A Complex Interplay between Nitric Oxide, Quorum Sensing, and the Unique Secondary Metabolite Tundrenone Constitutes the Hypoxia Response in Methylobacter

Abstract

Methylobacter species, members of the Methylococcales, have recently emerged as some of the globally widespread, cosmopolitan species that play a key role in the environmental consumption of methane across gradients of dioxygen tensions. In this work, we approached the question of how Methylobacter copes with hypoxia, via laboratory manipulation. Through comparative transcriptomics of cultures grown under high dioxygen partial pressure versus cultures exposed to hypoxia, 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 the hypoxia stress response. Through additional transcriptomic analyses, we uncovered a complex interconnection between the NO-mediated stress response, quorum sensing, the secondary metabolite tundrenone, and methanol dehydrogenase functions. This novel and complex hypoxia stress response system is so far unique to Methylobacter species, and it may play a role in the environmental fitness of these organisms and in their cosmopolitan environmental distribution. IMPORTANCE Here, we describe a novel and complex hypoxia response system in a methanotrophic bacterium that involves modules of central carbon metabolism, denitrification, quorum sensing, and a secondary metabolite, tundrenone. This intricate stress response system, so far unique to Methylobacter species, may be responsible for the persistence and activity of these species across gradients of dioxygen tensions and for the cosmopolitan distribution of these organisms in freshwater and soil environments in the Northern Hemisphere, including the fast-melting permafrosts.

Metagenomic Insight into Environmentally Challenged Methane-Fed Microbial Communities

“…The expression pattern for this system was of special interest to us, especially since the respective genes, hcp/hcr , were some of the most highly expressed genes in the microcosms, especially by the Methylobacter species (not shown), as noted previously for the hypoxic cultures [ 12 ]. Also, as noted previously for the hypoxic cultures [ 12 ], the expression of hcp/hcr had little correlation with the expression of the (partial) denitrification pathway in Methylobacter , with nitrate reductase and other denitrification genes being expressed at less than one order of magnitude compared to hcp/hcr (data for hcp , annotated as nitric oxide reductase, and narG expression are presented in Table S1 ). Interestingly, the hcp/hcr pair was also expressed in the Methylosarcina species ( Table S1 ), suggesting that the hypoxia stress response among the species of these two genera must overlap.…”

“…We recently described a unique hypoxia stress response system in Methylobacter that involves nitric oxide reductase (NOR), as well as a quorum sensing system regulating the synthesis of a so-far unique secondary metabolite tundrenone [ 12 ]. The expression pattern for this system was of special interest to us, especially since the respective genes, hcp/hcr , were some of the most highly expressed genes in the microcosms, especially by the Methylobacter species (not shown), as noted previously for the hypoxic cultures [ 12 ].…”

“…We recently described a unique hypoxia stress response system in Methylobacter that involves nitric oxide reductase (NOR), as well as a quorum sensing system regulating the synthesis of a so-far unique secondary metabolite tundrenone [ 12 ]. The expression pattern for this system was of special interest to us, especially since the respective genes, hcp/hcr , were some of the most highly expressed genes in the microcosms, especially by the Methylobacter species (not shown), as noted previously for the hypoxic cultures [ 12 ]. Also, as noted previously for the hypoxic cultures [ 12 ], the expression of hcp/hcr had little correlation with the expression of the (partial) denitrification pathway in Methylobacter , with nitrate reductase and other denitrification genes being expressed at less than one order of magnitude compared to hcp/hcr (data for hcp , annotated as nitric oxide reductase, and narG expression are presented in Table S1 ).…”

“…DNA and RNA were extracted following the published protocols, and their quantity and quality were monitored as previously described [ 12 , 15 , 16 , 17 , 18 ]. The preparations were mailed on dry ice to the DOE Joint Genome Institute (JGI) facility for analysis.…”

“…However, more recently, a concept of communal metabolism of methane has become prominent, providing a more complex outlook at the environmental function of the aerobic methanotrophs [ 6 , 7 , 8 ]. Another conceptual change in understanding the aerobic methanotrophy involves the realization that aerobic methanotrophs are likely dioxygen-limited in their natural environments, and, in many studies, they have been inferred to be active under hypoxic and even anoxic conditions [ 9 , 10 , 11 , 12 ]. Furthermore, oxygen sensitivity has been demonstrated for some aerobic methanotrophs [ 13 , 14 ].…”