Quorum sensing signals of the grapevine crown gall bacterium, Novosphingobium sp. Rr2-17: use of inducible expression and polymeric resin to sequester acyl-homoserine lactones

Bacterial strains, plasmids, growth media and biosensor strains

Novosphingobium sp. Rr2-17 was isolated from a nopaline grapevine tumor (Fig. 1) by Ernő Szegedi previously of the Research Institute for Viticulture and Enology, Kecskemét Hungary. Table one contains the bacterial strains, plasmids and primers used in this work (Table 1). Novosphingobium sp. Rr2-17 strain was grown in tryptone soy broth (TSB), potato dextrose (PD) or R2A medium (Difco Laboratories, Detroit, MI, USA) at 28 °C. Agrobacteria AB minimal medium (Chilton et al., 1974) at 28 °C was used to grow AHL-dependent biosensor strains Agrobacterium NTL4 and A136. For AHL signal induction bioassays, parental wild-type strain N. sp. Rr2-17 harboring empty vector pSRKKm or pSRKKm with cloned novI_spR1 gene were grown on Luria-Bertani broth (LB) medium at 28 °C and supplemented with kanamycin at 50 mg/ml, respectively. For AHL signal detection bioassays, Agrobacterium tumefaciens NTL4 (pZLR4) and A136 (pCF218, pMV26) were grown in AB medium supplemented with 0.2% (w/v) dextrose and 0.01% (w/v) yeast extract and gentamycin (10 μg/ml) for NTL4 (pZLR4) (Cha et al., 1998) and kanamycin (25 μg/ml) and tetracycline (5 μg/ml) for A136 (pCF218, pMV26) (Sokol et al., 2003; Zhu et al., 2003). Escherichia coli-based biosensors JM109 (pSB401), JM109 (pSB1075) and JM109 (pSB536) were grown in LB medium with the appropriate antibiotic for plasmid maintenance (Swift et al., 1997; Winson et al., 1998). Chromobacterium violaceum CV026 biosensor was grown in tryptone yeast extract/potato dextrose (1:1) agar medium for T-streak bioassays (McClean et al., 1997). Each AHL-dependent bacterial biosensor strain used in this work along with its AHL receptor protein, cognate AHL signal and reporter gene output is listed in Table S1. All media and growth conditions for AHL detection bioassays are as previously described by our laboratory (Scott et al., 2006; Gan et al., 2009, 2016; Lowe et al., 2009).

Protein alignment and phylogenetic tree analysis

LuxI homologs from the genus Novosphingobium were downloaded from the uniport database (as of 30^th October 2022). Alignment of the amino acid sequences was performed with MUSCLE v.3.8.1551 (default settings) followed by trimming with trimAl v1.4 (-automated1 option) that selects alignment sites based on similarity statistics optimized for maximum likelihood phylogenetic tree reconstruction (Capella-Gutiérrez, Silla-Martínez & Gabaldón, 2009; Edgar, 2004). Maximum likelihood tree was subsequently inferred using FastTree with the Le-Gascuel 2008 model (-lg option) (Price, Dehal & Arkin, 2010). Visualization of the amino acid alignment and phylogenetic tree used JalView (https://www.jalview.org/) and FigTree v1.4.4 (https://github.com/rambaut/figtree/), respectively.

Amplification and cloning of novI_Nsp.Rr2-17

Amplification of the novI_spR1 was performed using Q5 polymerase mastermix (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Approximately 150 ng of the purified PCR amplicons were mixed with 50 ng of pSRKKm vector (Khan et al., 2008) and double digested with NheI and NdeI (New England Biolabs, Ipswich, MA, USA) for 1 h. After heat inactivation, the digested products were purified using magnetic beads (Omega Biotek, Norcross, GA, USA) and ligated with Electroligase (New England Biolabs, Ipswich, MA, USA) for 30 min. The ligated products were transformed into wildtype N. sp. Rr2-17 and Escherichia coli JM109 using electroporation all as previously described by our laboratory (Scott et al., 2006; Gan et al., 2016).

Inducible expression of novI_Nsp.Rr2-17 and use of Amberlite XAD-16 resin for capture of AHLs

AHL extractions from induction assays Rr2-17 (pSRKKm:: novI_spR1) and Rr2-17 (pSRKKm) were grown in 20 mL of LB (50 mg/ml kanamycin) supplemented with different amount of isopropyl-D-1-thiogalactopyranoside (IPTG) inducer to final concentration of 0, 10, 100, or 1,000 μM and with resin at 2.0, 1.0, 0.5, 0.25 and 0.0 g (weight of resin per 100 mL of medium) of Amberlite XAD-16 resin (Rohm and Haas, Philadelphia, PA, USA) at 28 °C with shaking (150 r.p.m.) for 48 h (Khan et al., 2008; Gan et al., 2016). Amberlite XAD-16 is a non-ionic, hydrophobic, cross-linked polyaromatic resin. The media supernatants were separated from the resin by centrifugation and by transferring the supernatants to a new extraction flask. Supernatants and resin were extracted with acidified ethyl acetate (aEtOAc) 1:1 v/v (1 mL of glacial acetic acid per 200 mL of ethyl acetate) for 60 min with shaking (150 r.p.m.). The extracts were then centrifuged to separate the aqueous or resin and ethyl acetate phases. The ethyl acetate phase was recovered (1x extract) and dried in a Savant Speed Vac and one- and twenty-fold concentrated extracts were prepared and used in AHL detection bioassays and for LC-MS/MS analysis.

