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Received: 2 April 2018 Revised: 7 May 2018 Accepted: 19 May 2018 DOI: 10.1002/mbo3.674
ORIGINAL ARTICLE
CpxR negatively regulates the production of xenocoumacin 1, a dihydroisocoumarin derivative produced by Xenorhabdus nematophila Shujing Zhang1 | Xiangling Fang2,3 | Qian Tang1 | Jing Ge1 | Yonghong Wang1
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1
Xing Zhang
1 Research and Development Center of Biorational Pesticides, Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, Northwest A &F University, Yangling, Shaanxi, China 2
State Key Laboratory of Grassland Agroecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, China
3
School of Agriculture and Environment, Faculty of Science, The University of Western Australia, Crawley, Western Australia, Australia
Abstract Xenocoumacin 1 (Xcn1), a major antimicrobial compound produced by Xenorhabdus nematophila, has great potential for use in agricultural productions. In this study, we evaluated the effects of CpxR, a global response regulator associated with the mutualism and pathogenesis of X. nematophila, on the antimicrobial activity and Xcn1 production. The mutation of cpxR could promote the production of Xcn1 significantly with its level in ΔcpxR mutant being 3.07 times higher than that in the wild type. Additionally, the expression levels of xcnA-L genes, which are responsible for the production of Xcn1, were increased in ΔcpxR mutant while the expression levels of xcnMN, which are required for the conversion of Xcn1 into Xcn2 was reduced.
Correspondence Yonghong Wang, Research and Development Center of Biorational Pesticides, Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, Northwest A&F University, Yangling, Shaanxi, China. Email:
[email protected]
Noticeably, Xcn2 was also enhanced on account of the conversion of excessive Xcn1
Funding information National Natural Science Foundation of China, Grant/Award Number: 31171910; Natural Science Foundation of Shaanxi Province, Grant/Award Number: 2014JZ004
that CpxR negatively regulates xcnA-L genes expression while positively regulating
in spite of low expression levels of xcnM and xcnN in ΔcpxR mutant. The transcriptional levels of ompR and lrp, encoding the global response regulators OmpR and Lrp which negatively and positively regulate the production of Xcn1 were concurrently decreased and increased, respectively. Correspondingly, ΔcpxR mutant also exhibited increased antimicrobial activities in vitro and in vivo. Together, these findings suggest xcnMN expression in X. nematophila YL001, which led to a high yield of Xcn1 in ΔcpxR mutant. KEYWORDS
antimicrobial activity, biosynthesis regulation, CpxR, X. nematophila, xenocoumacin 1
1 | I NTRO D U C TI O N
genomic genes encode the proteins involving in secondary metabolism. The majority of these encoded molecules, however, are cryptic
Xenorhabdus nematophila, a mutualistic symbiont of the soil-dwelling
(Bisch et al., 2016; Chaston et al., 2011). Until now, X. nematophila
nematode Steinernema carpocapsae, is a potent producer of natural
has been known to produce several secondary metabolites with an-
bioactive compounds. Whole-genome sequencing programs have
timicrobial activity, including xenocoumacins (Xcns) (Huang et al.,
revealed that X. nematophila had great biosynthetic potential in
2005; Lang, Kalvelage, Peters, Wiese, & Imhoff, 2008; Yang et al.,
secondary metabolites. In X. nematophila ATCC 19061 7.5% of the
2011; Zhou, Yang, Qiu, & Zeng, 2017), indole derivatives (Li, Chen,
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. MicrobiologyOpen. 2018;e674. https://doi.org/10.1002/mbo3.674
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& Webster, 1997; Li, Chen, Webster, & Czyzewska, 1995; Sundar &
EnvZ/OmpR, are involved in responding to the nematode and
Chang, 1993), peptides (Boszormenyi et al., 2009; Fuchs, Proschak,
other insect environments, which regulate the mutualistic and
Jaskolla, Karas, & Bode, 2011; Gualtieri, Aumelas, & Thaler, 2009),
pathogenic interactions of X. nematophila with its host (Herbert &
benzylineacetone (Ji et al., 2004), and nematophin (Li et al., 1997).
