A transferred regulator that contributes to Xanthomonas oryzae pv. oryzicola oxidative stress adaptation and virulence by regulating the ...
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Journal of Integrative Agriculture 2021, 20(0): 2–11
Available online at www.sciencedirect.com
ScienceDirect
RESEARCH ARTICLE
A transferred regulator that contributes to Xanthomonas oryzae pv.
oryzicola oxidative stress adaptation and virulence by regulating
the expression of cytochrome bd oxidase genes
WANG Pei-hong1*, WANG Sai1*, NIE Wen-han1, WU Yan1, Iftikhar AHMAD1, 2, Ayizekeranmu YIMING1,
HUANG Jin1, CHEN Gong-you1, ZHU Bo1
1
Shanghai Yangtze River Delta Eco-Environmental Change and Management Observation and Research Station, Ministry of
Science and Technology, Ministry of Education/Shanghai Urban Forest Ecosystem Research Station, National Forestry and
Grassland Administration, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, P.R.China
2
Department of Environmental Sciences, COMSATS University Islamabad, Vehari-Campus, Vehari 61100, Pakistan
Abstract
Horizontal gene transfer (HGT) has been well documented as a driving force in the evolution of bacteria. It has been
shown that a horizontally acquired gene, xoc_2868, involved in the global response against oxidative stress and
pathogenicity of Xanthomonas oryzae pv. oryzicola strain BLS256. However, as a transcriptional factor (TF), the
regulatory mechanism of XOC_2868 has not yet been revealed. Here, evolutionary analysis suggested XOC_2868
might be co-transferred with its physically proximate downstream genes from a Burkholderiaceae ancestor. Interestingly,
RNA-seq data of wild-type (BLS256) and Δxoc_2868 strains under oxidative stress showed that XOC_2868 did not
regulate the expression of its adjacent genes, but remarkably influenced the expression of several genes involved in
the extracellular polysaccharide (EPS) production and xanthan biosynthesis. Chromatin immunoprecipitation-sequence
(ChIP-seq) combined with transcriptome analysis revealed that XOC_2868 directly regulates a cydAB operon, encoding
two subunits of cytochrome bd oxidase and involved in redox balance. Consistent with Δxoc_2868 strain, cydA and
cydAB knockout mutants also showed a higher sensitivity to H2O2 along with a reduced bacterial virulence compared with
the wild-type strain. Overall, our findings raise the possibility of regulatory circuit evolution shaped by HGT and driven
by selection and reveal a novel regulatory pathway that regulates the expression of cytochrome bd oxidase and thus
contributes to the virulence of BLS256.
Keywords: HGT, transcriptional factor, Xanthomonas oryzae pv. oryzicola, oxidative stress adaptation and virulence
Received 9 June, 2021 Accepted 7 July, 2021 1. Introduction
WANG Pei-hong, E-mail: wangpeihong@sjtu.edu.cn;
Correspondence ZHU Bo, Tel: +86-21-34205873, E-mail:
bzhu1981@sjtu.edu.cn Horizontal gene transfer (HGT), the acquisition of
*
These authors contributed equally to this study. genetic materials across species boundaries, is a
© 2021 CAAS. Published by Elsevier B.V. This is an open prevalent phenomenon across the biosphere. Numerous
access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
examples showed that HGT can serve as a major driver
doi: 10.1016/S2095-3119(21)63801-7 of the evolution in bacteria to provide new capabilitiesWANG Pei-hong et al. Journal of Integrative Agriculture 2021, 20(0): 2–11 3
and traits in various ecological niches (Ochman et al. therefore, it is interesting to unveil the proper response
2000; Bacciu et al. 2004; Goyal 2019). HGT is also the of the foreign XOC_2868 under oxidative stress. In this
main mechanism for spreading of genes associated study, we address the role of XOC_2868 in adaptation
with virulence and antibiotic resistance, contributing to evolution of Xoc via underlying its regulatory interaction
pathogenic bacteria to successfully colonize hosts and with its TGs, including figuring out its binding sites and
express virulence related genes (Lindsay 2010; Liu et al. functional analysis of the target genes.
