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.
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.
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-CÂ10-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:Multiple species of bacteria oxidize methane in the environment after it is produced by anaerobic ecosystems. These organisms provide a carbon and energy source for species that cannot oxidize methane themselves, thereby serving a key role in these niches while also sequestering this potent greenhouse gas before it enters the atmosphere. Deciphering the molecular details of how methane-oxidizing bacteria interact in the environment enables us to understand an important aspect that shapes the structure and function these communities. Here we show that many members of the Methylomonas genus possess a LuxR-type acyl-homoserine lactone (acyl-HSL) receptor/transcription factor highly homologous to MbaR from the quorum sensing (QS) system of Methylobacter tundripaludum, another methane-oxidizer that has been isolated from the same environment. We reconstitute this detection system in Escherichia coli and also use mutant and transcriptomic analysis to show that the receptor from Methylomonas species strain LW13 (LW13) is active and alters LW13 gene expression in response to the acyl-HSL produced by M. tundripaludum. These findings provide a molecular mechanism for how two species of bacteria that may compete for resources in the environment can interact in a specific manner through a chemical signal.
Project description:Acyl-homoserine lactone (acyl-HSL) quorum sensing was first discovered in Vibrio fischeri where it serves as a key control element of the seven-gene luminescence (lux) operon. Since this initial discovery, other bacteria have been shown to control hundreds of genes by acyl-HSL quorum sensing. Until recently, it has been difficult to examine the global nature of quorum sensing in V. fischeri. However, the complete genome sequence of V. fischeri is now available and this has enabled us to use transcriptomics to identify quorum-sensing regulated genes and to study the quorum-controlled regulon of this bacterium. In this study, we used DNA microarray technology to identify over two-dozen V. fischeri genes regulated by the quorum sensing signal N-3-oxohexanoyl-L-homoserine lactone (3OC6-HSL). Keywords: Comparison of transcriptome profiles
Project description:Methylobacter species, members of the Methylococcales, have recently emerged as some of the globally widespread, cosmopolitan species, appearing to play a key role in consumption of methane in a variety of environments and across gradients of dioxygen tensions. We approached the question of how Methylobacter copes with low dioxygen partial pressures that it encounters in its natural habitats, via laboratory manipulation. Through comparative transcriptomics of cultures grown under high dioxygen partial pressure and cultures starved for dioxygen, we identified a gene cluster encoding a hybrid cluster protein along with sensing and regulatory functions. Through mutant analysis we demonstrated that this gene cluster is involved in both oxidative and nitrosative stress responses and in NO reduction, while being responsive to NO, likely through direct sensing. Through additional transcriptomic analyses, we further uncovered a complex interconnection between the NO-mediated stress response and quorum sensing in turn controlling synthesis of the secondary metabolite tundrenone, these two systems being inversely regulated. Some of the key methylotrophy functions, such as the two alternative methanol dehydrogenases seem to be parts of the stress response regulatory cascade. One intriguing scenario that emerged from our analyses is that the main role of the respiratory denitrification pathway may be in producing the signaling molecule, NO, in response to hypoxia, rather than in serving as an energy-generating pathway. This novel and complex hypoxia stress response system is unique to Methylobacter tundripaludum, and it may play a role in the environmental fitness of these organisms and in their cosmopolitan environmental distribution.
Project description:Quorum sensing is a term used to describe cell-to-cell communication that allows cell density-dependent gene expression. Many Gram-negative bacteria use acyl-homoserine lactone (acyl-HSL) synthases to generate fatty acyl-HSL quorum sensing signals, which function with signal receptors to control expression of specific genes. The fatty acyl group is derived from fatty acid biosynthesis and provides signal specificity, but the variety of signals is limited. We have discovered that the photosynthetic bacterium Rhodopseudomonas palustris uses an acyl-HSL synthase to produce p-coumaroyl-HSL by using environmental p-coumaric acid rather than fatty acids from cellular pools. The bacterium has a signal receptor with homology to fatty acyl-HSL receptors that responds to p-coumaroyl-HSL to regulate global gene expression. We also found that p-coumaroyl-HSL is made by other bacteria including Bradyrhizobium BTAi1 and Silicibacter pomeroyi DSS-3. This discovery extends the range of possibilities for acyl-HSL quorum sensing and raises fundamental questions about quorum sensing within the context of environmental signaling. Keywords: Comparison of transcriptome profiles Transcriptome profiles between Rhodopseudomonas palustris cells grown in the in the presence or absence of pC-HSL were compared.