In this part all methods that were used for structure analysis and characterization of the novel QQ enzyme GqqA are described.
Mutagenesis of GqqA
For the mutagenesis of specific aa in GqqA divers quick exchange mutagenesis on nucleotide level were performed. For this purpose, the mutations were done by using the Phusion Polymerase, the mutation specific primers (Table 12) and the construct pET-21a::gqqA as template (Table 2). The PCR products were controlled on an Agarose gel and
the charging stock was digested with DpnI for removing the template DNA (30 min 37 °C, 10 min heat inactivation at 75 °C). After digestion, a phosphate group was added to the amplicon by using the polynucleotide phosphokinase under using manufacture protocol (Thermo Scientific, Bremen, Germany). With the added phosphate group, the linear amplicon could be ligated with the T4 ligase with the addition of 1 µL 10 mM ATP. Ligation took place at alternating 10 °C and 30 °C for 60 minutes. After a heat inactivation step a heat shock transformation in E. coli DH5α was done. The correctness of mutation was done with sequencing by using plasmid specific pET- primer (Table 21). The mutation construct with the right sequence was transformed in the overexpression strain E. coli BL21(DE3). The GqqA-Mut4 construct was generated by using a synthesized gene fragment (appendix, p. CXXXIII) containing the substitution and deletion side was used as primer.
Table 12: Overview of GqqA mutagenesis
Name Mutagenesis Primer I Primer II
GqqA-Mut1 F261S oKP221 oKP222
GqqA-Mut2 ΔR279; ΔK280; ΔP281 oKP223 oKP224
GqqA-Mut3 R25S oKP225 oKP226
GqqA-Mut4 P187A; ΔP188; ΔP189, ΔG190; P191S
--* --*
GqqA-Mut5 T118V oKP229 oKP230
* a synthesized gene fragment was used as primer for the amplification of GqqA-Mut4 (p. CXXXIII, appendix)
Overexpression and purification of GqqA
The overexpression of GqqA was done like previously published (Valera et al., 2016) with some modifications. The strain harboring the expression plasmid pET-21::gqqA was grown in 1 l LB medium supplemented with specific antibiotic. When the culture reached an OD600 of 0.6 – 0.8, the culture was induced with 1 µM IPTG and the expression of the target protein was carried out for 16 h at 22°C. The cells were harvested and resuspended in lysis buffer (QIAexpressionistTM) and were disrupted three times with a French pressure cell. The purification was performed according to the QIAexpressionistTM protocol no. 12. The protein eluates were buffered and concentrated using VivaspinTM sample concentrator and 100 mM potassium phosphate buffer (pH 8.0).
Materials and methods Structure analysis of GqqA
For identifying the structure of the novel class of QS degrading enzyme different approach were used. First the aa sequence of GqqA was compared to known QQ enzymes and for secondary structure and protein structure prediction. The purified protein was shipped to Collaboratory partner for crystallization and data analysis.
For similarity analysis of GqqA to other QQ enzymes, 22 sequences of characterized QQ enzymes were used and compared to GqqA sequence by using T- COFFEE multiple sequence alignment server (http://tcoffee.crg.cat/). The resulted alignment of these 23 sequences was phylogenetic analyzed using Mega X (https://www.megasoftware.net/) and the Maximum Likelihood method with JTT matrix-based model. The bootstrap was set to 500 replicates.
(Crystallization experiments and data analysis of GqqA were performed in cooperation with the working group of Prof. Dr. Christian Betzel from the Institute of Biochemistry and Molecular Biology of the University of Hamburg. Nadine Werner performed the following experiments and established the best conditions for GqqA crystallization).
Purified protein (10 mg/ml) was used for crystallization. For an initial screening, the sitting-drop vapor diffusion method was used in 96-well plates. Small crystals were identified after three days. These seedings were used to get larger crystals that can be used for X-ray analysis. Therefore, the hanging-drop vapor diffusion method was used.
The X-ray diffraction data were collected at the P11 beamline at Petra III Synchrotron (DESY Hamburg, Germany). The data were analyzed using XDS software package (Kabsch, 2010).
In vivo QQ enzyme assays
For a functional AHL- degrading assay the reporter strain C. violaceum CV026 was used.
Therefore, the E. coli BL21 (DE3) strains harboring 21a::gqqA wt or once of pET-21a::gqqA M1-M5 mutant constructs grew in a preculture. Fresh LB medium was inoculated with 1 % (vol/vol) of the precultures and cultured at 37 °C until an OD600 of 0.6 was reached.
