- Open Access
Regulation of antimicrobial activity and xenocoumacins biosynthesis by pH in Xenorhabdus nematophila
- Shuqi Guo†1, 5,
- Shujing Zhang†1,
- Xiangling Fang3, 4,
- Qi Liu1,
- Jiangtao Gao1,
- Muhammad Bilal5,
- Yonghong Wang1, 2Email author and
- Xing Zhang1, 2
© The Author(s) 2017
Received: 24 May 2017
Accepted: 8 November 2017
Published: 15 November 2017
Xenocoumacin 1 (Xcn1) and Xenocoumacin 2 (Xcn2) are the main antimicrobial compounds produced by Xenorhabdus nematophila. Culture conditions, including pH, had remarkably distinct effects on the antimicrobial activity of X. nematophila. However, the regulatory mechanism of pH on the antimicrobial activity and antibiotic production of this bacterium is still lacking.
With the increase of initial pH, the antimicrobial activity of X. nematophila YL001 was improved. The levels of Xcn1 and nematophin at pH 8.5 were significantly (P < 0.05) higher than that at pH 5.5 and 7.0. In addition, the expression of xcnA-L, which are responsible for the production of Xcn1 was increased and the expression of xcnMN, which are required for the conversion of Xcn1 to Xcn2 was reduced at pH 8.5. Also, the expression of ompR and cpxR were decreased at pH 8.5.
The alkaline pH environment was found to be beneficial for the production of Xcn1 and nematophin, which in turn led to high antimicrobial activity of X. nematophila at pH 8.5.
Xenorhabdus nematophila is a unique genus of Gram-negative bacteria, belonging to the family Enterobacteriaceae that is mutually associated with an infective dauer juvenile (IJ) insect-pathogenic nematode in the genus Steinernema (Steinernematidae) . The primary (I) phase of the bacteria is carried in the intestine of the infective dauer juvenile (IJ) developmental stage of the nematode. The IJ penetrates an insect host and releases the bacteria into the haemocoel. The bacteria rapidly produce various metabolites that can overcome the insect immune system , kill the insect, and inhibit the growth of various fungal and bacterial competitors [3–5]. The bacterial symbionts are believed to prevent putrefaction of the insect cadaver and establish conditions that favor the development of both the nematode and bacterial symbionts .
Currently, bio-pesticides based on living microbes and their bioactive compounds have been considered as an important component of environmentally-friendly pest management, with the aim to cope with the adverse effects of chemical pesticides on environment and human health. X. nematophila has been known to produce several compounds with antimicrobial activity, including indole derivatives, nematophin, benzylideneacetone, xenocoumacins and the major class of non-ribosomally produced secondary metabolites (Xenematides depsipeptides, lysine-rich cyclo PAX-peptides) [7–17]. These metabolites not only have diverse chemical structures, but also a wide range of bioactivities of medicinal and agricultural interest, such as antibiotic, antimycotic, insecticidal, nematicidal, antiulcer, antineoplastic and antiviral. These natural antibiotics provide useful clues in the research and development of drugs and agrochemicals.
Xenocoumacins (Xcn), including Xcn1 and Xcn2, are the antimicrobial compounds produced by X. nematophila. Xcn1 exhibits a broad-spectrum antibiotic activity against Gram-positive and Gram-negative bacteria and several fungal species, while Xcn2 is less active against the bacteria and inactive towards the fungal species examined . As Xcn1 is also active against the producer itself, it is detoxified and converted by its own resistance mechanism into the weak antibiotic Xcn2. Xcn1 and Xcn2 are composed of an arginine residue, a leucine residue and four acetate units creating a benzopyran-1-one (isocoumarin) ring structure . The 34-kb biosynthetic gene cluster associated with the production of Xcn contains 14 genes (xcnA–xcnN), which includes two nonribosomal peptide synthetases (NRPs), three polyketide synthetases (PKSs), and nine accessory genes [18, 19]. The PKSs and NRPs genes xcnAKFHL are necessary for Xcn1 synthesis, and the accessory genes xcnBCDE are specifically involved in the biosynthesis of the extender unit hydroxymalonyl-ACP [18, 19]. The conversion of Xcn1 into Xcn2 is due to the genes xcnM and xcnN, which encode proteins homologous to saccharopine dehydrogenases and fatty acid desaturases, respectively . When Xcn2 production is attenuated, an increase in Xcn1 is observed, along with a 20-fold reduction in cell viability. The conversion of Xcn1 to Xcn2 is a resistance mechanism utilized by the bacteria to avoid self-toxicity .