2014). As the causative agent of bacterial leaf streak,
Xanthomonas oryzae pv. Oryzicola (Xoc) would be 2. Materials and methods
exposed to a variety of harmful conditions in the host
environment and immune system during the invasion of 2.1. Phylogenetic analysis
rice. In the study of the molecular mechanisms of Xoc
responding to oxidative stress during invasion, over 30% For homologs searching of interested genes, their
of the coding genes in the Xoc representative strain nucleotide sequences and protein sequences were
BLS256 genome were identified as hypothetical genes compared against GenBank nonredundant nucleotide
(Fang et al. 2019). Among them, xoc_2868 gene was database and reference protein database, by using the
proven to participate in the oxidative stress response and BLASTN and BLASTP with the E value cutoff of 10–4. The
pathogenesis (Fang et al. 2019). Interestingly, xoc_2868 real homologue genes were selected based on the query
was a horizontally acquired gene encoding a transcription coverage and identity values over 80%. The multiple
factor and located adjacent to many genes encoding sequences were aligned online with MAFFT (http://mafft.
transposase, highlighting the importance of considering cbrc.jp/alignment/server/). Maximum likelihood were
its contribution to the oxidative stress adaption and the generated in the IQTree online tool (http://iqtree.cibiv.
evolution of bacterial regulatory networks. univie.ac.at/?user=guest&jobid=191204085022), and all
From an evolutionary point of view, HGT optimize phylogenetic trees were visualized with iTOL (http://itol.
existing abilities via shaping bacterial genomes. embl.de/itol.cgi). We performed 1000 bootstraps to gain
Specifically, the adaptation to a dynamic environment branch support values.
demands proper regulation of gene expression in bacteria
(Ishii et al. 2007; Perez and Groisman 2009; Price et al. 2.2. Bacterial strains, culture conditions, plasmids
2008), which is realized by transcription factors (TFs) and primers
that sense the cellular environments and bind to specific
DNA sequences or motifs (Price et al. 2008; Ali and The bacterial strains and plasmids used in this study are
Seshasayee 2020). Therefore, to unveil its regulatory listed in Appendix A. Primers are listed in Appendix B.
potential, it is important to locate TFs binding sites or Escherichia coli strains were cultivated at 37˚C on Luria-
motifs where TFs interact with DNA. Bertani (LB) agar plates or LB medium. Xanthomonas
Due to the rich genome sequence data and the wide oryzae pv. oryzicola (Xoc) strains were cultured at 28°C
application of computational analyses, the regulatory on nutrient agar (NA) plates, NA without sucrose (NAN)
features of horizontally acquired DNA has attracted an plates, NA with 10% sucrose (NAS) plates and in nutrient
increasing attention (Perez and Groisman 2009; Ali and broth (NB) medium. When required, kanamycin (Km)
Seshasayee 2020; Gelfand 2006; Lozada-Chávez et al. was added in growth media with the final concentration
2006; Madan Babu and Teichmann 2003). In the previous of 25 μg mL–1. DH5ɑ and BL21 (DE3) in this work were
literature, it has been revealed that most TFs (including used for plasmid construction and XOC_2868 purification,
putative regulators) regulate adjacent operons in E. coli respectively.
(Price et al. 2008; Hershberg et al. 2005). The proximity
of these regulators to their target genes were interpreted 2.3. Construction of in-frame deletion mutants
as co-transfer through HTG mechanism and maintained
by repeated transfers (Price et al. 2008). However, there Homologous double-crossover recombination was
is also evidence that TFs do not usually co-evolve with used to construct in-frame deletion mutations in this
their target genes in bacteria, because TFs evolve much study. In brief, two fragments flanking the left and right
faster than the target genes (TGs) (Lozada-Chávez et al. of target genes were amplified, sequenced, and ligated
2006; Babu et al. 2004; Madan Babu et al. 2006) and into the pKMS1 (Guo et al. 2012) suicide vectors at
show less conservation than their regulated genes (Ali BamHI and SalI sites. These recombinant plasmids
and Seshasayee 2020). were electroporated into BLS256 and screened on NAN
The genes are functionless without expression, plates supplemented with kanamycin to obtain single4 WANG Pei-hong et al. Journal of Integrative Agriculture 2021, 20(0): 2–11 homologous crossover colonies. NAS plates were with TMM normalization method was used to determine then used for selecting the second double-crossover the DEGs (differentially expressed genes). Significantly recombinants. Sucrose resistant and kanamycin-sensitive differentially expressed genes (FDR value
WANG Pei-hong et al. Journal of Integrative Agriculture 2021, 20(0): 2–11 5
R, listed in Appendix B). The DNA probe was labelled Bioscreen C (Labsystem, Helsinki, Finland) at 420–580
using EMSA Probe Biotin Labeling Kit (Beyotime). The nm, at regular intervals of 15 min for 40 h with continuous
protein-binding reactions were performed according to the shaking at 28°C.