Then the cells were induced with 0.1 mM IPTG. The proteins were now expressed recombinant for 16 h. Fresh LB medium with ampicillin and 10 µM of oxo-C8-HSL was inoculated with the expression strains in a ratio of 2 % (vol/vol) and incubated at 37 °C for 6 h. The culture supernatant was collected by centrifugation and 30 µL were used for the bioassay. Therefore, a grown CV026 culture was mixed with 24-fold volume of LB agar and poured in plates. Sterile filter papers were placed on the plates. The CV026 plates containing the supernatants of the cultures were incubated for 24 h at 28 °C. When CV026 cells become
violet, AI molecules are present, when CV026 cells remain colorless, the AI molecules are degraded.
In vitro enzyme assay and ESI-MS/MS analysis
(The enzyme assay and ESI-MS/MS measurements were performed in cooperation with Manuel Ferrer and Laura Fernandez-Lopez from Department of Applied Biocatalysis of the Institute of Catalysis (CSIC) in Madrid (Spain).
The in vitro enzyme assay was performed in triplicates and corrected for non-enzymatic transformation at 30°C. The substrate oxo-C8-HSL was added to a final concentration of 0.5 mg/ml (stock solution 100 mg/ml in DMSO) in one milliliter of 5 mM EPPS buffer, pH 8.0. A final concentration of 10 µg/ml GqqA (5 mg/ml stock solution in 40 mM HEPES, pH 7.0) was added immediately to the batch. Aliquots of the samples with and without protein were taken at different time intervals and the reaction products were analyzed by Mass spectrometry.
The MS analysis was performed in a hybrid quadrupole-time of flight mass spectrometer (QTOF, QSTAR pulsar I, ABSciex) supplied with a micro electrospray ion source for measurements in positive and negative mode. The assay products were dissolved in methanol in a 1:10 dilution and then introduced in the spectrometer. For identifying the products, the measurements were recorded in TOFMS mode. The parameters have been set as shown below: mass range 50-2000 Dalton; ion spray voltage (IS): 4500 V; ion source gas pressure (GS1): 10 psi; curtain gas pressure (Cur): 20 psi; declustering potential 30 V;
focusing potential: 210 V; declustering potential 2: 15 V; collision gas: 3.
Results
4. Results
NGR234 is able to fix nitrogen with more than 120 plant genera and is therefore a great model organism to study host-microbe interactions and rhizosphere processes. The genome of NGR234 consists of two genes coding for QS AI synthases (Schmeisser et al., 2009). In a previous work, several AI synthases mutants were constructed, including a NGR234 ΔngrIΔtraI_copy+ double deletion mutant (Krysciak et al., 2014; Grote et al., 2016).
Interestingly, the NGR234 ΔngrIΔtraI_copy+ double deletion mutant showed upregulation of almost all genes on the symbiotic plasmid. It has already been demonstrated that not only the transcription is higher, but also the translation of proteins is increased. For example, this mutant produces a higher number of NOPs proteins and secretes them (Grote et al., 2016).
Root hair curling in the Absence of Apigenin
Due to the upregulation of almost all genes on pNGR234a, the question arises whether this upregulation is sufficient to enable the production of Nod factors that are necessary for the establishment of symbiosis (Figure 2) and root hair curling of the host. Therefore, the Nod factors were extracted from culture supernatants of the NGR234 wt strain and the NGR234 ΔngrIΔtraI_copy+ mutant, which were incubated with and without apigenin. The cultures cultivated with apigenin were selected as a positive control known to produce Nod factors and
Figure 6: Root hair curling assay using Nod factor extracts of NGR234 wt strain and the NGR234ΔngrIΔtraI_copy+ mutant.
Root hairs of V. unguiculata were incubated with extracted supernatants of NGR234 cultures treated either with 1 µM apigenin (right) or the non-treated cultures (left). The curved root hairs of the NGR234ΔngrIΔtraI_copy+ where nothing was added are clearly recognizable, whereas the root hairs of the wt strain (NGR234) are straight. The control cultures, which were grown with apigenin, showed both the typical root hair curling. The images were taken with a Zeiss microscope and the provide software AxioVision.
trigger root hair curling. The extracted supernatants were incubated together with a germinated Vigna unguiculata bean for 24 h and then the root hairs were examined for their curling using a microscope. The root hairs incubated with the non-treated supernatant of the mutant show a distinct root hair curling while the wt supernatant without apigenin shows non-curled root hairs (Figure 6, left). Both extracted supernatants of the wt and the mutant strain, in which the cultures grew in the presence of 1 µM apigenin, trigger root hair curling after 24 h. (Figure 6; right).