Whole-genome sequencing programs highlight the great biosynthetic potential of X. nematophila for secondary metabolites, and a large number of biosynthesis gene clusters responsible for the production of secondary metabolites were revealed. Overall, 7.5% of the X. nematophila genome contains genes that encode enzymes involved in the synthesis of secondary metabolites, compared with 4.5% for Streptomyces coelicolor [20, 21]. The number of the known secondary metabolites was far less than the number of genes encoding biosynthetic enzymes in various bacteria including X. nematophila . Many biosynthesis genes remain silent and such cryptic pathways are tightly regulated, and are only activated under specific conditions and only a minority of potential chemical structures is produced under standard laboratory culture conditions. To gain insight to this untapped reservoir of potentially bioactive structures, the strategies to trigger biosynthetic pathways to yield cryptic natural products were developed, these included external cues, co-cultivation and genomic approaches such as genome-mining, epigenetic remodeling, and engineered pathway activation [22, 23]. For example, l-proline, an abundant amino acid in insect hemolymph, contributes to the bacterial proton motive force, up-regulates the production of secondary metabolites and reveals previously cryptic metabolites in X. nematophila. By a culture medium designed to mimic the amino acid content of wax moth larval circulatory fluid (a Galleria mellonella “hemolymph mimetic medium”, HMM), amicoumacin was found in X. bovienii .
Culture conditions are critical to the secondary metabolites production of microorganisms. Manipulating culture variables can promote biosynthesis of the secondary metabolite and thus facilitate the discovery of novel natural products . Therefore, it is a prerequisite to design proper culture conditions in an efficient fermentation process. In previous studies, it was found that pH had remarkably effects on the antibiotic activity of X. nematophila [25–28]. However, until now, there is no report about the regulatory mechanism of pH on antimicrobial activity and antibiotic production of this bacterium. Thus, it would be interesting to investigate whether the different initial pH will lead to a significantly change in antimicrobial activity and antibiotic production. The objective of this work was to evaluate the difference in antimicrobial activity, antibiotic production and the expression of related gene of antibiotic production of X. nematophila under different pH. The antimicrobial activity of the cell-free filtrate, ethyl acetate extract and methanol extract of X. nematophila culture under different pH were determined, the metabolomics profiling and the antibiotics production were measured. Also, the expression of xcn gene, which is necessary for the synthesis of the major antimicrobial compounds (Xcns) produced by X. nematophila, isn gene associated with the production of rhabduscin and the response regulator gene, ompR and cpxR were analyzed. Based on the correlation analysis of the results, the regulation mechanism of pH on antimicrobial activity and antibiotic production were determined.
Symbiotic bacterial strains and culture conditions
Xenorhabdus nematophila YL001 was isolated from its nematode symbiont, Steinernema sp. YL001 obtained from the soil from Yangling, China , and had been identified according to its morphological and molecular characteristics [29, 30]. The entomopathogenic nematode symbiotic bacteria including X. nematophila have two phenotypic (previous name: phase) variants, called primary (I) and secondary (II). Only primary (I) exhibits considerable antimicrobial activity . X. nematophila YL001 was maintained on nutrient agar (NA) medium and sub-cultured monthly. The primary-secondary switch is spontaneous and in case of X. nematophila irreversible. Consequently, the laboratory cultures should be scored after recovering from the 20% glycerol suspension in which they had been stored in the deep freezer. To ensure the predominance of phase I population, refrozen cells were seeded on the surface of NA media supplemented with 0.04% triphenyltetrazolium chloride (w/v) and 0.0025% bromothymol blue (w/v) (NBTA). Phase I is distinguished from II by its adsorption of NBTA to produce a red colony overlaid by dark blue and surrounded by a clear zone after 3–5 days of incubation, and fresh liquid culture were started from single blue (II) colonies . These cultures were scaled up for antibiotics production. Seed culture was prepared by inoculating a loopful of I forms of X. nematophila YL001 from a 72 h culture growing on an NBTA plate to a 250-mL flask containing 50 mL fresh NB (NA without agar) medium. The medium was adjusted to a final pH of 7.2 followed by cultivation in darkness at 28 °C on an Erlenmeyer rotary shaker at 180 rpm for 16–24 h, during which time the optical density (600 nm) was approximately 1.50 and 2.00.
The effect of initial pH on the antibiotic activity of the strain was studied using shake flask cultures at different initial pH values. A 250 mL Erlenmeyer flask contained 50 mL medium (WYH)  consisting of the following components (g/L): glucose 6.13, peptone 21.29, MgSO4·7H2O 1.50, (NH4)2SO4 2.46, KH2PO4 0.86, K2HPO4 1.11, and Na2SO4 1.72. The medium pH was adjusted to 5.5, 6.5, 7.0, 7.5 and 8.5 by adding 1 M NaOH or 1 M HCl. Ten percent (v/v) of the seed culture was used to inoculate the flasks. The culture was incubated on a rotary shaker at 28 °C and 150 rpm for 72 h. Three batches were repeated for each experiment.
