Agarose-Based Model Ecosystem for Cultivating Methanotrophs in a Methane-Oxygen Counter Gradient

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental ...

University of Utah

Multiple species of bacteria oxidize methane in the environment after it is produced by anaerobic ecosystems. These organisms provide reduced carbon substrates 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 structures and functions of these communities. Here we show that many members of the  genus possess a LuxR-type acyl-homoserine lactone (acyl-HSL) receptor/transcription factor that is highly homologous to MbaR from the quorum-sensing (QS) system of , another methane oxidizer that has been isolated from the same environment. We reconstitute this detection system in  and use mutant and transcriptomic analysis to show that the receptor/transcription factor from  sp. strain LW13 is active and alters LW13 gene expression in response to the acyl-HSL produced by  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. Methanotrophs are bacteria that sequester methane, a significant greenhouse gas, and thereby perform an important ecosystem function. Understanding the mechanisms by which these organisms interact in the environment may ultimately allow us to manipulate and to optimize this activity. Here we show that members of a genus of methane-oxidizing bacteria can be influenced by a chemical signal produced by a possibly competing species. This provides insight into how gene expression can be controlled in these bacterial communities via an exogenous chemical signal.

Interspecies Chemical Signaling in a Methane-Oxidizing Bacterial Community.

Biosynthesized small molecules known as specialized metabolites often have valuable applications in fields such as medicine and agriculture. Consequently, there is always a demand for novel specialized metabolites and an understanding of their bioactivity. Methylotrophs are an underexplored metabolic group of bacteria that have several growth features that make them enticing in terms of specialized metabolite discovery, characterization, and production from cheap feedstocks such as methanol and methane gas. This chapter will examine the predicted biosynthetic potential of these organisms and review some of the specialized metabolites they produce that have been characterized so far.

Specialized Metabolites from Methylotrophic Proteobacteria.

Natural products are an essential source of bioactive compounds. Isotopic labeling is an effective way to identify natural products that incorporate a specific precursor; however, this approach is limited by the availability of isotopically enriched precursors. We used an inverse stable isotopic labeling approach to identify natural products by growing bacteria on a C-carbon source and then identifying C-precursor incorporation by mass spectrometry. We applied this approach to methylotrophs, ecologically important bacteria predicted to have significant yet underexplored biosynthetic potential. We demonstrate that this method identifies acyl homoserine lactone quorum sensing signals produced by diverse methylotrophs grown on three different one-carbon compounds. We then apply this approach to simultaneously detect five previously unidentified signals produced by a methylotroph and link these compounds to their synthases. We envision that this method can be used to identify other natural product classes synthesized by methylotrophs and other organisms that grow on relatively inexpensive C-carbon sources.

Methylotroph Quorum Sensing Signal Identification by Inverse Stable Isotopic Labeling.

GNPS Dashboard: collaborative exploration of mass spectrometry data in the web browser.

Bacterial symbionts often provide critical functions for their hosts. For example, wood-boring bivalves called shipworms rely on cellulolytic endosymbionts for wood digestion. However, how the relationship between shipworms and their bacterial symbionts is formed and maintained remains unknown. Quorum sensing (QS) often plays an important role in regulating symbiotic relationships. We identified and characterized a QS system found in  sp. strain 2052S, a gill isolate of the wood-boring shipworm  cf. . We determined that 2052S produces the signal decanoyl-l-homoserine lactone (C-HSL) and that this signal controls the activation of a biosynthetic gene cluster colocated in the symbiont genome that is conserved among all symbiotic  isolates. We subsequently identified extracellular metabolites associated with the QS regulon, including ones linked to the conserved biosynthetic gene cluster, using mass spectrometry-based molecular networking. Our results demonstrate that QS plays an important role in regulating secondary metabolism in this shipworm symbiont. This information provides a step toward deciphering the molecular details of the relationship between these symbionts and their hosts. Furthermore, because shipworm symbionts harbor vast yet underexplored biosynthetic potential, understanding how their secondary metabolism is regulated may aid future drug discovery efforts using these organisms.  Bacteria play important roles as symbionts in animals ranging from invertebrates to humans. Despite this recognized importance, much is still unknown about the molecular details of how these relationships are formed and maintained. One of the proposed roles of shipworm symbionts is the production of bioactive secondary metabolites due to the immense biosynthetic potential found in shipworm symbiont genomes. Here, we report that a shipworm symbiont uses quorum sensing to coordinate activation of its extracellular secondary metabolism, including the transcriptional activation of a biosynthetic gene cluster that is conserved among many shipworm symbionts. This work is a first step toward linking quorum sensing, secondary metabolism, and symbiosis in wood-boring shipworms.

A Conserved Biosynthetic Gene Cluster Is Regulated by Quorum Sensing in a Shipworm Symbiont.

Natural product-encoding biosynthetic gene clusters (BGCs) within microbial genomes far outnumber the known natural products; chemical products from such BGCs remain cryptic. These silent BGCs hold promise not only for the elaboration of new natural products but also for the discovery of useful biosynthetic enzymes. Here, we describe a genome mining strategy targeted toward the discovery of substrate promiscuous natural product biosynthetic enzymes. In the genome of the methanotrophic bacterium  Sph1, we discover a transcriptionally silent natural product BGC that encoded numerous ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. These cryptic RiPP natural products were accessed using heterologous expression of the substrate peptide and biosynthetic enzyme-encoded genes. In line with our genome mining strategy, the RiPP biosynthetic enzymes in this BGC were found to be substrate promiscuous, which allowed us to use them in a combinatorial fashion with a similarly substrate-tolerant cyanobactin biosynthetic enzyme to introduce head-to-tail macrocyclization in the proteusin family of RiPP natural products.

