type I PKS
are typically encoded in close genomic vicinity and organized
in gene clusters. This type of enzyme is defined as multidomain
with a modular synthetic scheme, in which each module typically
has a minimal set of three core domains, namely acyltransferase
(AT), acyl carrier protein (ACP), and ketosynthase (KS), and is
responsible for the incorporation of a single building block of
acyl-CoA (Wang et al., 2015). The evolutionary origins of the
complex PKS biosynthetic pathways remain to be determined.
Previous studies on B. cocovenenans and BA have mostly
focused on clinical case reports (Falconer et al., 2017;Shi et al.,
2019), BA synthesis (Rohm et al., 2010;Moebius et al., 2012;
Matsumoto et al., 2015;Fujita et al., 2018), and apoptosis
inhibition (Matsumoto et al., 2015;Takeda et al., 2016;Kano
et al., 2017, 2019). However, the origin and evolution of BA
biosynthesis gene cluster, bon, in B. cocovenenans remain to
be elucidated. In this study, we performed whole genome
sequencing and determined the evolutionary origins of the bon
gene cluster in pathovar B. cocovenenans Co14, as well as in other
B. gladioli strains containing this gene cluster. We conducted
a genome-wide comparison of B. cocovenenans Co14 against
other B. gladioli genomes to provide deeper insights into the
genomic diversity and the evolutionary origins of the bon gene
cluster in this lethal foodborne pathogen. We showed evidence
for recombination events occurring in the BA biosynthesis gene
cluster evolution in B. cocovenenans.
MATERIALS AND METHODS
Bacterial Strain
In 1977, four people from one family died following the
consumption of contaminated fermented corn flour in Tonghe
County, Heilongjiang Province, China. A B. cocovenenans strain
(named B. cocovenenans Co14) and its toxic product BA were
identified from the remaining food and linked as the etiological
agent in this event. The serotype of B. cocovenenans Co14 was
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Peng et al. Genomic Analysis of B. cocovenenans
identified as O-IV (Meng et al., 1988a). This strain was the
first isolated and identified as a BA-producing pathogen causing
fermented corn flour poisoning in China (Meng et al., 1988b). It
was stored at −80◦C in the Microbiology Laboratory of the China
National Center for Food Safety Risk Assessment.
Genome Sequencing, Assembly and
Annotation
Burkholderia cocovenenans Co14 was subjected to genomic DNA
extraction using the E.Z.N.A. R
Bacterial DNA Kit (Omega
Bio-Tek, Norcross, GA, United States), in accordance with
the manufacturer’s protocol. B. cocovenenans Co14 underwent
genomic sequencing on both a Pacific Biosciences RS II
sequencing platform (Pacific Biosciences, Menlo Park, CA,
United States) and an Illumina Hiseq 2500 PE150 platform
(Illumina, San Diego, CA, United States). Briefly, single-molecule
real-time (SMRT R
) sequencing was conducted using the C4
sequencing chemistry and P6 polymerase with one SMRTR
cell. For Illumina sequencing, the template genomic DNA was
fragmented by sonication to a size of 350 bp using NEBNextR
UltraTM DNA Library Prep Kit for Illumina (NEB, United States)
and sequenced. The PacBio and Illumina sequencing reads
were assembled de novo using a hybrid assembly algorithm
implemented in Allpaths-LG software1(v44620) (Shibata et al.,
2013). The complete genome sequence of B. cocovenenans
Co14 was deposited in GenBank under the accession number
CP033430.1 (chromosome 1), CP033431.1 (chromosome 2) and
CP033429.1 (plasmid pCO1).
In total, 239 un-contaminated B. gladioli genomes and their
genome information were downloaded from the NCBI Genome
Database2and PATRIC 3.6.53. Characteristics of the single
isolates, including collection details, genome assembly statistics,
genomic features, origins etc are detailed in Supplementary
Dataset 1. Prokka v.1.14.0 (Seemann, 2014) was used to annotate
the 239 assembled genomes, including the B. cocovenenans Co14
genome. In addition, 4080 genus Burkholderia genomes and their
information (Supplementary Dataset 2) were also download
from the PATRIC Database.
Average Nucleotide Identity (ANI)
Analysis
Average nucleotide identity values were calculated between
the genomes of B. cocovenenans Co14 and the other 238
B. gladioli strains using FastANI v1.324(Jain et al., 2018). The
pheatmap package in R (v4.0.0) (R Core Team, 2020) was used
to perform hierarchical clustering and visualization5. FastANI
results for each genome pair was transformed to an ANI matrix.
Function pheatmap was applied with clustering distance method
“Euclidean” and the clustering method “complete.”
1http://www.broadinstitute.org/software/allpaths-lg/blog/
2https://www.ncbi.nlm.nih.gov/genome/genomes/3602?