Preparation of cell-free filtrate, ethyl acetate extract bioactive compounds (ethyl acetate extract) and methanol extract bioactive compounds (methanol extract)
Batch cultures were carried out in 250-mL Erlenmeyer flasks containing 50 mL medium with varying initial pH (5.5, 6.5, 7.0, 7.5 and 8.5). Ten percent (v/v) of the seed culture was used to inoculate the flasks. The culture was incubated on a rotary shaker at 28 °C and 150 rpm. After 72 h incubation, the cultures were centrifuged (12,000×g, 20 min, 4 °C) to separate the bacterial cells and the cell filtrate was stored at 4 °C until used. The cultures were vigorously extracted with equal volume of ethyl acetate three times, mixed, centrifuged, and the top organic layer was dried for analysis.
Liquid cultures were grown at 150 rpm on a shaker in 250 mL Erlenmeyer flasks containing 50 mL medium and 2% (v/v) of XAD-16 (Sigma-Aldrich). These cultures were inoculated with 10% (v/v) of a 24 h preculture in the same medium without XAD-16. Cultures were harvested after 72 h, and XAD beads were separated from the cells and supernatant by sieving. XAD beads were extracted with MeOH (25 mL), and the MeOH extract was concentrated to dryness on a rotary evaporator. The residue was re-dissolved in MeOH (1.5 mL) for HPLC/MS analysis.
Assay of antimicrobial activity
The antibacterial activity of cell-free filtrate of X. nematophila YL001 culture against six bacteria, including Bacillus subtilis, Bacillus thuringiensis, Bacillus cereus, Pseudomonas syringae pv. actinidiae, Xanthomonas campestris pv. Oryzae and Erwinia carotovora subsp. carotovora were determined using an agar diffusion plate assay . Briefly, 1 mL of medium containing 107–108 colonies of the tested bacterium was applied to an NA plate. After 2 h incubation at room temperature, the supernatants were filtered through a 0.22 μm-pore-size syringe microfilter. The filtrates (60 μL) were placed on 6-mm disc filters (Whatman 3-mm paper; Whatman, Clifton, NJ) and air dried. The dried discs were put on the NA plate and incubated at 28 °C for 48 h to determine the relationship between the size of the zones of inhibition of bacterial growth and the concentration of the antibiotic. Antibacterial activity was expressed as units of activity per mL of the supernatants, where 1 U was defined as a 1.0-mm annular clearing around the antibiotic disk. Wang et al. confirmed the assumption that the changes in the size of the zones of inhibition represented changes in antibiotic concentration [33, 34].
The antimicrobial activity of cell-free filtrates of X. nematophila YL001 against different plant pathogens at varying pH values
Fungi and oomycetes
Inhibition rate (%)1
26.86 ± 4.51d
34.69 ± 4.27c
61.78 ± 3.52b
61.36 ± 4.06b
99.28 ± 0.05a
35.61 ± 5.77d
54.84 ± 0.58c
60.71 ± 0.60c
70.28 ± 0.17b
97.84 ± 0.17a
74.47 ± 0.12b
100.00 ± 0.00a
100.00 ± 0.00a
100.00 ± 0.00a
100.00 ± 0.00a
23.26 ± 3.05d
73.88 ± 3.05b
76.92 ± 3.01ab
81.22 ± 3.02a
53.67 ± 3.01c
Curvularia lunata (Wakker) Boed
41.94 ± 4.01b
70.47 ± 5.01a
69.06 ± 0.29a
68.14 ± 2.01a
71.02 ± 3.56a
9.77 ± 3.45c
63.51 ± 3.11b
58.91 ± 2.09b
64.08 ± 4.05b
87.64 ± 4.32a
15.96 ± 2.89b
11.82 ± 1.74b
12.84 ± 2.32b
11.63 ± 2.33b
10.84 ± 2.66b
18.43 ± 4.02cd
25.31 ± 5.20bc
20.15 ± 3.01cd
30.71 ± 4.09ab
37.35 ± 3.01a
70.93 ± 0.05b
100.00 ± 1.28a
100.00 ± 0.71a
100.0 ± 1.00a
100.00 ± 0.79a
5.82 ± 0.09c
36.83 ± 0.04b
100.00 ± 1.36a
96.14 ± 0.29a
100.00 ± 1.82a
13.50 ± 0.90e
39.24 ± 1.24d
72.95 ± 3.00c
85.34 ± 0.34b
91.34 ± 1.00a
0.00 ± 0.00d
35.52 ± 5.01b
39.55 ± 3.02b
30.52 ± 2.68c
98.58 ± 0.35a
30.08 ± 4.04d
45.21 ± 5.10c
45.79 ± 4.01c
57.47 ± 4.05b
69.16 ± 4.01a
To determine the effect of the ethyl acetate extract and methanol extract on the mycelial growth of B. cinerea, a stock solution of the ethyl acetate extract and methanol extract (25 mg/mL) of the cell-free filtrate of X. nematophila YL001 culture was prepared by dissolving the dried ethyl acetate extract and methanol extract in distilled water. Twofold dilutions were made from the filter-sterilized stock solution. For each dilution, 1 mL was thoroughly mixed with 9 mL PDA and poured into a Petri dish plate. The final concentrations of the ethyl acetate extract and methanol extract in PDA were 0.039, 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 mg/mL, respectively. Then one mycelial disk (6-mm-diameter) from the edge of 3 to 5 day-old-colony of B. cinerea growing on PDA was put onto the center of each plate. For each concentration, there were three replicates (one plate per replicate), and the control plates for comparison were PDA only. The plates were maintained at 25 °C in darkness. The colony diameter of each plate was measured and the inhibitory rate for each concentration on each pathogen was determined after 7 days.