A Silent Biosynthetic Gene Cluster from a Methanotrophic Bacterium Potentiates Discovery of a Substrate Promiscuous Proteusin Cyclodehydratase.

Members of the genus  which are core components of the rhizosphere and specific symbionts of legume plants, possess genes for acyl-homoserine lactone (AHL) quorum sensing (QS). Here we show  MAFF 303099 (formerly ) synthesizes and responds to -[(2, 4)-2,4-dodecadienoyl] homoserine lactone (2 4C-HSL). We show that the 2 4C-HSL QS circuit involves one of four -type genes found in the sequenced genome of MAFF 303099. We refer to this circuit, which appears to be conserved among  species, as R1-I1. We show that two other  strains also produce 2 4C-HSL. The 2 4C-HSL is unique among known AHLs in its arrangement of two  double bonds. The R1 response to 2 4C-HSL is extremely selective in comparison with other LuxR homologs, and the  double bonds appear critical for R1 signal recognition. Most well-studied LuxI-like proteins use -adenosylmethionine and an acyl-acyl carrier protein as substrates for synthesis of AHLs. Others that form a subgroup of LuxI-type proteins use acyl-coenzyme A substrates rather than acyl-acyl carrier proteins. I1 clusters with the acyl-coenzyme A-type AHL synthases. We show that a gene linked to the  AHL synthase is involved in the production of the QS signal. The discovery of the unique I1 product enforces the view that further study of acyl-coenzyme A-dependent LuxI homologs will expand our knowledge of AHL diversity. The involvement of an additional enzyme in AHL generation leads us to consider this system a three-component QS circuit. IMPORTANCE We report a  quorum sensing (QS) system involving a novel acyl-homoserine lactone (AHL) signal. This system is known to be involved in root nodule symbiosis with host plants. The chemistry of the newly described QS signal indicated that there may be a dedicated cellular enzyme involved in its synthesis in addition to the types known for production of other AHLs. Indeed, we report that an additional gene is required for synthesis of the unique signal, and we propose that this is a three-component QS circuit as opposed to the canonical two-component AHL QS circuits. The signaling system is exquisitely selective. The selectivity may be important when this species resides in the complex microbial communities around host plants and may make this system useful in various synthetic biology applications of QS circuits.

A  quorum sensing circuit that involves three linked genes and an unusual acyl-homoserine lactone signal.

Bacteria known as pink-pigmented facultative methylotrophs colonize many diverse environments on earth, play an important role in the carbon cycle, and in some cases promote plant growth. However, little is known about how these organisms interact with each other and their environment. In this work, we identify one of the chemical signals commonly used by these bacteria and discover that this signal controls swarming motility in the pink-pigmented facultative methylotroph  DSM5686. This work provides new molecular details about interactions between these important bacteria and will help scientists predict these interactions and the group behaviors they regulate from genomic sequencing information.

A widespread methylotroph acyl-homoserine lactone synthase produces a new quorum sensing signal that regulates swarming in .

Connecting genes to phenotypic traits in bacteria is often challenging because of a lack of environmental context in laboratory settings. Laboratory-based model ecosystems offer a means to better account for environmental conditions compared with standard planktonic cultures and can help link genotypes and phenotypes. Here, we present a simple, cost-effective, laboratory-based model ecosystem to study aerobic methane-oxidizing bacteria (methanotrophs) within the methane-oxygen counter gradient typically found in the natural environment of these organisms. Culturing the methanotroph Methylomonas sp. strain LW13 in this system resulted in the formation of a distinct horizontal band at the intersection of the counter gradient, which we discovered was not due to increased numbers of bacteria at this location but instead to an increased amount of polysaccharides. We also discovered that different methanotrophic taxa form polysaccharide bands with distinct locations and morphologies when grown in the methane-oxygen counter gradient. By comparing transcriptomic data from LW13 growing within and surrounding this band, we identified genes upregulated within the band and validated their involvement in growth and band formation within the model ecosystem using knockout strains. Notably, deletion of these genes did not negatively affect growth using standard planktonic culturing methods. This work highlights the use of a laboratory-based model ecosystem that more closely mimics the natural environment to uncover bacterial phenotypes missing from standard laboratory conditions, and to link these phenotypes with their genetic determinants.

Linking methanotroph phenotypes to genotypes using a simple spatially resolved model ecosystem.

The sequencing of microbial genomes has far outpaced their functional annotation. Stable isotopic labeling can be used to link biosynthetic genes with their natural products; however, the availability of the required isotopically substituted precursors can limit the accessibility of this approach. Here, we describe a method for using inverse stable isotopic labeling (InverSIL) to link biosynthetic genes with their natural products. With InverSIL, a microbe is grown on an isotopically substituted medium to create a fully substituted culture, and subsequently, the incorporation of precursors of natural isotopic abundance can be tracked by mass spectrometry. This eliminates issues with isotopically substituted precursor availability. We demonstrate the utility of this approach by linking a luxI-type acyl-homoserine lactone synthase gene in a bacterium that grows on methanol with its quorum sensing signal products. In the future, InverSIL can also be used to link biosynthetic gene clusters hypothesized to produce siderophores with their natural products.

Aaron W. Puri

Department of Chemistry and the Henry Eyring Center for Cell and Genome Science

University of Utah

Agarose-Based Model Ecosystem for Cultivating Methanotrophs in a Methane-Oxygen Counter Gradient

Aaron W. Puri

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemistry and the Henry Eyring Center for Cell and Genome Science

University of Utah

Agarose-Based Model Ecosystem for Cultivating Methanotrophs in a Methane-Oxygen Counter Gradient

Department of Chemistry and the Henry Eyring Center for Cell and Genome Science