LuxS: its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanone

Institute of Infections and Immunity, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, UK 1 School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK 2 School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK 3 Immunology Research, R&D 21, VA Medical Center, Portland, OR 97201, USA 4 Author for correspondence: Klaus Winzer. Tel: +44 115 970 9907. Fax: +44 115 970 9923. e-mail: Klaus.Winzer{at}nottingham.ac.uk Many bacteria produce extracellular molecules which function in cell-to-cell communication. One of these molecules, autoinducer 2 (AI-2), was first described as an extracellular signal produced by Vibrio harveyi to control luciferase expression. Subsequently, a number of bacteria have been shown to possess AI-2 activity in their culture supernatants, and bear the luxS gene product, which is required for AI-2 synthesis. In Porphyromonas gingivalis , luxS and pfs , encoding a 5'-methylthioadenosine/ S -adenosylhomocysteine nucleosidase (MTA/SAH’ase), form an operon, suggesting that S -adenosylhomocysteine (SAH) or 5'-methylthioadenosine (MTA) serves as a substrate for AI-2 production. Cell-free extracts of Escherichia coli MG1655, but not DH5 (which carries a luxS frame-shift mutation) were capable of generating AI-2 activity upon addition of SAH, but not MTA. S -Ribosyl-homocysteine (RH) derived from SAH also served as a substrate in E. coli MG1655 extracts. RH-supplemented cell-free extracts of Pseudomonas aeruginosa , a bacterium that lacks luxS , only generated AI-2 activity following the introduction of a plasmid containing the Por. gingivalis pfs-luxS operon. In addition, defined in vitro systems consisting of the purified LuxS proteins from Por. gingivalis , E. coli , Neisseria meningitidis or Staphylococcus aureus converted RH to homocysteine and a compound that exhibits AI-2 activity.4-Hydroxy-5-methyl-3( 2H )-furanone was identified by mass spectrometry analysis as a major product formed in this in vitro reaction. In E. coli MG1655, expression of T3SH [the bacteriophage T3 S -adenosylmethionine (SAM) hydrolase] significantly reduced AI-2 activity in culture supernatants, suggesting that AI-2 production is limited by the amount of SAH produced in SAM-dependent transmethylase reactions. The authors suggest that the LuxS protein has an important metabolic function in the recycling of SAH. They also show that Ps. aeruginosa is capable of remo