In vivo efficiency of cell-free filtrate, ethyl acetate extract and methanol extract on B. cinerea were determined on tomato fruits. Fresh green tomato fruits with uniform size were used. To determine the therapeutic effect, three tomato fruits with the same size were placed at the bottom of each closed plastic container with moisture filter papers at the bottom to maintain high humidity. One mycelia agar disc (4 mm diameter) was taken from the edge of 3- to 5-day colony of B. cinerea on PDA and was placed in the middle side of each fruit with mycelia side facing the surface of each fruit. The containers were placed in a climate chamber at 25 °C. After 24 h, the fruits were sprayed immediately with cell-free filtrate and 2.5 mg/mL of ethyl acetate extract. There were three replications (eight fruits per replication) for each treatment. The control for comparisons was sprayed with water, and the 1000 × 80% carbendazim (Bianjing Plant Protection Technology Co., Ltd, suzhou, China) was used as a reference positive chemical control. To determine the protective effect, three tomato fruits were sprayed with each solution as above and kept under the same conditions as above. After 24 h, the fruits were inoculated with B. cinerea as described above. After 7 days, lesion diameter was measured in two perpendicular directions. The efficiency rate was determined according to the formula: [(average colony diameter of control − average colony diameter of treatment)/average colony diameter of control] × 100.
Metabolite analysis of X. nematophila YL001 at different pH
The metabolomic profiling of the ethyl acetate extracts from X. nematophila YL001 under different initial pH was analyzed by LC–MS. A certain amount dried ethyl acetate extract were re-suspended in 8 mL methanol. LC–MS/MS was performed on a Thermo ion trap LC–MS system (Thermo Scientific, USA) consisting of a surveyor auto sampler, a surveyor MS pump, and an LTQ XLTM linear ion trap mass spectrometer equipped with an ESI source that was operated in the positive mode. The data acquisition software used was Xcalibur 2.1. HPLC separation was carried out using an Agilent ZORBAX Eclipse XDB C18 column (250 mm × 4.6 mm, 3.5 µm particle sizes) with an acetonitrile/water gradient at 0.4 mL/min (gradient: 0–2 min, 10% MeCN; 2–20 min, 10–40% MeCN; 20–35 min; 40–100% MeCN, injection volume: 10 μL). The LC–MS/MS conditions were as follows: ESI spray voltage, 4 kV; sheath gas flow rate, 70 arb; auxiliary gas flow rate, 20 arb; capillary voltage, − 38 V; capillary temperature, 350 °C; and tube lens, 95 V. Collision-induced dissociation-MS/MS experiments were performed on the precursor ions selected from MS1 using the selected ion monitoring (SIM) mode: MS1 was performed in the full scan mode (m/z 100–1000); MS2 was performed in the SIM mode. The metabolites in ethyl acetate extract at different pH were identified based on LC–MS/MS. Relative amount of metabolites = [the peak area of extracted ion chromatograms (EIC) of metabolites at different pH/the peak area of extracted ion chromatograms (EIC) of metabolites at pH 8.5] × 100. The relative amount of metabolites at pH 8.5 refers to 100.