manufacture’s instruction (Chemiluminescent EMSA Kit,
Beyotime). Finally, the signals were detected using X-ray 2.10. Pathogenicity assays
film in a dark room.
Oryza sativa L. cultivar ‘Yuanfengzao’ plants, susceptible
2.8. Quantitative real-time PCR (RT-qPCR) to Xoc, were grown in our field stations (Shanghai
Jiaotong University) for one month. To prepare bacterial
The bacteria samples used for RT-qPCR were the same inoculum, Xanthomonas cells were grown overnight in
as those used for RNA-seq. Total RNA was purified NB broth, harvested at the exponential phase of growth
using EasyPure RNA Kit (Transgen Biotech, Beijing, by centrifugation, and resuspended in sterile water to
China). cDNA was synthesized from 1 μg total RNA reach OD600 of 0.8 after washing twice. The pathogenicity
with TransScript II One-Step gDNA Removal and cDNA of Xoc strains was examined by using leaf piercing and
Synthesis SuperMix (Transgen Biotech, Beijing, China). evaluating water-soaked symptoms based on lesion
RT-qPCR assay was conducted with ABI 7500 quantitative length measurement at 14 DAI. Ten fully expanded
PCR system (Applied Biosystems, Foster City, CA) using leaves were inoculated for each independent experiment.
transStart Tip Green qPCR SuperMix (TransGen Biotech,
Beijing, China). The gene-specific primers are listed in 3. Results
Appendix B and the relative expression of target genes
were normalized to gyrB using the 2−ΔΔCt method. 3.1. Physically proximate genes of xoc_2868 were
also transferred from a Burkholderiaceae ancestor
2.9. Bacterial growth analysis under H2O2 stress
As shown in Fig. 1-A, the other two hypothetical genes,
In this assay, all strains were grown overnight to an xoc_2866 and xoc_2867, present between xoc_2868
OD600 of 1.0 in fresh NB medium, diluted to OD600 ~0.1 and many transposase encoding genes. Notably, a
the next morning and then inoculated with a fresh NB comprehensive BLAST sequence analysis showed that
medium without or with the addition of H2O2 0.075 mmol both of them were only present in Xoc and absent in
L–1. Bacterial growth was automatically determined on other Xanthomonas. Based on the phylogenetic analysis
A Genetic structure
0.1 kb
xoc_2068 xoc_2067 xoc_2066 xoc_2065 xoc_2064 xoc_2063 xoc_2061 xoc_2062 xoc_2060 xoc_2059
Hypothetical proteins Transposases
L8
B 100
BXOR1
C 0-9
100
GX01
0-9
GX01 BXOR1
85
B8-12 L8
Xanthomonas oryzae pv. oryzicola
RS105
94.6 BLS279
BLS279 Xanthomonas oryzae pv. oryzicola
YM15 RS105
75.9
CFBP2286 B8-12
BLS256
90.6 YM15
92.4 Burkholderia lata 89.3
Burkholderia cepacia CFBP2286
97.1
86.9
Burkholderia seminalis BLS256
98.5
96.6 Burkholderia pseudomultivorans Burkholderia
84 83.8
Ralstonia
Burkholderia pseudomallei Cupriavidus
77.2 33.5 56.1
37.7 Bradyrhizobium sp. Bradyrhizobium
Cupriavidus sp. 81.9
Ralstonia
49.3
Rhizobium leguminosarum
Anabaena
unclassified Burkholderia
Tree scale: 0.1 Burkholderia anthina Tree scale: 0.1 Merismopedia
Fig. 1 Evolution of the physically proximate genes of XOC_2868. Genes are represented by boxes, black boxes corresponding to
genes coding for unknown proteins and grey boxes indicating genes coding for transposase. A, genetic structure of the co-linearly
arranged genes adjacent to XOC_2868. B and C, phylogeny of the xoc_2867 and xoc_2866. The phylogenetic tree shown was
calculated using the maximum likelihood (ML) program in the IQTree online tool and visualized with iTOL. Only the ML values
≥50% were shown. Bar=0.1 substitution per site.6 WANG Pei-hong et al. Journal of Integrative Agriculture 2021, 20(0): 2–11
with high bootstrapping support (Fig. 1-B and C), it can (PTS fructose transporter subunit IIBC), xoc_1974
be further inferred that xoc_2866 and xoc_2867 in Xoc (1-phosphofructokinase), mtp (xoc_1975) and xoc_1976
might transfer from a Burkholderiaceae ancestor over the (LacI family transcriptional regulator). In addition, their
course of evolutionary history, which is consistent with the expression profile was examined through RT-qPCR with
analysis of xoc_2868 in the previous study (Fang et al. the specific primers (Appendix B). Consistent with the
2019). RNA-seq results, all of them were indeed significantly
downregulated (Fig. 2-A).
