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.

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.
Competing Interests: The authors declare that they have no competing interests. Han Ming Gan is employed by Patriot Biotech Sdn Bhd.
(© 2024 Gan et al.)