3https://www.patricbrc.org/
4https://github.com/ParBLiSS/FastANI
5https://www.rdocumentation.org/packages/pheatmap/versions/1.0.12/topics/
pheatmap
Core- and Pan-Genome and
Phylogenetic Analyses
The core- and pan-genome of the B. gladioli genomes were
estimated using Roary v3.12.06(Page et al., 2015). A 95%
identity cutoff was used, and core genes were defined as those
contained in 99% of the genomes analyzed (≥236). Roary
provided three figures using the roary_plots.py script7, which
summarized the output: a graph showing how the number of
genes changed in the core/pan genome; a pie chart summarizing
the conservation of genes and the number of genomes where
they were present; a figure showing the presence and absence of
core and accessory genes. A maximum-likelihood phylogenetic
tree was constructed with the single nucleotide polymorphisms
(SNPs) of the core gene alignment using FastTree 2.1 with the
Jukes-Cantor + CAT nucleotide substitution model (Price et al.,
2010). The core SNPs were extracted using snp-sites v2.5.18
(Keane et al., 2016). The phylogenetic tree was visualized with
the gene presence/absence. The B. glumae ASM993137 (accession
number: GCA_009931375.1) sequence was used as an outgroup
for phylogenetic tree rooting.
BA Biosynthetic Gene Cluster bon
Analysis
The 67.5-kbp BA biosynthesis gene cluster bon (GenBank
Accession JX173632) from B. cocovenenans DMSZ11318 was
used as the reference sequence. BLASTn+ (v. 2.9.0) was
used to align the B. cocovenenans Co14, 239 B. gladioli and
4080 Burkholderia spp. genomes individually to the reference
sequence, with a minimum percentage identity of 90%. The open
reading frames (ORFs) of the bon gene cluster in the 36 B. gladioli
genomes were annotated according to the previously reported
BA biosynthesis gene cluster of B. cocovenenans DMSZ11318 and
B. gladioli BSR3 (Moebius et al., 2012). Also, the BLAST tool from
NCBI aligned the ORFs of the bon gene cluster of B. cocovenenans
Co14 and each coding ORF sequence in the Non-redundant
protein sequences (nr) Database to search the most highly related
proteins in non-B.gladioli species. Easyfig9(Sullivan et al., 2011)
was used to create a linear comparison figure depicting the
bon gene clusters of B. gladioli along with homologous flanking
sequences identified from GenBank. antiSMASH 5.010 (Blin et al.,
2019) was also used to identify, annotate, and analyze the bon
gene cluster in the B. cocovenenans Co14 genome using the
“relaxed” detection strictness mode.
Genomic Island (GI) Analysis
Three GI prediction methods, IslandPick, SIGI-HMM, and
IslandPath-DIMOB, integrated by IslandViewer 4 (Bertelli et al.,
2017) were used as guides for the identification of GIs in
the three complete bon gene cluster-containing chromosomes
B. cocovenenans Co14, B. gladioli BSR3, and B. gladioli
6https://github.com/sanger-pathogens/Roary
7https://github.com/sanger-pathogens/Roary/tree/master/contrib/roary_plots
8https://github.com/sanger-pathogens/snp- sites
9http://mjsull.github.io/Easyfig/
10https://antismash.secondarymetabolites.org/
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Peng et al. Genomic Analysis of B. cocovenenans
3723STDY6437373. The location of the bon gene cluster
of B. cocovenenans Co14, B. gladioli BSR3, and B. gladioli
3723STDY6437373 were compared with the predicted GI.
RESULTS
Features of the B. cocovenenans Co14
Genome
The complete genome sequence of B. cocovenenans Co14
consisted of two independent closed circular chromosomes and
one circular plasmid. Chromosome 1 contained 4,171,651 bp
with 3,729 protein-coding ORFs, while chromosome 2 contained
4,002,946 bp with 3,290 protein-coding ORFs. The GC content
of chromosomes 1 and 2 were 67.82% and 68.32%, respectively.
Both showed vague GC skew at their origins of replication. The
size of the plasmid pCO1 was found to be 145,928 bp with a GC
content of 63.25%, encoding 139 ORFs.
Distribution and Genome Characteristics
of B. gladioli
Burkholderia gladioli are widely distributed and commonly found
in different environments (Supplementary Dataset 1). The 239
strains included in this study were isolated throughout the
world, including North America (United States and Canada),
South America (Brazil and Colombia), Asia (China, South Korea,
Indonesia, and India), Europe (United Kingdom and Italy)
and Africa (Zimbabwe). The origin of these strains included
plants, such as corn, rice, gladiolus and onion; human clinical
samples, including cystic fibrosis, sputum, nose-ethmoid sinus
and ethmoid sinus; animal samples including beetle eggs; and
environment samples including water and soil. The most studied
strain B. gladioli ATCC R
10248 was originally isolated from
Gladiolus sp. in the United States in 1913. Seven of the 239
strains had complete whole genome sequences published in the
NCBI Genome Database. All possessed 2 chromosomes and 1–4
plasmids. The sizes of the 239 genomes were between 7.33–
9.31 Mbp (Mean: 8.31 Mbp; Median: 8.32 Mbp) with GC contents
of 67.34%–68.30% (Mean: 68.05%; Median: 68.21%).