The analysis of the production of Xcns was performed by a liquid chromatography–tandem mass spectrometry (LC–MS/MS, API 2000, AB Sciex, USA). A certain amount dried methanol extract was re-suspended in 8.0 mL methanol for analysis. For the determination of Xcn1 and Xcn2, the molecular ions m/z [M+H]+ 466.3 and 407.3 were quantified, respectively, using the API2000 LC–MS system equipped with an ESI source that was operated in the positive mode. HPLC separation was carried out using an Agilent C18 column (150 mm × 4.6 mm, 5 µm) with an acetonitrile/water gradient (+ 0.1% formic acid) at 0.4 mL/min (gradient: 0–22 min, 5–95% MeCN; 22–24 min, 95–5% MeCN; 24–30 min, 5% MeCN; 40–45 min, 100–10% MeCN; 45–55 min, 10% MeCN, injection volume: 10 μL). The LC–MS/MS conditions: auxiliary gas flow rate, 20 arb; sheath gas flow rate, 70 arb; capillary voltage, − 38 V; ESI spray voltage, 4 kV; and tube lens, 95 V; capillary temperature, 350 °C. The full scan mode (m/z 100–1000) was applied for MS1. MS/MS experiments were performed on the precursor ions selected from MS1 using the selected ion monitoring (SIM) mode. The relative amount of Xcns was calculated by the following equation: the relative amount of Xcns = [the peak area of extracted ion chromatograms (EIC) of Xcns at different pH/the peak area of extracted ion chromatograms (EIC) of Xcns at pH 8.5] × 100, the relative amount of Xcns at pH 8.5 was referred to 100. All analyses were performed in triplicate.
RT-PCR and qRT-PCR analysis
Standard conditions for isolating total RNA from cells grown in WYH medium were as follows: X. nematophila YL001 strains were initially grown overnight in LB, Ten percent (v/v) of the seed culture was used to inoculated into WYH media with different initial pH in a 250 mL flask and incubated to desired cell density. For RT-PCR analysis, exponentially growing cells were used to extract total RNA use total RNA isolation system (Promega). RT-PCR was performed using RevertAiD First Strand cDNA Synthesis Kit (Thermo). The RT-PCR reaction (25 μL) contained the following components: 300 ng of total RNA, 20 pmol each of forward and reverse primer and 200 units of Revert Aid M-MuLV Reverse Transcriptas. The cDNA synthesis was conducted at 42 °C for 60 min. The following cycle condition was used for PCR reaction: 30 s at 94 °C, 30 s at 55 °C and 60 s at 72 °C for extension. Annealing temperature varied depending on which primer was used. For RT-PCR analysis, 26–30 cycles of PCR reaction was used for all xcn genes. 16S rDNA was used as the internal control gene to confirm that equal amounts of total RNA was used in each reaction. Quantitative RT-PCR was performed using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa) and SYBR® Premix ExTaq™ II (TliRNaseH plus) (TaKaRa) and method using Thermal Cycler Dice Real Time System. Equal amounts of DNase-treated total RNA (300 ng) were used to generate cDNA. The resultant cDNA was diluted ten times with sterilized distilled water. The diluted cDNA (2.0 μL) was subsequently used in 25 μL qRT-PCR reaction, which was carried out in triplicate on cDNA Iq™5. Sequences of primers used in RT-PCR and qRT-PCR are shown (Additional file 1: Table S1). Cycle threshold (Ct) results and melting curve for each sample were generated by Opticon MonitorTM software (Version 1.0). The fold changes in the amount of xcnA, xcnM and xcnN transcripts (target genes) relative to the recA transcripts (control gene) were determined by the following equation: fold change = 2−Δ(ΔCt); ΔCt = Cttarget − Ctcontrol; and Δ(ΔCt) = ΔCttreatment1 − ΔCttreatment2; final mean values of Δ(ΔCt) and fold changes were obtained from three independent RNA samples.
Data analyses were conducted using the SPSS statistical package (version 18.0 for windows; SPSS Inc., Chicago, IL) and Origin Pro 9.1. Analyses of variance were carried out to determine the effects of different treatments at different initial pH. Subsequent multiple range comparisons between means were determined based on least significant differences (l.s.d) at P < 0.05.
In vitro antimicrobial activity of the cell-free filtrate, ethyl acetate extract and methanol extract
The cell-free filtrate of X. nematophila YL001 under different initial pH showed an extensive antimicrobial activity against the fungal and oomycete pathogens tested, with a range of 70.93–100% for M. grisea, 35.61–97.84% for A. solani, 26.86–99.28% for A. brassicae, 74.47–100.00% for B. cinerea, 5.82–100.00% for P. piricola and 0.00–98.58% for S. sclerotiorum (Table 1). In particular, the cell-free filtrates of X. nematophila YL001 under initial pH 8.5 exhibited an inhibitory effect greater than 90% on A. solani, A. brassicae, B. cinerea, M. grisea, P. piricola, P. capsici and S. sclerotiorum. Among these, B. cinerea was selected as the pathogens for subsequent studies.