Goodrich-B lair, 2009; Herbert et al., 2007; Park & Forst, 2006). As
These metabolites not only have diverse chemical structures but also
a sensor histidine kinase, CpxA and EnvZ can sense diverse signals
a wide range of bioactivities of medicinal and agricultural interests.
including the changes in pH and osmolarity. Upon recognition of
Xenocoumacins, including Xcn1 and Xcn2, are the major antimi-
these signals, CpxA and EnvZ autophosphorylate and then donate
crobial compounds produced by X. nematophila. Xcn1 exhibits a broad
their phosphoryl groups to CpxR and OmpR, respectively. After
antimicrobial activity against Gram-positive bacteria and a strong
being phosphorylated, the cognate response regulator can bind to
antifungal activity (Alternaria alternata, Botrytis cinerea, Rhizoctonia
a specific promoter sequence of a target gene and subsequently
solani, and Phytophthora species) (Huang, Yang, & Yang, 2006; Huang
regulate its expression (Jubelin et al., 2005). The increased os-
et al., 2005; Zhou et al., 2017). Xcn2, however, shows substantially
motic stress has been found to generally stimulate metabolite pro-
reduced bioactivities (Mcinerney, Taylor, Lacey, Akhurst, & Gregson,
duction of X. nematophila (Crawford, Kontnik, & Clardy, 2010). As
1991; Yang et al., 2011; Zhou et al., 2017). It has been proved that 14
EnvZ senses high osmolarity of hemolymph, EnvZ phosphorylates
genes (xcnA-xcnN) involved in Xcn synthesis in X. nematophila. The
OmpR to activate this response regulator (Forst & Boylan, 2002).
genes (xcnA-L) are responsible for Xcn1 synthesis and the conversion
It was shown that OmpR repressed flagella synthesis, exoenzyme,
of Xcn1 into Xcn2 is controlled by xcnM and xcnN that encode pro-
and antibiotic production of X. nematophila by negatively regulat-
teins homologous to saccharopine dehydrogenases and fatty acid
ing the flhDC operon (Park & Forst, 2006). Moreover, OmpR also
desaturases, respectively (Park et al., 2009). When Xcn2 production
negatively regulates the expression of xcnA-L and positively regu-
is attenuated, an increase in Xcn1 is observed, along with a 20-fold
lates the expression of xcnMN in X. nematophila (Park et al., 2009).
reduction in cell viability, suggesting that the conversion of Xcn1
As a response regulator of great importance, CpxR is also in-
into Xcn2 is a resistance mechanism utilized by the bacteria to avoid
volved in regulating the mutualism and pathogenesis of X. nematoph-
self-toxicity (Park et al., 2009). Although Xcn1 has great potential for
ila. It can positively influence the motility, secreted lipase activity,
using as a new biopesticide in agricultural productions, its low yield
and transcription of lrhA necessary for the virulence as well as the
in X. nematophila wild strains is a substantial limitation for its prac-
expression of nil genes responsible for mutualistic colonization of
tical applications.