ANI Analysis of B. gladioli
To assess similarity among B. gladioli genomes, ANI values were
calculated, and a clustered heatmap was generated. As shown in
Figure 1, all B. gladioli genomes exhibited pairwise ANI values of
97.29%∼100.00% (mean 98.25%), above the 95% suggested cut-
off for species identification (Goris et al., 2007;Jain et al., 2018).
Burkholderia cocovenenans Co14 shared the greatest ANI
(99.42%) with B. gladioli BCC1661 (isolated from a clinical
sample, United States), followed by B. gladioli BCC1686 and
BCC1665 (both isolated from clinical samples, United States)
and B. gladioli UCD-UG_CHAPALOTE (isolated from chapalote
corn, Canada, 2008). In contrast, B. cocovenenans Co14 only
shared 97.42% and 97.47% identity with B. gladioli BCC1828
and B. gladioli BCC1684 (isolated from clinical samples from
Canada and United States, respectively). A previously reported
BA biosynthesis gene cluster-containing strain B. gladioli BSR3,
which was isolated from a rice sheath in South Korea in 2011,
was most similar to B. gladioli 579, a strain isolated from mature
oil palm fruits in Brazil in 2015, with an ANI value of 99.16%.
Core- and Pan-Genome of B. gladioli
Insights into the pan- and core-genome properties of all 239
B. gladioli sequences were obtained using Roary (Figure 2).
A total of 36,950 genes were identified in the pan-genome
of B. gladioli, 4,127 of which were considered to be core
genes (present in ≥99% of strains, Figure 2A). The numbers
of soft-core genes (95% ≤strains <99%), shell genes
(15% ≤strains <95%) and cloud genes (0 ≤strains <15%) were
819, 3,412 and 28,592, respectively (Figure 2B).
A maximum-likelihood phylogenetic tree was constructed
based on the core genome SNPs of B. gladioli isolates (Figure 2C
and Supplementary Figure 1). As shown in Supplementary
Figure 1 and Supplementary Dataset 1,B. gladioli isolated from
human clinical samples, food, plants, animals and environmental
sources were distributed across multiple clusters. B. cocovenenans
Co14 had the closest phylogenetic relationship with B. gladioli
BCC1686, followed by B. gladioli BCC1661, B. gladioli BCC1665,
and B. gladioli UCD-UG_CHAPALOTE, similar to the ANI
results. B. gladioli BSR3 was found to have close phylogenetic
relationships with B. gladioli BCC1735, B. gladioli BCC1780, and
B. gladioli BCC1675.
BA Biosynthesis Gene Cluster Analysis
Based on the results of BLASTn+, a 66,726-bp sequence (located
between positions 2,961,077∼3,027,802 bp in the genome) was
found in chromosome 1 of B. cocovenenans Co14, with an
identity of 99.39% to the BA biosynthesis gene clusters bon
of B. cocovenenans DMSZ11318 (67,546-bp). In addition, this
bon gene cluster of B. cocovenenans DMSZ11318 was also
found in 36 genomes of B. gladioli with an identity range
of 97.93%–99.95%, respectively (Supplementary Dataset 3 and
Supplementary Table 1). Twenty-six of the 36 B. gladioli
genomes (72.2%) originated from human clinical samples from
the United States. In addition, among the 4080 Burkholderia spp.
genomes (Supplementary Dataset 2), the BA biosynthesis gene
cluster bon was found only in the above 36 B. gladioli genomes.
Interestingly, the 36 genomes containing the BA biosynthesis
gene cluster formed three clusters in the ANI heatmap (Figure 1)
and three clades in the core SNPs phylogenetic tree (Figure 2C
and Supplementary Figure 1). The ANI range of the 36 BA
biosynthesis gene cluster-containing B. gladioli genomes was
97.45%∼100.00% with an average value of 98.28%, slightly higher
than the average value of 329 genomes (98.25%). The average
identity of the 36 bon gene clusters was 98.53%, which was
higher than the average genome-wide ANI value (98.28%) of
the 36 bon gene cluster-containing genomes. These 36 genomes
contain the intact BA biosynthesis gene cluster bon (Table 1
and Supplementary Dataset 4). The bon gene cluster-containing
B. cocovenenans DMSZ11318 and B. cocovenenans Co14 were
proved to produce BA by experiment. This suggested that these
36 genomes are likely to produce BA. As such, we theorize
that these strains can produce BA and are able to cause food
poisoning. However, at the present experimental work on these
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FIGURE 1 | Heatmap of the average nucleotide identity (ANI) of Burkholderia gladioli genomes. Pairwise comparison was applied between 239 genomes. The 36
B. gladioli genomes containing the BA biosynthesis gene cluster bon are shown in red color and bold text. B. cocovenenans Co14 forms a cluster with B. gladioli
BCC1661, B. gladioli BCC1686 and B. gladioli BCC1665 genomes with an alignment fraction over 99.41%, while B. gladioli ISTR5 and B. gladioli BCC1829 showed
an identity of 99.02% and 98.95%, respectively.