3.2. Transcriptome analysis of Δxoc_2868 strain
and wild-type strain under oxidative stress 3.3. Genome-wide transcriptional and binding pro-
files of XOC_2868
To explore the regulatory interaction between XOC_2868
and the possible TGs, we next carried out RNA-seq To identify genes directly regulated by the XOC_2868,
analysis of Δxoc_2868 strain and wild-type strain we performed ChIP-seq experiment using a recombinant
(BLS256) under the treatment of H2O 2, each with two strain, a C-terminal His6 tagged xoc_2868 in BLS256.
samples. The comparative analysis indicated that The ChIP-seq results were shown in Appendix
xoc_2866 and xoc_2867 were not directly or indirectly D, including the DNA binding sites of XOC_2868
regulated by XOC_2868 under oxidative stress (data throughout the genome, loci and enrichment fold. The
shown in Appendix C). In addition, five genes associated integrated analysis of RNA-seq data and ChIP-seq
with fructose and glucose metabolism were down- data was conducted to identify direct regulatory targets
regulated at least 4-fold in mutant strain (Appendix C), of XOC_2868. As shown in the Fig. 2B, a total of 229
These genes included oprB (xoc_1972), xoc_1973 and 479 genes were differentially regulated or specially
A 1.5 WT C
2
Δxoc_2868
Bits
(in NB +0.1 mMH2O2)
Relative expression
1
1.0
0
1
2
3
4
5
6
7
8
0.5
cydAB … T T CG G G T T CG T CAT CG CG T T CCA C…
0.0
D 1 2 3
oc 76
rB
)
73
7)
tp
74
26
42
m
op
19
19
19
dB _14
Labeled cydAB_BS + + +
_1
c_
c_
c_
oc
xo
xo
xo
(x
(x
dA
6His-XOC_2868 – + +
cy
cy
B
RNA-seq DEGs ChIP-seq TGs
Protein-DNA complex
213 16 463
(30.8%) (2.3%) (66.9%)
Free probe
Fig. 2 Integrated analysis and validation of the results of RNA-seq and ChIP-seq. A, verification of RNA-seq results by RT-qPCR.
Wild type BLS256 and the mutant were grown in nutrient broth to an OD600 of 1.0, then treated with 0.1 mmol L-1 H2O2 for 15 min,
and were subjected to RNA isolation. Fold change values are shown and normalized to gyrB. B, integrated analysis of differentially
expressed genes (DEGs) in RNA-seq and potential target genes (TGs) in ChIP-seq. C, potential XOC_2868 binding motifs were
identified by MEME from the ChIP-seq peak regions and present within protein-coding sequences of xoc_1426. D, EMSA experiments
were conducted in the presence of purified 6His-XOC_2868 and the corresponding probe listed in Appendix C. Labeled cydAB_BS,
free probe labeled with biotin; lane 1, probe only; lanes 2 and 3, probe with purified 6His-XOC_2868.WANG Pei-hong et al. Journal of Integrative Agriculture 2021, 20(0): 2–11 7
bound by XOC_2868 from the results of RNA-seq and (UTR) of oprB (Appendix E) and within the coding
ChIP-seq, respectively. In addition, 16 potential TGs of sequences of cydAB (Fig. 2-C). EMSA was thus only
XOC_2868 shared by them and three of those genes carried out to verify whether XOC_2868 binds to cydAB.
