Project description:Aerobic methanotrophic bacteria use methane as their sole source of carbon and energy and serve as a major sink for the potent greenhouse gas methane in freshwater ecosystems. Despite this important environmental role, little is known about the molecular details of how these organisms interact in the environment. Many bacterial species use quorum sensing systems to regulate gene expression in a density-dependent manner. We have identified a quorum sensing system in the genome of Methylobacter tundripaludum, a dominant methane-oxidizer in methane enrichments of sediment from Lake Washington (Seattle, WA, USA). We determined that M. tundripaludum primarily produces N-3-hydroxydecanoyl-L-homoserine lactone (3-OH-C10-HSL) and that production is governed by a positive feedback loop. We then further characterized this system by determining which genes are regulated by quorum sensing in this methane-oxidizer using RNA-seq, and discovered this system regulates the expression of a novel nonribosomal peptide synthetase biosynthetic gene cluster. These results identify and characterize a mode of cellular communication in an aerobic methane-oxidizing bacterium. Samples are 2 sets of biological replicates of a Methylobacter tundripaludum strain 21/22 mutant where the acyl-homoserine lactone (AHL) synthase gene mbaI (T451DRAFT_0796) has been deleted. The mutant strain was grown to log (48 hours) or stationary (68 hours) phase in the absence or presence of the AHL 3-OH-C10-HSL.
Project description:Supernatant from a stationary phase culture of Methylobacter tundripaludum was run over a C18 solid phase extraction column. The column was washed with 10% methanol in water and then eluted with 90% methanol in water. The eluate was dried and run over a standard LC-MS/MS gradient (ACN in H2O with 0.1% formic acid) focusing on ions with a parent m/z of 150-600.
The excreted metabolome of the methane-oxidizing bacterium Methylobacter tundripaludum
21/22 and mutants related to the production of the natural product tundrenone. Supernatant from a stationary phase culture of Methylobacter tundripaludum 21/22 was extracted twice with an equal volume of ethyl acetate containing 0.01% acetic acid. The organic extract was then dried and analyzed over a standard LC-MS/MS gradient (ACN in H2O with 0.1% formic acid). mbaI: Quorum sensing deficient mutant. mbaIplusSig: Quorum sensing deficient mutant grown in the presence of 1 uM exogenous quorum sensing signal 3-OH-C10-HSL. tunJ: Tundrenone deficient mutant lacking the annotated acyl-CoA ligase TunJ.