strains is lacking, and it remains unknown whether they can
produce BA or cause any human foodborne disease.
ORFs of BA Biosynthesis Gene Cluster
bon
As shown in Table 1 and Figure 3, 12 ORFs designated
bonLJKFGABDEHIM in B. cocovenenans Co14 were annotated
as being involved in BA biosynthesis. The ORFs in the bon gene
cluster of B. cocovenenans Co14 that were present or absent in
B. cocovenenans DMSZ11318, as well as the 35 other B. gladioli
genomes, are shown in Table 1 and Supplementary Dataset 4.
Further analysis of the ORFs of the bon gene cluster in the
B. gladioli core- and pan-genomes showed that the 12 genes
bonLJKFGABDEHIM belonged to shell genes of pan-genome.
All the bon gene clusters displayed greater synteny with
B. cocovenenans Co14 than with B. cocovenenans DMSZ1131,
although from PKS assembly lines, only three ORFs (BonABD)
are encoded on B. cocovenenans Co14 while four ORFs
(BonABCD) are on B. cocovenenans DMSZ1131 (Figure 3 and
Supplementary Dataset 4). The differences in the amino acid
sequences found in PKS assembly lines were primarily due to
single nucleotide substitutions that are more prominent in the
PKS part of the bon gene cluster of B. cocovenenans DMSZ11318,
and made a stop codon in bonD. Similarly, a single nucleotide
substitution is present in the bonA of B. gladioli 579 and formed
two ORFs in this gene.
The antiSMASH program facilitates rapid genome-wide
identification, annotation and analysis of secondary metabolite
biosynthesis gene clusters in bacterial and fungal genomes.
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FIGURE 2 | Core- and pan-genome analysis of Burkholderia gladioli based on 239 genomes. (A) Core- and pan-genome profile curves of B. gladioli reporting how
the core- and pan- genes vary as genomes are added in a random order. The solid line and dotted line denote the size of the core- and pan-genome size of
B. gladioli, as well as the relationship between core- and pan-genome size and genome number, respectively. On the yaxes, the number of genes is reported, while
on the xaxes, the number of strains considered is shown. (B) The pie chart illustrates the number of genes belonging to the core, the soft core, the shell, or the
cloud of the B. gladioli species. (C) Core and accessory genes and the core-genome SNPs phylogenetic relationship of B. gladioli. The left tree was constructed
based on the core-genome SNPs of B. gladioli species using a maximum-likelihood method, and the right matrix plot denotes the presence and absence of every
gene over all strains. The 36 strains containing the bongkrekic acid biosynthetic gene cluster bon are formed three clades and shown in pink background color on
the tree. The B. glumae ASM993137 genome was used as an outgroup. The tree scale was shown in the bottom.
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TABLE 1 | Comparison of the bongkrekic acid biosynthesis gene clusters of B. cocovenenans DMSZ11318, B. cocovenenans Co14, B. gladioli BSR3, B. gladioli 3723STDY6437373, B. gladioli MSMB1756, B. gladioli
BCC1650, and B. gladioli BCC1837.