Effect of ethyl acetate extract and methanol extract of the cell-free filtrate of X. neamatophila YL001 culture at pH 8.5 on mycelial growth of B. cinerea
95% confidence interval
Ethyl acetate extract
y = − 0.185 + 1.653x
y = 0.462 + 0.299x
The inhibitory effect of cell-free filtrates of X. nematophila YL001 on the selected bacterial pathogens at varying initial pH
Antibiotic activity (U/mL)1
96.0 ± 17.0c
194.0 ± 6.0b
205.0 ± 13.0a
205.0 ± 6.0a
210.0 ± 29.0a
8.0 ± 0.6e
77.0 ± 1.2d
73.0 ± 1.2c
81.0 ± 6.0b
92.0 ± 1.2a
17.0 ± 1.2d
44.0 ± 6.0c
84.0 ± 6.0b
84.0 ± 3.0b
95.0 ± 1.7a
Pseudomonas syringae pv. actinidiae
13.0 ± 6.0d
84.0 ± 2.3c
107.0 ± 12.0b
128.0 ± 2.9a
132.0 ± 6.0a
Xanthomonas campestris pv. oryzae
15.0 ± 1.2c
26.0 ± 1.2b
24.0 ± 6b
32.3 ± 2ab
34.0 ± 3.0a
Erwinia carotorora subsp. carotovora
18.0 ± 0.6b
9.0 ± 2.9c
13.0 ± 0.6b
20.0 ± 0.6a
22.7 ± 1.5a
In vivo efficiency of the cell-free filtrate, ethyl acetate extract and methanol extract on tomato fruits infected with B. cinerea
Effect of pH on gene expression
Rhabduscin takes an important part in the melanization pathway of the insect’s immune system . The rhabduscin gene cluster (isn) has been identified in genomes from both Xenorhabdus and Photorhabdus [21, 36]. RT-PCR analysis revealed that the expression level of isnA, isnB and isnC were increased with the increase of the initial pH (Fig. 3b), this was also supported by qRT-PCR analysis that isnA, isnB and isnC expression were 0.53-, 2.18- and 2.71-fold higher at pH 8.5 relative to pH 5.5, respectively (Fig. 4B).
CpxR and OmpR are response regulators involved in the regulation of bacterial pathogenicity and mutualism, which negatively regulate antibiotic activities of X. nematophila. RT-PCR analysis showed that cpxR and ompR expressed at lower levels with the increase of the initial pH from 5.5 to 8.5 (Fig. 3c). The result was also supported by qRT-PCR analysis that cpxR and ompR expression decreased 0.64- and 0.71-folds at pH 8.5, compared with that at pH 5.5 (Fig. 4C, D).
Metabolites analysis of X. nematophila at different pH
The relative amount of rhabduscin, nematophin and indole derivatives in ethyl acetate extract of the cell-free filtrate of X. neamatophila YL001 at pH 5.5, 7.0 and 8.5
Relative amount of metabolitesa
Indole derivative 2
Indole derivative 3
Indole derivative 4
In order to further evaluate the effect of pH on the production of metabolites, we characterized several compounds from X. nematophila. After purification of the metabolites, NMR and GC–MS experiments were performed, identifying one of them as nematophin (Additional file 1: Table S3; Figures S6–S8). HPLC analysis shown that the concentration of nematophin produced over 72 h by X. nematophila Yl001 at pH 5.5, 7.0 and 8.5 were 33.20, 146.42 and 228.12 μg/mL, respectively (Additional file 1: Figure S5).
The relative amount of xenocoumacins (Xcn1 and Xcn2) in methanol extract of the cell-free filtrate of X. neamatophila YL001 at pH 5.5, 7.0 and 8.5
Relative amount of Xcns1
In vitro and in vivo, analysis of the antimicrobial activity of X. nematophila YL001 at different initial pH indicated that the alkaline pH conditions were beneficial to antibiotic production. Previous studies have reported the variation in antimicrobial activities of different Xenorhabdus spp. strains to fungal pathogens at different pH [25, 26]. The higher and lower initial pH value (e.g., pH 4.0 and 1.0) were not beneficial to cell growth and antibiotic production of X. nematophila YL001, and the influence of pH on cell growth was not significant as compared to that of antibiotic production within the range of 5.5–8.5 . Similar results appeared in the other work [25, 26] in which it was reported that the antibiotic production by X. nematophila BJ and Xenorhabdus sp. D43 were affected by the initial pH within the range of 4.5–8.5 and optimal initial pH for cell growth and antibiotic production occurred at 6.0–8.0. These results could also be validated by the profiles of pH variation of the culture broth of X. nematophila YL001 at different initial pH. At initial pH 5.5, 7.0 and 8.5 with the use of glucose as sole carbon source, the pH of the culture broth has a natural tendency to change along with the bioprocess, the culture broth pH of YL001 decline from 5.5, 7.0 and 8.5 to pH 5.08, 6.1 and 6.45 during the first few hours of growth, and then increased throughout the course of the experiment reaching 8.4, 8.79 and 8.57 at 72 h, respectively (data not shown). Similar results were obtained in the reports of Yang et al. [25, 26], the culture broth pH of X. nematophila BJ varied from 6.5 to 8.5.