are annotated genes (shown in Appendix E). Of note, Band shifts were observed with using the purified 6His-
no significant enrichment of DNA binding sites was found XOC_2868 and the probes containing the potential
for all the above down-regulated genes except for oprB. binding motif (Fig. 2-D). Taken together, the above results
The oprB mentioned earlier encodes an outer membrane revealed that XOC_2868 directly controls the expression
protein B, which functions as a carbohydrate-selective of xoc_1426 and xoc_1427.
porin in Xoo (Bae et al. 2018), Xcc (Ficarra et al. 2017)
and Pseudomonas species (Wylie and Worobec 1995; 3.4. Roles of XOC_2868 regulated genes under oxi-
Chevalier et al. 2017). The other two genes are cydAB dative stress and in the bacterial virulence
and mutM, which also have been demonstrated to be
essential for bacterial cellular survival (Endley et al. 2001; The mutant strains ΔcydA (with the deletion of xoc_1426)
Landova and Silhan 2020). The mutM gene encodes a and ΔcydAB (with the deletion of xoc_1426 and
bacterial DNA glycosylase that is important in repairing xoc_1427) were generated via double crossover using the
the oxidized DNA (Landova and Silhan 2020). suicide vector pKMS1 to evaluate the contributions of the
The cydAB operon encodes two subunits (CydA and XOC_2868 regulated genes under oxidative stress. The
CydB) of cytochrome bd ubiquinol oxidase that is one kind growth curves of WT and three mutant strains (Δxoc_2868,
of the terminal oxidases and is widespread in bacteria ΔcydA and ΔcydAB) under oxidative stresses were
and involved in redox balance (Degli Esposti et al. 2015; measured in four repeats and representative curves
Safarian et al. 2016). RT-qPCR was firstly utilized out to were shown in Fig. 3. Obviously, all the strains showed
examine the regulatory role of XOC_2868 for xoc_1426 similar growth patterns in the absence of H2O2 in nutrient
and xoc_1427 during infection. Compared with the wild broth medium. When cultures were added with 0.075
type strain BLS256, obvious decreased expression of mmol L–1 H2O2, these mutant strains displayed different
xoc_1426 and xoc_1427 were observed in the Δxoc_2868 lags compared to the wild type, curves corresponding to
mutant strain under H 2O 2 treatment (Fig. 2-A), which the minimum lag time of 2 h (ΔcydA), 6 h (Δxoc_2868),
was consistent with the RNA-seq data and confirmed the and 18 h (ΔcydAB). This indicated that the lack of cydA
reliability of sequencing results again. or cydAB also increased the sensitivity of BLS256 to
The MEME analysis on the ChIP-seq data revealed H2O2. It led us speculated that they might also play a
the potential XOC_2868 binding motif (5’-TTCGACAT-3’, role in pathogenesis of BLS256. Not surprisingly, these
Fig. 2-C), which was within the 3′ untranslated region three knockout mutants in this study showed decreased
NB NB + 0.075 mM H2O2
1.0 0.8 WT
Δxoc_2868
Absorbance (420–580 nm)
Absorbance (420–580 nm)
0.8 ΔcydA
0.6 ΔcydAB
0.6
0.4
0.4
0.2
0.2
0 0
0 6 12 18 24 30 36 0 6 12 18 24 30 36
Time (hours) Time (hours)
Fig. 3 Comparison of the H2O2 resistance of wild type (Xoc) and knockout mutant strains in nutrient broth (NB) medium. All strains
were grown overnight to an OD600 of 1.0 in fresh NB medium, diluted to OD600 ~0.1 the next morning and then inoculated with a
fresh NB medium without or with the addition of 0.075 mmol L-1 H2O2. Growth was monitored at 420-580 nm during 0-40 h using a
Bioscreen C (Growth Curves) maintained at 28°C with continuous shaking. The data shown in the figure are the mean OD values
(quadruplicate). Shaded areas show the standard error of the means.8 WANG Pei-hong et al. Journal of Integrative Agriculture 2021, 20(0): 2–11
pathogenicity when compared with the wild-type strain response to the host environment stimuli (André et al.