B. cocovenenans DMSZ11318 B. cocovenenans Co14 B. gladioli BSR3 B. gladioli
3723STDY6437373
B. gladioli
MSMB1756
B. gladioli
BCC1650
B. gladioli
BCC1837
Protein Size
(aa)
Predicted function#Accession
number
Size
(aa)
Role in BA
biosynthesis*
Present/
absent (±)
Size
(aa)
Present/
absent (±)
Size
(aa)
Present/
absent (±)
Size
(aa)
Present/
absent (±)
Size
(aa)
Present/
absent (±)
Size
(aa)
Present/
absent (±)
BonL 417 Cytochrome P450
monooxygenase
AFN27475 417 Addition + 410 + 417 + 417 + 417 + 417 +
BonJ 337 Acyl transferase AFN27476 337 Core + 337 + 337 + 337 + 337 + 337 +
BonK 377 Acyl transferase AFN27477 377 Core + 377 + 377 + 377 + 377 + 377 +
BonF 416 KS (involved in
β-branching)
AFN27478 416 Core + 416 + 416 + 416 + 416 + 416 +
BonG 424 3-Hydroxy-3-
methylglutaryl-CoA
synthase
AFN27479 424 Core + 419 + 419 + 419 + 424 + 424 +
BonA 7908 Polyketide synthase AFN27480 8093 Core + 8130 + 8122 + 8121 + 6805 + 8112 +
BonB 3582 Polyketide synthase AFN27481 5303 Core + 5299 + 5297 + 5297 + 5301 + 5297 +
BonC †1705 Polyketide synthase AFN27482 – – – – – – – – – – – – –
BonD 4096 Polyketide synthase AFN27483 3986 Core + 4072 + 4099 + 3602 + 4103 + 3995 +
BonE 450 Enoyl reductase AFN27484 450 Addition + 450 + 450 + 450 + 450 + 450 +
BonHU276 Enoyl-CoA hydratase 272 Addition + 272 + 272 + 272 + 276 + 272 +
BonI 249 Enoyl-CoA hydratase AFN27485 249 Addition + 249 + 249 + 249 + 249 + 249 +
BonM 269 O-methyltransferase AFN27486 269 Addition + 269 + 269 + 269 + 269 + 269 +
#The predicted function referred to the work of Moebius et al. (2012).
*The protein role in BA biosynthesis was predicted by antiSMASH 5.0 (https://antismash.secondarymetabolites.org/ ).
†The polyketide synthase was encoded on four ORFs BonABCD in B. cocovenenans DMSZ11318 since single nucleotide substitutions made a stop codon in bonB, in contrast with that of only three ORFs BonABD in
other bon gene cluster-containing genome.
UBonH of B. cocovenenans DMSZ11318 was missing by annotation error.
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FIGURE 3 | Linear alignment and detailed structure of the 11 typical bongkrekic acid (BA) biosynthetic gene cluster bon and its flanking homologous sequences.
The gene product names are labeled on the top of B. cocovenenans DMSZ11318 bon gene cluster. The red arrows represented the ORFsin the bon gene cluster,
the orange arrows represent the flanking sequences, the blue arrows represent non-bon genes between the flanking homologous sequences, and the green arrows
represent the transposase encoding sequences.
Atrans-AT PKS responsible for BA production was identified
in this region of the B. cocovenenans Co14 chromosome 1. The
genes bonJKFGABD were identified as the core BA biosynthetic
genes, while bonLEHIM were identified as additional biosynthetic
genes (Table 1).
Genetic Recombination Identification
By aligning the two flanking homologous sequences of the
B. cocovenenans Co14 bon gene cluster with other bon
gene cluster-containing or non-containing B. gladioli genomes,
it was found that the down flanking sequence showed
more diversity than that of the upper flanking sequence
(Figure 3 and Supplementary Figure 2). The sequence
between both homologous flanking sequences may represent
a gene recombination hotspot. The two flanking homologous
sequences of the B. cocovenenans Co14 bon gene cluster
were conserved in B. gladioli, but additional sequences were
found to be inserted between them. A 67,680-bp locus
encoding 65 ORFs was inserted downstream of the bon gene
cluster in B. gladioli BSR3. Most of the inserted encoding
genes were of unknown function. A high identity gene
sequence was also found in B. gladioli FDAARGOS_389,
a strain originating from an onion, but the bon gene
cluster was absent upstream of this sequence. However, in
B. gladioli ATCC_10248 and B. gladioli KACC_11889, a 128-
kbp high-identity sequence was inserted between the two
flanking homologous sequences and encoded 35 ORFs. As in
B. gladioli BSR3, most encoding genes in this region were of
unknown function.
Notably, in some genomes of bon-containing B. gladioli
(BCC1650, BCC1675, BCC1678 et al.), adjacent to the bonM
gene, ISNCY family transposase ISBcen27 was found, while in
the genome of B. gladioli BCC1870, it was IS3 family transposase
ISBp1 instead. It showed that this cluster may have been acquired
by horizontal gene transfer (HGT).
Among the 36 BA biosynthesis gene cluster-containing
B. gladioli genomes, the B. cocovenenans Co14, B. gladioli
BSR3, and B. gladioli 3723STDY6437373 genome were
closed. As shown in Supplementary Figure 3, the bon gene
cluster was located within one large GI (located between
positions 2,962,767∼3,025,227 bp) of B. cocovenenans Co14
chromosome 1 which was identified by IslandPick (Langille
et al., 2008). This region included the ORFs bonLJKFGABDE.
Interestingly, the bon gene cluster was located within one
large GI (2,430,789∼2,496,466 bp) in one chromosome of
B. gladioli 3723STDY6437373. This region included the ORFs
bonLJKFGABDEHIM. In contrast, the bon gene cluster was not
located in any GI in B. gladioli BSR3 chromosome 1.
