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

PeerJ. 2024 Dec 20:12:e18657. doi: 10.7717/peerj.18657. eCollection 2024.

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.

MeSH terms

  • Acyl-Butyrolactones* / metabolism
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression Regulation, Bacterial
  • Plant Tumors / microbiology
  • Quorum Sensing* / genetics
  • Sphingomonadaceae* / genetics
  • Sphingomonadaceae* / metabolism
  • Vitis* / microbiology

Substances

  • Acyl-Butyrolactones
  • Bacterial Proteins

Grants and funding

Funding was provided by the American Society for Microbiology through an Undergraduate Research Fellowship to Lucas Dailey. Lucas Dailey and Rochester Institute of Technology (RIT) provided the use of facilities and supplies to perform experiments. Paul Williams’ laboratory at the University of Nottingham, UK, provided the LC-MS/MS analysis and synthesis of AHL standards carried out by Alex Truman. Han Ming Gan received support from the Monash University Malaysia Tropical Medicine and Biology Multidisciplinary Platform. DNA primers, DNA isolation and DNA purification kits, enzymes and other expendables were provided by the Thomas H. Gosnell School of Life Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.