(Fig. 4). 2005; Kim et al. 2016). For example, The Xanthomonas
citri ssp. citri(Xcc)lacking OprB and Xanthomonas
4. Discussion oryzae pv. oryzae(Xoo)lacking OprB are both also highly
susceptible to H2O2 (Bae et al. 2018; Ficarra et al. 2017).
In light of the above-mentioned fact that most bacterial Further, the OprB family is also known to be involved
regulators are adjacent to operons that they regulate, we in the biosynthesis of the extracellular polysaccharide
combined a comprehensive BLAST sequence analysis (EPS) xanthan (Vojnov et al. 2001; Slater et al. 2000),
and the phylogenetic analysis to characterize genes probably by modulating the rate of glucose transport,
xoc_2866 and xoc_2867 that lie downstream of xoc_2868. which has knock-on effects on biofilm formation and
Based on their physically-linked character and the same virulence within Xoo (Bae et al. 2018) and Xcc (Ficarra
evolutionary source, we hypothesized that the three et al. 2017). Previous studies have shown that BLS256
genes were likely acquired as an unit and XOC_2868 lacking XOC_2868 showed enhanced sensitivity to
might be a neighbor regulator. However, RNA-seq H 2O 2 (Fang et al. 2019), which is also verified in this
analysis suggested that xoc_2866 and xoc_2867 were not study. Accordingly, we hypothesized that oprB in
the regulatory genes of xoc_2868 under H2O2 treatment. BLS256 is also involved in the tolerance to H 2O 2. In
One possible explanation is that these regulatory proteins addition, the XOC_2868 mutant also showed obviously
can act at distant sites, and the close proximity of TFs and reduced virulence. Thus, it can be postulated that the
their TGs is not essential for the regulatory mechanism downregulation of these genes involved in fructose and
(Lawrence and Roth 1996). However, an alternative glucose metabolism caused by the loss of XOC_2868
explanation is that bacterial TFs have a tendency to in a xoc_2868 mutant, which, in return, influenced
evolve faster than their regulated genes (Lozada- xanthan production and impaired H 2O 2 tolerance and
Chávez et al. 2006; Babu et al. 2004; Madan Babu et al. pathogenicity, although this needs to be investigated
2006). Thus, we reasoned that xoc_2868, xoc_2866 and further. At this point, it was clear that XOC_2868 was
xoc_2867 might diverge in Xoc after the co-transfer event indeed functional in cellular survival. Collectively, these
via HGT, enabling it to adapt some specific niches. The findings indicate that XOC_2868 suggest the existence
absence of xoc_2868, xoc_2866 and xoc_2867 in its of novel and potential binding sites for XOC_2868 after
closely related strains, Xoo, can also be a circumstantial transferring under oxidative stress.
evidence to support our inference. The genes directly regulated by the XOC_2868 were
Notably, the regulatory genes involved in fructose and identified by the ChIP-seq assay and their interaction
glucose metabolism have previously been reported in were substantiated by EMSA. In addition, we examined
68
A B
28
B
56
dA
dA
c_
S2
xo
cy
cy
BL
***
Δ
Δ
Δ
5 ***
***
4
Lesion Length (cm)
3
2
1
0
dA
56
68
B
dA
S2
28
cy
cy
Δ
c_
BL
Δ
xo
Δ
Fig. 4 Pathogenicity assays for BLS256, Δxoc_2868, ΔcydA and ΔcydAB. A, water-soaked symptoms of inoculated leaves
(Yuanfengzao, one-month-old) at 14 days after inoculation (DAI). B, box blots display the respective distributions of lesion lengths
on the adult inoculated with the Xoc strains. Statistical analysis was done by a GraphPad Prism 8.0, significant values are marked
with asterisks * PWANG Pei-hong et al. Journal of Integrative Agriculture 2021, 20(0): 2–11 9
the roles of the XOC_2868 regulated genes, cydAB, in the virulence was observed for a mutant strain defective in
oxidative stress response and pathogenicity. In this study, expressing cytochrome bd (Shi et al. 2005). The mutation
the deletion of cydA or cydAB increased the sensitivity of cytochrome bd in Brucella abortus also resulted in a
of BLS256 to H2O2 along with decreased pathogenicity. attenuated bacterial virulence in a murine infection model
Consistently, mutants defective in cytochrome bd also (Endley et al. 2001). All these evidences strongly indicate
display a hypersensitivity to H 2O 2 in Escherichia coli that bd-type oxidases play a critical role in facilitating
(Lindqvist et al. 2000), Azotobacter vinelandii (Edwards bacterial survival during successful pathogen infection
et al. 2000) and Porphyromonas gingivalis (Leclerc and pathogenesis.