Origin Analysis of the BA Biosynthesis
Gene Cluster
A search using the complete bon gene cluster of B. cocovenenans
Co14 with BLASTn showed that this sequence region had a
high query coverage and identity with that found in the 36 BA
biosynthesis gene cluster containing B. gladioli genomes (Table 2
and Supplementary Table 2). ORFs found in non-B. gladioli
species (BonG, BonE, BonI and BonM) showed higher query
coverage (>90%) and identities (>70%) with proteins from
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Peng et al. Genomic Analysis of B. cocovenenans
Pseudomonas sp. R26(2017), Podila verticillate CPC16_007748
and Trinickia diaoshuihuensis, which showed that these genes
may originate from non-B. gladioli species or inverse (Table 2).
By contrast, BonL, BonJ, BonK, BonF, BonA, BonB, BonD, and
BonH showed lower identities with proteins from other non-
B. gladioli species, which may show that these genes may originate
via an intra-species inheritance pathway.
DISCUSSION
Burkholderia species are incredibly diverse and versatile bacteria
that possess large multi-replicon chromosomes along with
several large plasmids (>100 kbp), which enable these bacteria
survive different environmental conditions (De Felice et al.,
2016;Mullins et al., 2020). The majority of life essential genes
in multiple-chromosome bacteria are usually located on one
primary chromosome, with additional chromosomes containing
fewer essential genes, being mainly composed of niche-specific
genes (Losada et al., 2010). In the case of Burkholderia species,
most genes necessary for the basic life processes were located on
chromosome 1 (Spring-Pearson et al., 2015).
Burkholderia species are also known as prolific producers
of secondary metabolites with potent biological and
pharmacological properties (Ross et al., 2014;Mullins et al.,
2020). BA is an important secondary metabolite produced by
B. cocovenenans, a pathovar belonging to B. gladioli (Ross et al.,
2014). Besides being recognized as a harmful toxin, BA is also
associated with inhibiting apoptosis and can be applied to protect
neuronal death in the cortex (Okazaki et al., 2015). BA plays this
role in coordinated (apoptosis) and uncoordinated (necrotic) cell
death by inhibiting the mitochondrial permeability transition
pore (MPTP) via binding to the ANT in mitochondria (Anwar
et al., 2017). This compound is synthesized by a multi-modular
type I PKS (also called trans-AT PKS) along with accessory
enzymes that function to catalyze assembly the polyunsaturated
tricarboxylic acid (Moebius et al., 2012).
Another intensive studied Burkholderia toxin, TF, revealed
that both regulatory genes and biosynthesis genes were important
for toxin production (Seo et al., 2015;Lee et al., 2016).
Moebius et al. (2012) predicted that two putative regulatory
genes bonR1-R2 were located upstream of the bon genes.
However, the function of these two genes have not been proven
experimentally. In contrast with TF, which were produced by
both B. gladioli and B. glumae, BA biosynthesis gene clusters were
only found in some B. gladioli genomes in this study.
Previous phylogenetic studies indicated that Burkholderia
species constituted two distinct lineages: the larger cluster
included mainly plant growth-promoting bacteria and the other
cluster was dominated by human, animal and plant pathogens
(Baldeweg et al., 2019). In our study, B. gladioli strains
originating from human, plant and environmental sources were
distributed across multiple clusters. Moreover, the bon gene
cluster-containing B. gladioli strains of human, plant, food,
and environmental origins from different countries were found
to have a close phylogenetic relationship. The 36 B. gladioli
strains formed three clades on the phylogenetic tree using core
SNPs. This may be caused by that the toxin gene cluster was
acquired from a common ancestor B. gladioli species, and then
inherited vertically, or that this gene cluster was gained in specific
B. gladioli genomes via HGT. Apart from the gain of this gene
cluster, it may also be possible that the ancestral gene losses of
this cluster during the inheritance resulted in the absence of
the other strains.
Pangenomic diversity in B. gladioli was high and their
pangenomes were “open” as revealed by core- and pan-genome
analysis. The core genes of B. gladioli comprised 11.17% of the
complete set of genes and this proportion would likely reduce
with a greater number of samples. However, though the genomes
of B. gladioli have large diverse pan-genome, our results also
revealed high ANI values shared by B. gladioli, far above the
95% intra-species cutoff value (Jain et al., 2018). Interestingly,
the BA biosynthesis genes of B. gladioli were all defined as
shell genes, which are those genes often present but lacking in
subsets of genomes and also thought to be more recently acquired
(Deschamps et al., 2014;Snipen and Liland, 2015).
Surprisingly, 36 of the 329 published B. gladioli genomes,
isolated from food, plants, soil and patients globally from
1977 to 2014, contained the BA biosynthesis bon gene
cluster, which implicated that the prevalence of this foodborne
TABLE 2 | Closest relative protein in non-Burkholderia gladioli species of the core and additional bongkrekic acid biosynthesis genes.