nematodes (Herbert & Goodrich-Blair, 2009). In addition, CpxR nega-
X. nematophila can adapt to the changing environmental condi-
tively influences the production of antibiotic activities, protease, and
tions to modulate the pathogenic and mutualistic behaviors to its
secreted hemolysin. The ΔcpxR1 mutant strain exhibited increased
host, which is closely associated with the production of bioactive
antimicrobial activity against B. subtilis, while the antimicrobial activ-
substances (Herbert, Cowles, & Goodrich-B lair, 2007; Herbert &
ity of complementary strain ΔcpxR1 (Tn7/cpxRA) was restored to the
Goodrich-B lair, 2007). Many previous researches have confirmed
comparative level of the wild type (Herbert et al., 2007). However, to
that the antimicrobial activity of X. nematophila varies according to
our best knowledge, there is no report about the regulatory effects
the fermentation conditions (Cowles, Cowles, Richards, Martens,
of CpxR on the Xcn1 production and the antimicrobial activity of
& Goodrich- B lair, 2007; Furusawa et al., 2008; Goodrich- B lair,
X. nematophila against phytopathogens. Therefore, in this study, we
2007; Wang, Fang, An, Wang, & Zhang, 2011; Wang, Fang, Li, &
constructed a ΔcpxR mutant of X. nematophila YL001 to determine
Zhang, 2010; Wang, Li, Zhang, & Zhang, 2008). But it still remains
the influences of CpxR on the Xcn biosynthesis and the antifungal
unclear how X. nematophila recognizes these changes or how these
activities of X. nematophila against Botrytis cirerea, in vivo and in
signals are associated with the antimicrobial activity. Lrp (leucine-
vitro.
responsive protein), a global regulator of transcription, serves as a sensor of intracellular metabolic status and thus generally associates with the response to nutrient availability (Brinkman, Ettema, De Vos, & Van Der Oost, 2003; Hart & Blumenthal, 2011). In X. nematophila, as a global regulator as well, Lrp can regulate
2 | E X PE R I M E NTA L PRO C E D U R E S 2.1 | Bacterial strains and growth conditions
the pathogenic and mutualistic interactions, especially affect
X. nematophila YL001 was isolated from its nematode symbiont,
the production of secondary metabolites (Cowles et al., 2007;
Steinernema sp. YL001 obtained from the soil from Yangling, China,
Goodrich- B lair, 2007; Hussa, Casanovatorres, & Goodrichblair,
and had been identified according to its morphological and molecu-
2015). Lrp is predominantly a positive regulator of secondary me-
lar characteristics.
tabolite production. The mutation of lrp could significantly reduce
Details of the strains and plasmids used in this study were pro-
the production of antibiotics, including xenortides, xenematides,
vided in Table 1. X. nematophila strain was grown in TSB medium
and Xcn1. Correspondingly, its mutant exhibited no antimicrobial
(tryptic soy broth) at 28°C. Escherichia coli was grown at 37°C either
activities against Micrococcus luteus and Bacillus subtilis (Cowles
in Luria-Bertani medium (LB: 1.0% Bacto tryptone, 0.5% yeast ex-
et al., 2007; Engel, Windhorst, Lu, Goodrichblair, & Bode, 2017;
tract, and 1% NaCl) by shaking at 180 rpm or on corresponding solid
Goodrich-B lair, 2007). Both two-component systems, CpxRA and
agar media (1.5% agar) as needed.
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TA B L E 1 Bacterial strains and plasmids used in this studya
Relevant genotype, phenotype, or characteristic (s)
Source
YL001
Wild-t ype, phase I variant; Ampr
Laboratory stock
ΔcpxR
YL001ΔcpxR::Kmr
This study
DH5α (λpir)
General cloning strain
TAKARA
S17-1(λpir)
recA, thi, pro, hsdR-M + . RP4-2Tc::Mu Km::Tn7 in the chromosome; donor strain for conjugations
Laboratory stock
Suicide vector; Cmr, Suc s, oriR6K, mobRP4
Laboratory stock
Plasmid pDM4 carrying 1,105-bp cpxR 5′ upstream region, 973-bp kanamycin- resistant cassette, and 1,192-bp cpxR 3′ downstream region
This study
Cloning vector; Ampr
TAKARA
pMD19T carrying 1,105-bp cpxR 5′ upstream region, 973-bp kanamycin-resistant cassette, and 1,192-bp cpxR 3′ downstream region
This study
Source of Kmr gene
Laboratory stock
Strains X. nematophila
E. coli
Plasmids pDM4 pDM4cpxRKm
r
pMD19T pMD19TcpxRKm
r
pJCV53 a
r
r
r
Amp , Ampicillin resistance; Km , Kanamycin resistance; Cm , Chloramphenicol resistance.