et al. 2015). Morover, disruption of Mycobacterium In conclusion, our result confirmed that although the
tuberculosis cytochrome c maturation (CCM) would high correlation between co-transfer and regulatory
induce the overexpression of cytochrome bd and the relationships between TFs and their TGs was detected
hyper-resistance to H2O2 (Small et al. 2013). Along the in the previous study (Price et al. 2008), however, it is
same line, exposure to external H2O2 could increase the not a 100% reliable indicator to predict the function of
expression of genes encoding cytochrome bd in E. coli an acquired or uncharacterized regulator. Based on our
(Lindqvist et al. 2000). Those observations point to a results, we have summarized the acquisition, evolution
crucial role for cytochrome bd in protecting bacteria cells and function of XOC_2868 in a model (Fig. 5). As shown
from oxidative stress. This possibility may be due to the in Fig. 5-A, XOC_2868 as well as the two physically-
extraordinarily high oxygen affinity of CydAB (Borisov linked genes were likely transferred as a unit, however,
et al. 2011). This oxidase is employed for reducing evolved separately and maintained by selection to adapt
molecular oxygen or dioxygen to water (Safarian et al. the oxidative stress and other possible competitive
2016) and thus for respiratory protection (Poole and Hill microenvironments. During infection, the production of
1997) and even the colonization of O2-limited niches by reactive oxygen species (ROS) activates the transcription
pathogenic bacteria (Endley et al. 2001; Baughn and of stress response genes in BLS256. XOC_2868 binds
Malamy 2004; Shi et al. 2005). This hypersensitivity of to cydAB operon to protect cells from oxidative stress
mutants may be due to the inability to remove superoxide via removing H2O2 and other ROS and thus contribute to
anion and H2O2 in the periplasm (Borisov et al. 2010). bacterial virulence. The oprB and Fru-PTS related genes
As outlined above, several lines of evidence point have been shown to be associated with EPS production
to the positive correlation between the cytochrome bd and xanthan biosynthesis (Bae et al. 2018; Ficarra
expression and virulence in bacteria. For example, M. et al. 2017), which are crucial for the biofilm formation
tuberculosis cytochrome bd was observed to be up- of the Xanthomonadaceae family (Katzen et al. 1998).
regulated in vivo during the transition from acute to The indirect activation of the expression of oprB and
chronic infection of mouse lungs, inversely, reduced Fru-PTS related genes thus assist in colonization and
A B
Host environment
Selection pressure
(oxidative stress, ROS
carbonsource, etc.)
BLS256
Horizontal gene transfer xoc_2868 Operon (xoc_2867/2866)
Stress response and
transcription activation
Gene duplication
Gum
XOC_2868
Time (+)
cydAB Gum production
(Respiratory chain) (+)
Fr
HO H O+O Fructose
u-
2 2 2 2
PT
op
S
rB
xoc_2868 Operon (xoc_2867/2866) Glucose
cydAB
Fig. 5 Evolutionary hypothesis for the early diversification and functional mechanism of XOC_2868. A, hypothetical model of
acquisition and evolution of XOC_2868. B, functional mechanism of XOC_2868 mediating the survival determinants under oxidative
stress. Dotted lines indicate indirect activation. More details of the model were discussed in the text.10 WANG Pei-hong et al. Journal of Integrative Agriculture 2021, 20(0): 2–11
dissemination within the host. in transcription factors and its implications for the evolution
of regulatory networks in bacteria. Nucleic Acids Research,
48, 4100–4114.
Acknowledgements
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This work was supported by National Key R&D Program
Structural Biology, 16, 420–429.
of China (2018YFD0201202 and 2017YFD0201108),
Lozada-Chávez I, Janga S C, Collado-Vides J. 2006. Bacterial
the Agri-X Interdisciplinary Fund of Shanghai Jiao Tong
regulatory networks are extremely flexible in evolution.
University (Agri-X2017010), the Shanghai Committee of
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organization is shaped by the transcription regulatory
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