Genome-scale modeling of light-driven reductant partitioning and carbon fluxes in diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142.

Genome-scale metabolic models have proven useful for answering fundamental questions about metabolic capabilities of a variety of microorganisms, as well as informing their metabolic engineering. However, only a few models are available for oxygenic photosynthetic microorganisms, particularly in cyanobacteria in which photosynthetic and respiratory electron transport chains (ETC) share components. We addressed the complexity of cyanobacterial ETC by developing a genome-scale model for the diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142. The resulting metabolic reconstruction, iCce806, consists of 806 genes associated with 667 metabolic reactions and includes a detailed representation of the ETC and a biomass equation based on experimental measurements. Both computational and experimental approaches were used to investigate light-driven metabolism in Cyanothece sp. ATCC 51142, with a particular focus on reductant production and partitioning within the ETC. The simulation results suggest that growth and metabolic flux distributions are substantially impacted by the relative amounts of light going into the individual photosystems. When growth is limited by the flux through photosystem I, terminal respiratory oxidases are predicted to be an important mechanism for removing excess reductant. Similarly, under photosystem II flux limitation, excess electron carriers must be removed via cyclic electron transport. Furthermore, in silico calculations were in good quantitative agreement with the measured growth rates whereas predictions of reaction usage were qualitatively consistent with protein and mRNA expression data, which we used to further improve the resolution of intracellular flux values.

Feedback-controlled LED photobioreactor for photophysiological studies of cyanobacteria.

A custom photobioreactor was designed to enable automatic light adjustments using computerized feedback control. The system consisted of a 7.5-L cylindrical vessel and an aluminum enclosure housing quantum sensors and light-emitting diode arrays, which provide 630 or 680 nm light to preferentially excite the major cyanobacterial pigments, phycocyanin and/or chlorophyll a, respectively. Custom-developed software rapidly measures light transmission and subsequently adjusts the irradiance to maintain a defined light profile to compensate for culture dynamics, biomass accumulation, and pigment adaptations during physiological transitions, thus ensuring appropriate illumination across batch and continuous growth modes. In addition to chemostat cultivation, the photobioreactor may also operate as a turbidostat, continuously adjusting the media dilution to achieve maximal growth at a fixed culture density. The cultivation system doubles as an analytical device, using real-time monitoring to avoid sampling bias (e.g., in-situ light-saturation response), determine conditions for optimal growth, and observe perturbation responses at high time-resolution.

Methylophilaceae link methanol oxidation to denitrification in freshwater lake sediment as suggested by stable isotope probing and pure culture analysis.

In this work we assessed the potential for the denitrification linked to methanol consumption in a microbial community inhabiting the top layer of the sediment of a pristine lake, Lake Washington in Seattle. Stable isotope probing with (13) C methanol was implemented in near in situ conditions and also in the presence of added nitrate. This revealed that the bacterial population involved in methanol uptake was dominated by species belonging to the Methylophilaceae, most prominently species belonging to the genus Methylotenera. Based on relative abundance of specific phylotypes in DNA clone libraries generated from (13) C labelled DNA, some of these species appear not to require nitrate to assimilate methanol while others assimilate methanol in a nitrate-dependent fashion. A pure culture of Methylotenera mobilis strain JLW8 previously isolated from the same study site was investigated for denitrification capability. This culture was demonstrated to be able to grow on methanol when nitrate was present, in aerobic conditions, while in media supplemented with ammonium it did not grow on methanol. The denitrifying capability of this strain was further demonstrated in defined laboratory conditions, by measuring accumulation of N2 O. This study provides new insights into the potential involvement of Methylophilaceae in global nitrogen cycling in natural environments and highlights the connection between global carbon and nitrogen cycles.

Methylosarcina lacus sp. nov., a methanotroph from Lake Washington, Seattle, USA, and emended description of the genus Methylosarcina.

An obligately methanotrophic bacterial strain, LW14T, isolated from the sediment of Lake Washington, Seattle, USA, is described taxonomically. The isolate is an aerobic, Gram-negative, non-motile bacterium capable of growth on methane, and possesses type I intracytoplasmic membranes (i.e. it is a type I methanotroph). The strain possesses particulate methane monooxygenase (MMO) and has no soluble MMO. Formaldehyde is assimilated via the ribulose monophosphate cycle. The isolate grows within a pH range of 4-8, with the optimum between pH 5.5 and 6.5. The cellular fatty acid profile is dominated by C(16 : )omega18c, C(16 : 1)omega7c and C(16 : 1)omega5t fatty acids. The DNA G+C content is 53.3+/-0.4 mol%. On the basis of sequence analysis of the 16S rRNA gene, isolate LW14T is related most closely to representatives of the genus Methylosarcina. However, DNA-DNA hybridization analysis reveals only a distant relationship between isolate LW14T and the previously described Methylosarcina species. On the basis of its phenotypic and genotypic characteristics, LW14T represents a novel species of the genus Methylosarcina, for which the name Methylosarcina lacus sp. nov. is proposed, with LW14T (=ATCC BAA-1047T=JCM 13284T) as the type strain.