Antibiotics were used when needed at the following concen-
into a pMD19T via the SphI and SacI restriction sites to create a pM-
trations: ampicillin (150 μg/ml for X. nematophila and 50 μg/ml
D19TcpxRKmr. The fused fragment of upstream and downstream
for E. coli), chloramphenicol (25 μg/ml for both X. nematophila and
of cpxR and Kmr was PCR screened from pMD19TcpxRKmr and
E. coli), and kanamycin (50 μg/ml for both X. nematophila and E. coli).
cloned into the suicide vector pDM4 using SacI and SphI sites, creating pDM4cpxRKmr. The resulted plasmids were transformed into
2.2 | DNA manipulation DNA and plasmid isolation, restriction digests, PCR, ligation reac-
E. coli S17-λpir and conjugally transferred into the wild-t ype strain of X. nematophila YL001. The mutant strain was identified based on the described method (Park et al., 2009).
tions, and gel electrophoresis were conducted according to the standard protocols of molecular Cloning. PCR amplification was conducted using Ex Taq (Takara Otsu, Shiga, Japan) according to the
2.4 | Measurement of the growth rate
manufacturer’s directions. Prime STAR® Max was used as the DNA
YL001 and ΔcpxR cells were grown overnight in TSB medium at
Polymerase (Takara Otsu, Japan). PCR-amplified fragments were
28°C by shaking at 180 rpm. Then, the cells were resuspended in
recovered from agarose gels using the Mini-Best DNA Fragment
TSB medium with an initial OD600 value of 0.01, respectively. Under
Purification Kit (Takara Otsu, Japan). Primers for PCR amplifica-
the identical conditions, the growth rates were monitored every
tion were designed using Primer Premier 5.0 software (Table S1).
6 hr during the 72-hr period using a UV-3310 spectrophotometer
Recombinant constructs were verified by DNA sequencing.
(Hitachi, Japan). Before testing, each sample solution was fully dispersed using a moderate ultrasonic amplitude (50%) for 5 min
2.3 | Construction of cpxR mutant strain
to avoid the probable cell aggregation. Each experiment was performed in triplicate.
Fragments, carrying upstream (1105-bp) and downstream (1192-bp) of cpxR, were amplified with primer pairs cpxR-up-F/cpxR-up-R and cpxR-down-F/cpxR-down-R , which contained engineered restriction enzyme sites, from YL001 chromosomal DNA. The PCR fragment containing kanamycin- resistant cassette (973- bp) was amplified
2.5 | Cell-free filtrate and methanol extract preparation Nine percent (v/v) of the seed culture was used as the inoculum.
using compatible restriction sites with primer pairs Km-F/Km-R from
Cultures of the wild type and ΔcpxR mutant of X. nematophila
pJCV53. Fused PCR was performed in an additional amplification
YL001 were conducted in 250 ml flask containing 100 ml TSB
step via complementary DNA regions and its product was cloned
medium (Wang et al., 2010, 2011). Each flask was incubated on a
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TA B L E 2 The relative amount of xenocoumacins (Xcn1 and Xcn2) in the wild type and the ΔcpxR mutant Relative amount of Xcnsa (Arbitrary units OD −1) Strain
Xcn1
Wild type
1
ΔcpxR
3.07 ± 0.52*
Xcn2 1 10.05 ± 1.97*
a The peak area of extracted ion chromatogram (EIC) of Xcn in ΔcpxR mutant/the peak area of extracted ion chromatogram (EIC) of Xcn in the wild type, the peak area was calibrated by its OD600 value, and the relative amount of Xcn of the wild type was referred to 1. Data are presented as the average ± SD for three replicates. An asterisk indicates a significant difference in Xcn level between the wild type and the ΔcpxR mutant (p