Biosensor detection and one- and two-dimensional thin layer chromatography for AHL visualization

Reverse-phase (RP) one-dimensional (1-D) TLC plates were used to determine AHL signal profiles. Concentrated acidified ethyl acetate (aEtOAc) extracts were spotted on to the C18 RP-TLC plate (EMD Chemicals Inc., Gibbstown, NJ, USA) origin in 2-mL volumes and representing from 0.5 to 2-mL supernatant equivalents. Plates were developed in a 70:30 (v/v) methanol: water mobile phase, dried and AHLs were detected as previously described (Scott et al., 2006; Gan et al., 2016). Bioluminescence produced by the A. tumefaciens A136 traR, PtraI::luxCDABE-based biosensor strain (Bernier, Beeston & Sokol, 2008) overlaid on the chromatograms was detected with a Bio-Rad charge coupled device (ccd) ChemiDoc MP system at different sensitivity settings to detect AHL signals with appropriate reference compounds. This involves determining and comparing retardation factors (Rf) of unknown samples with AHL reference compounds (Shaw et al., 1997).

RP two-dimensional (2-D) TLC for AHL separation and detection was developed in our laboratory (Gan et al., 2016) and performed by initially spotted one sample onto the bottom left corner of the C18 RP-TLC plate. The amount needed was estimated based on the AHL signal strength obtained from independent 1-D RP-TLC runs. The spotted TLC plate was eluted with 70:30 (v/v) methanol: water as the first mobile-phase in a glass tank. The mobile-phase was allowed to rise to the top of the TLC plate before removing the plate to dry overnight. Then, the TLC plate was rotated 90° counterclockwise, placed into a tank with 25:75 (v/v) 2-propanol: water as the second mobile-phase until it reached the top of the TLC plate as previous described by our laboratory (Gan et al., 2016). After drying, the TLC plate was overlaid with TraR-dependent Agrobacterium biosensor strain A136 using the process as used for 1-D TLCs (Scott et al., 2006; Gan et al., 2009, 2016).

AHL identification and quantification by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)

The Shimadzu series 10AD VP LC system was used to carry-out chromatography. A Phenomenex Gemini C18 HPLC column (3.0 μm, 100 × 3.0 mm) with an appropriate guard column was used and the column oven was set at 50 °C. Phase A (mobile) was 0.1% (v/v) formic acid in water, and phase B (mobile) consisted of 0.1% (v/v) formic acid in methanol. The chromatographic separation was carried-out at a flow rate of 450 μL/min, with a gradient initially at 10% B and increased linearly to 99% B over 12 min and remained at 99% B for 1 min. A rapid decrease to 10% B occurred over 0.1 min, and stayed at this composition for 1.9 min. Total run time per sample was 15 min. All methods are as previously described (Gan et al., 2016).

An Applied Biosystems Qtrap 4000 hybrid triple-quadrupole linear ion trap mass spectrometer was used with an electrospray ionization (ESI) interface. Analyst software was used to control the instrument, and for data collection and analysis. The following parameters were set and included: auxiliary gas: 15.0, curtain gas: 20.0, ion source potential: 5,000 V, nebulizer gas: 20.0 and temperature: 450 °C (Gan et al., 2016).

Sample preparation and AHL standards

Synthetic standards of C4, C6, C8, C10, C12, C14, 3-oxo-C4, 3-oxo-C6, 3-oxo-C8, 3-oxo-C10, 3-oxo-C12, 3-oxo-C14, 3-OH-C4, 3-OH-C6, 3-OH-C8, 3-OH-C10, 3-OH-C12 and 3-OH-C14 AHLs were synthesized according to established procedures (Chhabra et al., 1993; Gan et al., 2016). Dried extracts were stored at −20 °C. Prior to analysis, each sample extract was reconstituted in 100 μl of methanol +0.1% (v/v) formic acid. The injection volume was 5 μl (Gan et al., 2016).

