Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath.

The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO2 was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth's biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2. IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.

Fast and easy quantitative characterization of methanotroph-photoautotroph cocultures.

Recent research has demonstrated that synthetic methanotroph-photoautotroph cocultures offer a highly promising route to convert biogas into value-added products. However, there is a lack of techniques for fast and accurate characterization of cocultures, such as determining the individual biomass concentration of each organism in real-time. To address this unsolved challenge, we propose an experimental-computational protocol for fast, easy, and accurate quantitative characterization of the methanotroph-photoautotroph cocultures. Besides determining the individual biomass concentration of each organism in the coculture, the protocol can also obtain the individual consumption and production rates of O2 and CO2 for the methanotroph and photoautotroph, respectively. The accuracy and effectiveness of the proposed protocol was demonstrated using two model coculture pairs, Methylomicrobium alcaliphilum 20ZR-Synechococcus sp. PCC7002 that prefers high pH high salt condition, and Methylococcus capsulatus-Chlorella sorokiniana that prefers low salt and neutral pH medium. The performance of the proposed protocol was compared with a flow cytometry-based cell counting approach. The experimental results show that the proposed protocol is much easier to carry out and delivers faster and more accurate results in measuring individual biomass concentration than the cell counting approach without requiring any special equipment.

From micelles to bicelles: Effect of the membrane on particulate methane monooxygenase activity.

Particulate methane monooxygenase (pMMO) is a copper-dependent integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. Studies of isolated pMMO have been hindered by loss of enzymatic activity upon its removal from the native membrane. To characterize pMMO in a membrane-like environment, we reconstituted pMMOs from Methylococcus (Mcc.) capsulatus (Bath) and Methylomicrobium (Mm.) alcaliphilum 20Z into bicelles. Reconstitution into bicelles recovers methane oxidation activity lost upon detergent solubilization and purification without substantial alterations to copper content or copper electronic structure, as observed by electron paramagnetic resonance (EPR) spectroscopy. These findings suggest that loss of pMMO activity upon isolation is due to removal from the membranes rather than caused by loss of the catalytic copper ions. A 2.7 Å resolution crystal structure of pMMO from Mm. alcaliphilum 20Z reveals a mononuclear copper center in the PmoB subunit and indicates that the transmembrane PmoC subunit may be conformationally flexible. Finally, results from extended X-ray absorption fine structure (EXAFS) analysis of pMMO from Mm. alcaliphilum 20Z were consistent with the observed monocopper center in the PmoB subunit. These results underscore the importance of studying membrane proteins in a membrane-like environment and provide valuable insight into pMMO function.

Characterization of the pyrophosphate-dependent 6-phosphofructokinase from Methylococcus capsulatus Bath.

An active pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) from the thermotolerant methanotroph Methylococcus capsulatus Bath, containing a six-residue polyhistidine tag, was characterized. The enzyme was homodimeric (2 x 45 kDa), nonallosteric and most active at pH 7.0. PPi-PFK catalyzed reactions of PPi-dependent phosphorylation of fructose-6-phosphate (F-6-P) (K(m) 2.27 mM and V(max) 7.6 U mg(-1) of protein), sedoheptulose-7-phosphate (K(m) 0.027 mM and V(max) 31 U mg(-1)) and ribulose-5-phosphate. In the reaction with F-6-P, the apparent K(m) for PPi was 0.027 mM, while in the reverse reaction, K(m) for orthophosphate was 8.69 mM and that for fructose-1,6-bisphosphate 0.328 mM (V(max) 9.0 U mg(-1)). Phylogenetically, M. capsulatus PPi-PFK was most similar to PPi-PFKs from the lithoautotrophic ammonia oxidizers Nitrosomonas europaea (74.0%), Nitrosospira multiformis (73.6%) and Betaproteobacterial methylotroph Methylibium petroleiphilum PM1 (71.6% identity). Genes coding PPi-PFK and a putative V-type H(+)-translocating pyrophosphatase (H(+)-PPi-ase) were cotranscribed as an operon. The potential significance of the PPi-PFK for regulation of carbon and energy fluxes in M. capsulatus Bath is discussed.