Product Role in BA biosynthesis Closest relative of non-B. gladioli species (sequence ID) Query cover (%) Identity (%)
BonL Addition Acidobacteria bacterium 13_2_20CM_56_17 (OLB28637.1) 97 46.94
BonJ Core Burkholderia cepacia (WP_105393823.1) 91 54.87
BonK Core Pseudomonas sp. MSSRFD41 (WP_185699837.1) 100 60.16
BonF Core Pseudomonas aestus P308_15900 (ERO59990.1) 97 69.14
BonG Core Pseudomonas sp. R26(2017) (WP_085653298.1) 98 77.43
BonA Core Methylomusa anaerophila (WP_126310258.1) 91 47.65
BonB Core Pseudomonas acidophila BWP39_00030 (PCE28611.1) 85 50.41
BonD Core Chitinophaga pinensis (WP_012792895.1) 98 42.35
BonE Addition Podila verticillate CPC16_007748 (KAF9395587.1) 93 70.85
BonH Addition Candidatus Desulfosporosinus infrequens (WP_106800322.1) 93 58.43
BonI Addition Trinickia diaoshuihuensis (WP_116137735.1) 97 72.84
BonM Addition Trinickia diaoshuihuensis (WP_116137737.1) 99 72.01
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Peng et al. Genomic Analysis of B. cocovenenans
pathogen was wider than that of only in Asia and Africa,
as previously thought. Notably, the United States did not
report any food poisoning caused by bon-containing B. gladioli
strains, though several such strains were isolated. However,
B. cocovenenans-food poisoning outbreaks have only been
reported in China, Indonesia and Africa which may be
attributed to the following: (1) Region-specific food products.
The consumption of some local traditional food, such as tempe
bongkrek in Indonesia, fermented corn products and Tremella
fuciformis mushrooms in China and fermented corn flour-
based beverages in Mozambique, which were contaminated by
B. cocovenenans and BA, caused food poisoning and death.
(2) Sanitary conditions. BA is produced in warm environments
(22∼30◦C) with a neutral pH. The presence of fatty acids
in coconut and corn will promote the production of BA
in B. cocovenenans. High-risk foods are more likely to be
contaminated by B. cocovenenans under unsanitary conditions.
(3) Under-reporting. A lack of confirmatory testing capacity for
detection of B. cocovenenans or BA or a failure to consider the
diagnosis could be contributing to misdiagnosis in other parts
of the world, as B. cocovenenans is ubiquitous in plants and soil
(Anwar et al., 2017).
Recombination plays an important role in the evolution of
niche-specific gene pools thereby facilitating genome flexibly in
the ecological speciation of bacteria. Recombination events can
be classically divided into different types: homologous versus
non-homologous or illegitimate recombination, the latter often
being termed HGT (Mostowy et al., 2017). In B. gladioli,
some transposases were found adjacently to bon clusters, which
suggested that both recombination and HGT events may have
affected to this cluster formation and spread.
Genomic islands are genomic regions that can serve as a
driving force to virulence. They may contain a few or many
genes which can be acquired through recombination from
other bacteria (Patil et al., 2017). In B. pseudomallei, GIs
were a key feature of the genome, accounting for a major
source of genomic diversity, as well as being associated with
pathogenicity in humans (Sim et al., 2008). High rates of
gene transfer and recombination events occurred within GIs,
which were found to be incompatible in retaining gene order
unless these processes were either highly localized to specific
sites within the genome, or characterized by symmetrical gene
gain and loss (Spring-Pearson et al., 2015). In our study, bon
gene clusters in the 36 B. gladioli strains and B. cocovenenans
DMSZ11318 demonstrated a conserved gene organization and
order. Moreover, some flanking sequences of the bon gene cluster
insertion region were homologous, which is suggestive of site-
specific recombination events involved in the formation of the
bon gene cluster.
The acquisition of foreign genes can contribute to bacteria’s
quick adaptation to a new environment (Bachert et al., 2015).
Clustered arrangements of genes are more easily transferred to
other species, thus improving the prospects for survival when
under selection (Ziemert et al., 2014). The bon gene clusters
within the 36 B. gladioli genomes are so similar to each other
indicated that some vertical and horizontal gene transfer events
may emerge during this toxin cluster inheritance and spread.
