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

Abstract

Background: A grapevine crown gall tumor strain, Novosphingobium sp. strain Rr2-17 was previously reported to accumulate copious amounts of diverse quorum sensing signals during growth. Genome sequencing identified a single luxI homolog in strain Rr2-17, suggesting that it may encode for a AHL synthase with broad substrate range, pending functional validation. The exact identity of the complete suite of AHLs formed by novIspR1 is largely unknown.

Methods: This study validates the function of novIspR1 through inducible expression in Escherichia coli and in the wild-type parental strain Rr2-17. We further enhanced the capture of acyl homoserine lactone (AHL) signals produced by novIspR1 using polymeric resin XAD-16 and separated the AHLs by one- and two-dimensional thin layer chromatography followed by detection using AHL-dependent whole cell biosensor strains. Lastly, the complete number of AHLs produced by novIspR1 in our system was identified by LC-MS/MS analyses.

Results: The single LuxI homolog of N. sp. Rr2-17, NovIspR1, is able to produce up to eleven different AHL signals, including AHLs: C8-, C10-, C12-, C14-homoserine lactone (HSL) as well as AHLs with OH substitutions at the third carbon and includes 3-OH-C6-, 3-OH-C8-, 3-OH-C10-, 3-OH-C12- and 3-OH-C14-HSL. The most abundant AHL produced was identified as 3-OH-C8-HSL and isopropyl-D-1-thiogalactopyranoside (IPTG) induction of novIspR1 expression in wild type parental Rr2-17 strain increased its concentration by 6.8-fold when compared to the same strain with the vector only control plasmid. Similar increases were identified with the next two most abundant AHLs, 3-OH-C10- and unsubstituted C8-HSL. The presence of 2% w/v of XAD-16 resin in the growth culture bound 99.3 percent of the major AHL (3-OH-C8-HSL) produced by IPTG-induced overexpression of novIspR1 in Rr2-17 strain. This study significantly adds to our understanding of the AHL class of quorum sensing system in a grapevine crown gall tumor associated Novosphingobium sp. Rr2-17 strain. The identity of nine AHL signals produced by this bacterium will provide a framework to identify the specific function(s) of the AHL-mediated quorum-sensing associated genes in this bacterium.

Keywords: Acyl-homoserine lactones; Agrobacterium vitis tumor; Grapevine crown gall tumor; Inducible expression; NovI; Novosphingobium. sp.; Quorum sensing; Resin.

Figure 1. Crown gall tumors.

Figure 2. In-silico validation of the NovI_spR1 based on maximum likelihood tree inference.

Evolutionary relationships among putative LuxI homologs from the genus Novosphingobium. The maximum likelihood tree was rooted with LuxI homologs from R. radiobacter and A. fabrum as the outgroups. Node labels indicate SH-like branch support values and branch lengths represent number of substitutions per site.

Figure 3. Acyl homoserine lactones (AHL) produced by wild type strain Rr2-17 in chromatograph of extract and AHL standards and detection with Agrobacterium biosensor strain NTL4(pZLR4) overlay.

AHL standards include: substituted AHL signals: 3-hydroxy-C6-AHL (OH-C6, lane 1), 3-hydroxy-C8-AHL (OH-C8, lane 3) and unsubstituted AHL signals C6-AHL (C6, lane 1) and C8-AHL (C8, lane 3) as previously described (Gan et al., 2009). Ethyl acetate extract (EtOAc) of culture supernatants of strain Rr2-17 (1.0 ml of 2.5 × EtOAC extract). E.coli JM109 (pSRKKm::novI_SpR1 liquid cultures were grown overnight in the presence of 0, 10, 100 or 1,000 μM of IPTG inducer and culture supernatants were extracted with EtOAc. Lanes 5 to 8 each represent 1 μl of 1x extracted overnight broth cultures. Each dot represent the center of signal. The sample loading origin, (O) is indicated at bottom of chromatogram.

Figure 4. Acyl homoerine lactone (AHL) profiles after TLC-based chromatography and AHL detection with biosensor NTL4(pZLR4).

Extracts were recovered from broth cultures of Rr2-17 (pSRKKm::novI-Sp-R1) and Rr2-17 containing empty control plasmid pSRKKm (pSRKKm only). Extracts were recovered from broth cultures of Rr2-17 (pSRKKm::novI_SpR1) and Rr2-17 containing empty control plasmid pSRKKm (pSRKKm only) containing increasing concentrations of binding resin XAD-16. Extracts of the broth culture media without binding resin (A) and extracts of the binding resin after separated from the broth culture (B). Binding resin XAD-16 was added to broth culture media at 0, 0.5, 1.0 and 2.0%, (white triangles), inoculated and grown overnight. Standards on TLC chromatograph included: unsubstituted AHLs: C6, C8, C10, and C12 (S1), and of hydroxy substituted OH-C6 and OH-C8 (S2) as previously described (Gan et al., 2009).

Figure 5. Concentration of the three most abundant AHL signals produced from NovI_SpR1.

Overexpression of novI_SpR1 in parental strain Rr2-17 and recovery of the acyl homoserine lactones (AHLs) sequestered in resin during culture growth culture. Concentration of the three most abundant AHL signals, OH-C8, OH-C10 and C8, are shown after recovery from resin and culture media extracts prepared from cultures of Rr2-17 (pSRKKm::novI_SpR1 containing XAD-16 resin at 0.25%, 0.5%, 1.0%, and 2.0%. Controls include: culture extracts from growth of Rr2-17 (pSRKKm::novI_SpR1) and Rr2-17(pSRKKm) in the absence of resin.

Figure 6. Two-dimensional thin layer chromatography (2-D TLC) of Rr2-17 (pSRKKm::novIspR1) resin extracts (A), and unsubstituted (B) and OH substituted.

2-D TLCs were dried and overlayed with a medium-agar culture of the TraR-dependent biosensor A136, incubated for 36 h and imaged by a charge coupled device (Bio-Rad). 2-D TLC of the Rr2-17 (pSRKKm::novI) extract after imaging with the A136 biosensor (A) and the same with black and white detection (B) and at the same sensitivity used for the color detection shown in panel A. 2-D TLC of C6, C8, C10, and C12 as unsubstituted standards (C). 2-D TLC of 3-OH-C6, 3-OH-C8, 3-OH-C10, and 3-OH-C12 as OH substituted standards (D). The green circle in each panel represents the origin of the resin extract (A, B) and origin of pure standards in 2-D TLC bioassay (C, D).