[Features of solid materials' colonization by pure and mixed cultures of methanotrophs]. / Osobennosti kolonizatsii tverdykh materialov chistymi i smeshannymi kul'turami metanotrofov.

The process of colonization of hydrophilic (glass) and hydrophobic (polysterene) carriers by pure cultures of methanotrophs Methylocystis parvus UCM B-3490T, Methylococcus capsulatus UCM B-3030, as well as by their cultures mixed with Bacillus megaterium UCM B 5723T and Pseudomonas putida VKPM B-4188 under the conditions efficient for methanotrophic bacteria. M. parvus demonstrated the highest intensity of this process on the above carriers owing to high hydrophobic cell surface. Both methanotrophs colonized the glass surface more quickly with formation of microcolonies on carriers after 6 days of incubation in pure and mixed cultures with B. megaterium. The number of bacilli on these carriers quickly decreased. In the mixed cultures with P. putida the glass and polysterene colonization intensity decreased, while the amount of pseudomonas on carriers increased.

Production of high-quality particulate methane monooxygenase in high yields from Methylococcus capsulatus (bath) with a hollow-fiber membrane bioreactor.

In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from Methylococcus capsulatus (Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme. The optimal copper concentration in the growth medium was found to be 30 to 35 micro M. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant. The copper stoichiometry is approximately 13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes. Further purification by membrane solubilization in dodecyl beta-D-maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an alphabetagamma protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme.

The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath.

Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.

The surface-associated and secreted MopE protein of Methylococcus capsulatus (Bath) responds to changes in the concentration of copper in the growth medium.

Expression of surface-associated and secreted protein MopE of the methanotrophic bacterium Methylococcus capsulatus (Bath) in response to the concentration of copper ions in the growth medium was investigated. The level of protein associated with the cells and secreted to the medium changed when the copper concentration in the medium varied and was highest in cells exposed to copper stress.

[Effect of gas mixture component composition on the process of hard materials colonization by methanotrophic bacteria]. / Vliianie komponentnogo sostava gazovoi smesi na protsess kolonizatsii tverdykh materialov metanotrofnymi bakteriiami.

A device has been proposed which allows decreasing a possibility to contaminate the inoculum of methane-using bacteria (methanotrophs) under its growing. The gas mixture composition has been investigated for the intensity of grass and polysterene colonization by two species of methanotrophs: Methylococcus capsulatus UCM B-3030, possessing hydrophilic surface and hydrophobic Methylocystis parvus UKM B-3490T. It has been shown that immobilized methanotrophic bacteria can colonize the above materials in the wide range of methane and oxygen concentrations in gas mixture. The process of hard materials colonization was most intensive when the mixture contained 17-28% of methane and 5-28% of oxygen. No essential differences have been registered under colonization of hydrophilic (glass) and hydrophobic (polysterene) materials by the both cultures.

Heterotrophic bacteria growing in association with Methylococcus capsulatus (Bath) in a single cell protein production process.

The methanotrophic bacterium Methylococcus capsulatus (Bath) grows on pure methane. However, in a single cell protein production process using natural gas as methane source, a bacterial consortium is necessary to support growth over longer periods in continuous cultures. In different bioreactors of Norferm Danmark A/S, three bacteria consistently invaded M. capsulatus cultures growing under semi-sterile conditions in continuous culture. These bacteria have now been identified as a not yet described member of the Aneurinibacillus group, a Brevibacillus agri strain, and an acetate-oxidiser of the genus Ralstonia. The physiological roles of these bacteria in the bioreactor culture growing on natural, non-pure methane gas are discussed. The heterotrophic bacteria do not have the genetic capability to produce either the haemolytic enterotoxin complex HBL or non-haemolytic enterotoxin.

The ribulose-1,5-bisphosphate carboxylase/oxygenase gene cluster of Methylococcus capsulatus (Bath).

The genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Methylococcus capsulatus (Bath) were localised to an 8.3-kb EcoRI fragment of the genome. Genes encoding the large subunit ( cbbL), small subunit ( cbbS) and putative regulatory gene ( cbbQ) were shown to be located on one cluster. Surprisingly, cbbO, a second putative regulatory gene, was not located in the remaining 1.2-kb downstream (3') of cbbQ. However, probing of the M. capsulatus (Bath) genome with cbbO from Nitrosomonas europaea demonstrated that a cbbO homologue was contained within a separate 3.0-kb EcoRI fragment. Instead of a cbbR ORF being located upstream (5') of cbbL, there was a moxR-like ORF that was transcribed in the opposite direction to cbbL. There were three additional ORFs within the large 8.3-kb EcoRI fragment: a pyrE-like ORF, an rnr-like ORF and an incomplete ORF with no sequence similarity to any known protein. Phylogenetic analysis of cbbL from M. capsulatus (Bath) placed it within clade A of the green-type Form 1 Rubisco. cbbL was expressed in M. capsulatus (Bath) when grown with methane as a sole carbon and energy source under both copper-replete and copper-limited conditions. M. capsulatus (Bath) was capable of autotrophic growth on solid medium but not in liquid medium. Preliminarily investigations suggested that other methanotrophs may also be capable of autotrophic growth. Rubisco genes were also identified, by PCR, in Methylococcus-like strains and Methylocaldum species; however, no Rubisco genes were found in Methylomicrobium album BG8, Methylomonas methanica S1, Methylomonas rubra, Methylosinus trichosporium OB3b or Methylocystis parvus OBBP.

[Physiological activity of mixed cultures of Methylcoccus capsulatus UKM B-3030 with Bacillus megaterium UKM B-5723T and Bacillus subtilis VKPM B-1489 on solid surface colonization]. / Fiziologicheskaia aktivnost' smeshannykh kul'tur Methylococcus capsulatus UKM B-3030 s Bacillus megaterium UKM B-5723T i Bacillus subtilis VKPM B-4189 pri kolonizatsii tverdoi poverkhnosti.

Physiological activity of monoculture of Methylococcus capsulatus UCM B-3030 and its mixed cultures with two bacilli species distinguished by proteolytical properties--Bacillus megaterium UCM B-5723T and Bacillus subtilis BK[symbol: see text]M B-4189 in terms of long-duration cultivation is investigated. It is shown, that the most active methane consumption by methanotrophic bacteria and their mixed cultures with bacilli under solid surface colonization occurred on the second-eighth day of cultivation. The obtained results permit one to evaluate intensity of CH4 oxidation process by typical microflora of some econiches (depleted coal mines, in particular).

Expression of individual copies of Methylococcus capsulatus bath particulate methane monooxygenase genes.

The expression of the two gene clusters encoding the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus Bath was assessed by analysis of transcripts and by use of chromosomal gene fusions. The results suggest that the two clusters are functionally redundant but that relative expression alters depending on the copper levels available for growth.

Molecular analysis of an outer membrane protein, MopB, of Methylococcus capsulatus (Bath) and structural comparisons with proteins of the OmpA family.

The gene encoding a major outer membrane protein (MopB) of the methanotroph Methylococcus capsulatus (Bath) was cloned and sequenced. The cloned DNA contained an open reading frame of 1044 bp coding for a 348-amino-acid polypeptide with a 21-amino-acid leader peptide. Comparative sequence analysis of the predicted amino acid sequence revealed that the C-terminal part of MopB possessed sequences that are conserved in the OmpA family of proteins. The N-terminal half of the protein had no significant sequence similarity to other proteins in the databases, but the predicted secondary structure showed stretches of amphipathic beta-strands typical of transmembrane segments of outer membrane proteins. A region with four cysteines similar to the cysteine-encompassing region of the OprF of Pseudomonas aeruginosa was found toward the C-terminal part of MopB. Results from whole-cell labeling with the fluorescent thiol-reacting reagent 5-iodoacetamidofluorescein indicated a surface-exposed location for these cysteines. A probe consisting of the 3'-end of the mopB gene hybridized to the type I methanotroph Methylomonas methanica S in Southern blots containing DNA from nine methanotrophic strains representing six different genera.