In our previous studies, it was found that at initial pH 7.0, the pH of the fermentation broth of X. nematophila YL001 varied between 5.90 and 9.75 . Low and high pH values (e.g., constant pH 4.5 and 9.5) were disadvantageous to cell growth and antibiotic production. The result indicated that the pH variation values were near the extreme values at initial pH 7.0 . In this study, the pH variation values proceeds within the acceptable ranges at initial pH 5.5, 7.0 and 8.5.
Manipulating nutritional or environmental factors can promote secondary metabolite biosynthesis of microorganisms enabling the discovery of new natural products. Molecules present in the insect hemolymph can selectively stimulate the secondary metabolic pathways in the Photorhabdus and Xenorhabdus [17, 36, 37]. Recently, Crawford group designed a culture medium to mimic the amino acid content of wax moth larval circulatory fluid (a Galleria mellonella “hemolymph mimetic medium”), which stimulated X. bovienii to produce amicoumacins . Due to the structural-relationship between amicoumacins and xenocoumacins (Xcns), this novel nutritional stimulation adjustment approach may offer a new route to regulate xenocoumacins and other antibiotics production in X. nematophila. In present study, the influence of an external environmental stimulus (pH shock) on the production of antimicrobial compounds in X. nematophila was explored. Similar to nutrient stimulation signals, pH shock not only enhanced the levels of some antibiotics but also induced the production of several cryptic metabolites by activating the related secondary metabolic pathways (Fig. 5). In this study, the increased antimicrobial activity of X. nematophila YL001 under pH 8.5 (Figs. 1, 2) suggested that antibiotics might be produced at elevated levels or the cryptic biosynthetic pathway of secondary metabolites were induced in this condition. To address this possibility, the metabolomics profiling of organic extracts from X. nematophila YL001 under different initial pH and the antibiotics production were analyzed by HPLC–MS [17, 18]. The previously described molecules exhibited a range of biological activities, and their regulation by pH underscores the connection between recognition of the pH of the environment and subsequent bacterial metabolic adjustment. The pH of the environment significantly altered indole-containing metabolites (structure 1–4), rhabduscin (structure 5) and xenocoumacins (Xcn). At pH 8.5, the production of nematophin (structure 1) and its reduction product (structure 3) increased, which illustrates that the pH of the environment regulates a general up-regulation rather than a metabolic shift. Nematophin production was 1.57- and 2.02-folds higher at pH 8.5 relative to that at pH 7.0 and 5.5, respectively (Table 4). These may explain why the antibacterial and antifungal activity of ethyl acetate extracts against B. subtilis and B. cinerea were 5.51- and 0.33-folds higher at pH 8.5 than pH 5.5, respectively (Fig. 1). Also, the production of the water-soluble peptide antimicrobial compound Xcn1, the major antibiotics produced by X. nematophila, was 16.36-fold higher at pH 8.5 relative to that at pH 5.5. The result may explain why the antibacterial and antifungal activity of methanol extracts against B. subtilis and B. cinerea were 1.188- and 0.343-folds higher at pH 8.5 relative to that at pH 5.5, respectively (Fig. 1). Meanwhile, alkaline pH (pH 8.5) could induce the production of previously cryptic metabolites (Fig. 5, peaks 7, 8, and 9). However, the biological activities of these induced cryptic metabolites need further validated by purification and characterization. Taken together, the increased antimicrobial activity of X. nematophila YL001 under pH 8.5 was likely due to the combined effect of elevated Xcn1 and nematophin or the cryptic antibiotics induced in this condition. Differential metabolomics profiling with relevant environment factors provides a system-wide approach to coupling small-molecule metabolites to the bacteria’s physiological state.