Analysis method

Initial analysis was conducted with the MS operating in precursor ion scan mode screening for precursor ions that give rise to a product ion of m/z = 102 (a fragment ion that is common to all AHLs), upon collision induced fragmentation (Table S2). Comparison of detected peak areas with an AHL mix sample of known concentration was used to gauge a useful calibration range for the subsequent quantification of detected AHLs. Samples were re-run with the MS in MRM (multiple reaction monitoring) mode, analyzing the LC eluent for specific AHLs detected in the previous analysis. The quantification was conducted by comparing peak areas of detected peaks with a six-point calibration curve constructed by analyzing (in triplicate) mixed AHL calibration samples containing C8, 3-OH-C8 and 3-OH-C10 AHLs at 0.5, 1.0, 2.0, 5.0, 10 and 20 μM (Gan et al., 2016).

Whole genome sequencing of N. sp. Rr2-17 identified a single luxI/ luxR pair homologs (novI, novR)

The draft genome of strain Novosphingobium. sp. Rr2-17 (Gan et al., 2012) consists of 4,539,029 bps (148 × coverage), has a GC content of 62.7% and consists of 166 contigs (N50 of 130 kb with largest being 318 kb) (Table S3). The nucleotide sequence accession number is GenBank: AKFJ00000000.1 (Gan et al., 2012). Anti-SMASH analysis (Blin et al., 2019) of N. sp. Rr2-17 identified a single luxI homolog and its luxR cognate receptor was found canonical luxI luxR pair (novI, novR) genetically linked to each other (Table S4). By performing BLAST searches against the curated LuxI homologs, a putative AHL synthase was identified in strain Rr2-17, locus tag WSK_3264. We propose the name novI_spR1 for locus tag WSK_3264 of N. sp. Rr2-17.

NovI_spR1 alignment with LuxI homologs of the Novosphingobium genus and phylogenetic tree analysis

Alignment of the NovI_spR1 protein sequence with known LuxI homologs showed that all the homologs, including the luxI homolog of Rr2-17 strain, NovI_spR1, contain the highly conserved amino acid signatures which are required for the function of AHL synthases (Fig. S1, asterisk marks) and includes three highly conserved amino acids, Arg24, Phe28 and Trp34. Among some autoinducer proteins within the genus previously identified, NovI_spR1 share identity with three LuxI homologs of N. subterraneums DSM 12447, NovI_Nsub1, NovI_Nsub2 and NovI_Nsub3 at 39.6%, 49.5% and 50.3% for locus tags NJ75_2841, NJ75_2498 and NJ75_4146, respectively (Gan et al., 2016).

Phylogenetic analysis of publicly available Novosphingobium LuxI homologs, including NovI_spR1, revealed that NovI_spR1 forms a sister group with the LuxI homologs of N. pentaromativorans and N. sp. TCA1 (Uniprot accession codes A0A2W5NAM5 and A0A6H9HEL6, respectively), with strong support (Fig. 2). While certain Novosphingobium species possess multiple copies of LuxI homologs, our findings indicate that these homologs are often distantly related, occupying different phylogenetic clades. An intriguing observation is the putative LuxI homolog of N. resinovorum (A0A1D8AGG5), which, despite sharing the signature conserved amino acids of an acyl-homoserine lactone synthase (Fig. 2), appears basal to the other Novosphingobium LuxI homologs, showing a relatively long branch length. This highlights a potential unique evolutionary trajectory for homolog novI_spR1.

NovI_spR1 protein of epiphytic crown gall strain N. sp. Rr2-17 produces multiple AHL signals

Culture extracts prepared from wild-type N. sp. Rr2-17 activated two of the four AHL-dependent whole cell bacterial biosensors tested (Table 2). 1-D RP-TLC separation of the culture extract followed by AHL detection using the Agrobacterium-based TraR-based bioluminescence biosensor led to the detection of four putative AHL signals (Fig. 3, lane 4).

Further, dose-dependent accumulation of AHLs was observed from growth culture supernatants of JM109 (pSRKKm::novI_spR1) at different IPTG-inducer concentrations. Culture supernatants from JM109 (pSRKKm::(novI_spR1) showed an increase in AHLs from background levels (no inducer IPTG) with as little as 10 μM of IPTG and continued to increase when exposed to 100 and 1,000 μM of IPTG inducer (Fig. 3, lanes 5–8). As expected, the highest accumulation of AHLs was observed at 1,000 μM of inducer IPTG and this level of inducer was used in all further experiments.