To date, 239 genomes of this species have been published and,
of those, B. cocovenenans Co14 was the only one which had been
tested to be positive for the production of BA. Genomic analysis
of the BA biosynthesis gene cluster bon of B. cocovenenans
at species-wide level revealed complex recombination and
HGT events contributed to this toxin cluster evolution and
formation. Our data also suggests that further sequencing of
the Burkholderia genome and toxin production testing may lead
to a better understanding of the origin and evolution of this
lethal foodborne pathovar. These findings can then be used to
limit the risk to public health of B. cocovenenans food poisoning
events in the future. Additionally, as an ANT inhibitor and an
ADP/ATP translocator, BA can be used as a pharmacological tool
to modulate the properties of the MPTP or ANT in mitochondria,
as well as a tool in the elucidation of apoptosis mechanisms. In
this respect, a deep understanding for the origin and evolutionary
pathway of BA biosynthesis gene cluster would be beneficial for
mining other potential biological activities of BA and develop this
compound as a drug target candidate.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories
and accession number(s) can be found in the
article/Supplementary Material.
AUTHOR CONTRIBUTIONS
ZP, JX, and FL contributed to the conceptualization, project
administration, and resources. ZP, TD, and YH contributed to
the methodology. TD and YH contributed to the software. ML
and MB contributed to the validation. ZP, SY, TD, YH, ML,
and MB contributed to the formal analysis. ZP contributed to
the investigation. ZP and MB contributed to the data curation.
ZP, SY, and SF contributed to the writing – original and draft
preparation. MB, SF, JX, and FL contributed to the writing –
review and editing. YH contributed to the visualization. JX and
FL contributed to the supervision. ZP, TD, JX, and FL contributed
to the funding acquisition. All authors have made substantial
contributions to and have approved the final manuscript.
FUNDING
This work was financially supported by National Key Research
and Development Program of China (2018YFC1604303); and
Ministry of Science and Technology of P. R. China under
Grant Key Project of International Scientific and Technological
Innovation Cooperation Between Governments (number
2018YFE0101500) and the Innovate UK grant FARMWATCH:
Fighting ABR with a wide repertoire of sensing technologies
(104986); and Chinese Academy of Medical Science (CAMS)
Innovation Fund for Medical Science (CIFMS 2019-12M-5-
024); and China Food Safety Talent Competency Development
Initiative: CFSA 523 Program.
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Peng et al. Genomic Analysis of B. cocovenenans
ACKNOWLEDGMENTS
The authors would like to express their gratitude to Andrew
Warry of digital research service of University of Nottingham for
his helpful recommends.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2021.628538/full#supplementary-material
Supplementary Figure 1 | Maximum-likelihood phylogenetic tree based on
core-genome SNPs of the 239 Burkholderia gladioli genomes originating from
human clinic, plant, environment, food, and animal samples. The sample sources
are shown in solid circles with different colors at the end of each branch. The 36
strains containing the bongkrekic acid biosynthetic gene cluster bon are formed
three clades and shown in pink background color on the tree. The B. glumae
ASM993137 genome was used as an outgroup. The tree scale was shown
on the left top.
Supplementary Figure 2 | Linear alignment and detailed structure of all the 23
complete bongkrekic acid (BA) biosynthetic gene cluster bon and its flanking
homologous sequences. The gene product names are labeled on the top of
B. cocovenenans DMSZ11318 bon gene cluster. The red arrows represented the
ORFsin the bon gene cluster, the orange arrows represent the flanking
sequences, the blue arrows represent non-bon genes between the flanking
homologous sequences, and the green arrows represent the transposase
encoding sequences.
Supplementary Figure 3 | Genomic island of B. cocovenenans Co14 (A),B.
gladioli BSR3 (B) and B. gladioli 3723STDY6437373 (C) identified by IslandViewer
4. The bongkrekic acid biosynthetic gene cluster bon regions of B. cocovenenans
Co14, B. gladioli BSR3 and B. gladioli 3723STDY6437373 are framed by a
blue rectangle.
Supplementary Table 1 | The query coverage and nucleotide identity of the
complete bongkrekic acid biosynthesis gene clusters to that of Burkholderia
gladioli pv. cocovenenans DMSZ11318 in GenBank.
Supplementary Table 2 | The query coverage and nucleotide identity of the
complete bongkrekic acid biosynthesis gene clusters to that of Burkholderia
gladioli pv. cocovenenans Co14 in GenBank.
Supplementary Dataset 1 | Information of Burkholderia gladioli strains and their
genomes.
Supplementary Dataset 2 | Information of Burkholderia spp. strains and their
genomes.
Supplementary Dataset 3 | Information of 36 BA biosynthesis gene clusters bon
containing Burkholderia gladioli strains and their genomes.
Supplementary Dataset 4 | Comparison of the bongkrekic acid biosynthesis
gene clusters of the 36 bon-containing Burkholderia gladioli genomes.
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2021 Peng, Dottorini, Hu, Li, Yan, Fanning, Baker, Xu and Li. This is an
open-access article distributed under the terms of the Creative Commons Attribution
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Frontiers in Microbiology | www.frontiersin.org 12 May 2021 | Volume 12 | Article 628538