In present study, under pH 8.5 the transcription levels of xcnA-L genes, which were responsible for Xcn1 synthesis, were significantly higher than that at other pH condition, but xcnM and xcnN, which responsible for the conversion of Xcn1 into Xcn2, were expressed at lower levels. These finding were supported by the result of LC–MS analysis of methanol extracts showing that Xcn1 increased 16.36-fold at pH 8.5 relative to that at pH 5.5. Thus, the 16.36-fold increase in Xcn1 production at pH 8.5 was likely due to the combined effect of elevated expression of the xcnA-L genes and reduced xcnMN expression. Also, under pH 8.5, the transcription levels of ompR and cpxR gene, encoding the response regulator (OmpR and CpxR) of CpxRA and EnvZ/OmpR two-component signal transduction system, were significantly lower than that at other pH condition. In X. nematophila CpxRA and EnvZ/OmpR are used to sense environmental stresses and transduce the information inside the cells to regulate the pathogenic and mutualistic interactions of X. nematophila for the adaptation of the nematode and insect environments [38–40]. Crawford et al.  found that increased osmotic stress generally stimulated metabolite production in X. nematophila cultures, and l-proline provided further stimulation. When X. nematophila multiply in the insect hemolymph, which has long been known to contain high solute (e.g., l-proline, sugars, polyols and derivatives) and inorganic salt concentrations to manage water losses due to environmental osmotic stresses , EnvZ senses high osmolarity of hemolymph and activates OmpR to coordinately repress flagella synthesis, exoenzyme and antibiotic production in X. nematophila by negatively regulating the flhDC operon . Also, OmpR was found to negatively regulate xcnA-L genes expression and positively regulate xcnMN expression [18, 19]. In ompR mutant strain Xcn1 levels and expression of xcnA-L were increased while Xcn2 levels and xcnMN expression were reduced . Our findings further validated the notion of Park et al. (2009) that OmpR negatively regulate the production of Xcn1 and positively regulate the production of Xcn2.
Upon recognition of the signal of the change in pH, CpxA phosphorylates CpxR, activating this response regulator, and regulate the expression of its regulon which include CpxR . In this study, cpxR expressed at lower levels with the increase of the initial pH from 5.5 to 8.5, indicating that at pH 8.5 CpxR repress its own expression. Also, it is interesting to find that at pH 8.5, Xcn1 levels and xcnA-L expression were increased while xcnMN expression was decreased. As OmpR, CpxR was found to negatively regulate xcnA-L gene expression, and positively regulate xcnMN expression . In cpxR mutant strain Xcn1 levels and expression of xcnA-L were increased while Xcn2 levels and xcnMN expression were reduced . A similar regulatory overlap appears to occur in E. coli the CpxR regulon overlaps the regulons of the two-component regulator OmpR [43, 44]. The uniform effects of X. nematophila CpxR and OmpR suggest that these factors directly regulate similar genes in uniform fashions or that one of these regulators positively regulates the other, indirectly affecting downstream genes. The conclusion was supported by the results that in the cpxR mutant the expression of ompR decreased compared to the wild strain . Together, these findings support a model in which at pH 8.5, CpxA senses high pH, activates CpxR, and CpxR repress its own expression, and CpxR either directly or indirectly positively regulates ompR expression and ompR expression was decreased, however OmpR negatively regulate the production of Xcn1 and positively regulate the production of Xcn2. So, at pH 8.5 Xcn1 was shown to be produced at high levels and X. nematophila YL001 exhibited high antimicrobial activity.
In summary, with the increase of initial pH, the antimicrobial activity of X. nematophila YL001 increased. At pH 8.5, nematophin and Xcn, the major antibiotics produced by X. nematophila, were increased remarkably. So, the increased antimicrobial activity might be even synergistic effects of these compounds acting together, but we did not exclude to the production of a second and probably still unknown compound at pH 8.5. Further work will be focused to isolate and characterize the antimicrobial compounds at pH 8.5 to clarify the problem.
YHW designed and supervised the experiments. SQG, SJZ, XLF, QL and JTG conducted the experiments. SQG, SJZ, and YHW performed the data analyses and interpreted the results. SQG, XLF, MB, YHW and XZ wrote and revised the paper. All authors read and approved the final manuscript.
We would like to thank “Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, Northwest A & F University, Shaanxi, China for HPLC–MS analyses.
The authors declare that they have no competing interests.
Availability of supporting data
The dataset(s) supporting the conclusions of this article is (are) included within the article [and its additional file(s)].
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All authors read and approved the final manuscript.
Ethics approval and consent to participate
We state this is not applicable. The manuscript does not report on or involve the use of any animal or human data or tissue.
This work was supported by the Natural Science Foundation of China (No. 31171910) and Chinese Universities Scientific Fund (No. ZD2013003) and Natural Science Foundation of Shaanxi Province (No. 2014JZ004).
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