Inducible over-expression of novI_spR1 in wild type parental strain and accumulation of AHLs using binding resin Amberlite XAD-16

Inducible over-expression of novI_spR1 in the wild-type parental strain which contains the native novI_spR1 was expected to enhance the biosynthesis and allow detection of additional AHLs signals. To test this, plasmid pSRKKm::novI_spR1 was introduced into the parental strain. Overexpression of an additional copy of the novI_spR1 gene combined with varying amounts of XAD-16 resin in the growth cultures influenced AHL accumulation and recovery (Fig. 4). IPTG induction consistently increased AHL levels in a concentration-dependent manner, and higher resin concentration enabled recovery of more AHL signals (Figs. 3 and 4). An example of the AHL signals separated by 1-D TLC shows a significant increase in the intensity and diversity of AHL signals produced, extracted and visualized from homologous over expression of novI_spR1 (Fig. 4B). Saturating levels of resin occurred at 1% as determined by similar detection intensities of AHL signals recovered and visualized by TLC biosensor overlay from extracted broth (Fig. 4A) and extracted resin (Fig. 4B). This may indicate that the AHL-dependent biosensor is near or at a saturated response, and that any additional signal has little effect on the response / output, when using the lacZ as the reporter gene.

To validate these findings, a second Agrobacterium-based biosensor, A136 (pCF218) (pMV26), that also uses the identical AHL receptor, TraR, but provides an output response signal of bioluminescence was employed. Dis-diffusion experiment confirmed that increasing resin concentrations enhanced recovery of total AHLs. (Fig. 2A). Quantification of the total AHLs show a decrease in the amount of AHL signals remaining in the culture media, indicating that as they were captured by the resin (Figs. S2B, S2C).

LC-MS/MS, 2D-TLCs and XAD-16 resin to determine AHL signals

A complement of nine AHL signals were found to be produced by the wild type strain and five were identified by over expression in E. coli JM109 strain (Table 3). The LC-MS/MS output traces from this analysis are shown (Fig. S3) and retention times expected and observed are shown in Table 3. LC-MS/MS analysis revealed that the most abundant AHL produced by novIspR1 was 3-OH-C8, followed by 3-OH-C10 and C8. The ratio of 3-OH-C8 to 3-OH-C10 differed by at least 5-fold in control cultures (no resin) and varied between 5.8 and 11.5 in resin extracts, depending on resin concentration (Fig. 5, Table S5). Increasing the resin concentration from 0.25% to 2.0% reduced the 3-OH-C8 to 3-OH-C10 ratio, with a decrease to 5.8 and 8.0 at 2.0% resin for Rr2-17 (pSRKKm::novIspR1) and Rr2-17 (pSRKKm), respectively. Overexpression of novIspR1 in wild-type Rr2-17 combined with 2.0% resin increased AHL recovery by 7- to 9.3-fold for C8, 3-OH-C8, and 3-OH-C10 compared to the control strain (Table S5).

Table 3:

Acyl-homoserine lactone species, acyl chain lengths and observed retention time of the characteristic total molecular ions of m/z for wild type Rr2-17 strain and DH5α (pSRKkm::novI_SpR1).

Strain or luxI homolog	Number of AHLs detected	R1 Substitution at the 3rd carbon	R2 Acyl chain length (total number of carbons)	Retention time/min of signal standard	Retention time/min of signal observed
Rr2-17	9	OH	C6	3.93	3.97
		Unsubstituted, OH	C8	5.20 4.49	5.20 4.49
		Unsubstituted, OH	C10	5.87 5.05	5.83 5.05
		Unsubstituted, OH	C12	6.65 5.72	6.65 5.72
		Unsubstituted, OH	C14	7.44	7.44
				6.50	6.50
DH5a (pSRKkm::NovIspR1)	5	Unsubstituted, OH	C8	5.20 4.49	5.16 4.49
		OH	C10	5.05	5.05
		OH	C12	5.72	5.72
		OH	C14	6.50	6.50

DOI: 10.7717/peerj.18657/table-3

2-D RP-TLC for enhanced signal detection

The application of 2-D RP-TLC facilitated the separation and detection of multiple AHLs from Rr2-17 (pSRKKm::novIspR1). A total of eleven AHL spots were identified, with seven clearly visible and four faint signals detected in the novIspR1 resin extracts (Fig. 6A, Fig. S3). In contrast, only one faint signal was found in the culture extracts of Rr2-17 (pSRKKm) (Fig. 6B), with no additional spots in its resin extracts, highlighting the impact of novIspR1 overexpression.

Saturation at 1% resin was indicated by similar detection intensities of AHL signals from both extracted broth (Fig. 6C) and resin (Fig. 6D). The varying visibility of signal spots in resin extracts from Rr2-17 (pSRKKm::novIspR1) compared to those from Rr2-17 (pSRKKm) is further illustrated in Fig. S4. LC-MS/MS analyses confirmed that overexpression of novIspR1 in the wild-type strain produced at least nine AHLs, including C8-C10, C12, C14, and five hydroxyl-substituted AHLs (3-OH-C6, -C8, -C10, -C12, -C14). Additionally, two potentially uncharacterized signals activated the TraR receptor in whole cell biosensor overlays, indicating a diverse array of AHLs greater than typically observed in bacteria with a single luxI homolog.