Methanotrophic Bacterium Methylococcus Capsulatus Research Articles

Transcriptional Regulation of Methanol Dehydrogenases in the Methanotrophic Bacterium Methylococcus capsulatus Bath by Soluble and Insoluble Lanthanides.

The effects of soluble and insoluble lanthanides on gene expression in Methylococcus capsulatus Bath were investigated. Genes for lanthanide-containing methanol dehydrogenases (XoxF-MDHs) and their calcium-containing counterparts (MxaFI-MDHs) were up- and down-regulated, respectively, by supplementation with soluble lanthanide chlorides, indicating that M. capsulatus has the "lanthanide switch" observed in other methanotrophs. Insoluble lanthanide oxides also induced the lanthanide switch and were dissolved by the spent medium of M. capsulatus, suggesting the presence of lanthanide-chelating compounds. A transcriptome ana-lysis indicated that a gene cluster for the synthesis of an enterobactin-like metal chelator contributed to the dissolution of insoluble lanthanides.

Alternative protein sources: bacteria Methylococcus capsulatus concentrate, characteristic of composition and biological value

A promising growth vector of food protein production in the context of the Russian Federation's food sovereignty security is the use of microbial synthesis. Taking into consideration the proven promising use of biotechnological processes in the production of alternative protein sources, modern scientific research is focused, among other issues, on improving the technology of obtaining food microbial protein using a variety of substrates and strains-producers, as well as evaluating the consumer properties, food, biological value and safety of such products. The purpose of the research was to study and comparatively evaluate protein concentrate (PC) from bacteria Methylococcus capsulatus and basic food of animal and plant origin within the development of the technology of optimal in nutritional and biological value PC production. Material and methods. Analysis of the nutritional and biological value of PC obtained from denucleinized and purified from cell walls biomass of methanoxidizing bacteria Methylococcus capsulatus (strain GSB-15) was carried out on 46 indicators, including estimation of protein content and amino acid composition, fat content and fatty acid composition, ash and moisture. Biological studies based on measuring of net protein ratio / net protein utilization were performed on 28 growing (between 25-50 days of life) male Wistar rats. Rats in the control group (n=14) received a semi-synthetic casein diet with a protein content of ~12% in calories, the test group (n=14) received a diet including an equivalent amount of PC protein. Body weight, feed intake, and fecal and urine nitrogen losses were measured during the experiment. The biological value and digestibility of protein were judged by coefficients of protein efficiency ratio, net protein ratio, true protein digestibility, true protein biological value, true net protein utilization. Results. The nutritional value study of PC showed high protein content - 69.0%, the share of fat, moisture and ash, accounted for 0.17, 9.5 and 14.4%, respectively. The carbohydrate content was 7.0% (of which mono- and disaccharides were <0.1%). The results of a comparative assessment of Methylococcus capsulatus protein amino acid profile and basic food of animal and plant origin showed a balanced content of the most amino acids, the level of which is comparable with the protein of chicken egg, which is traditionally a standard of quality of complete protein. At the same time, the content of the essential amino acid tryptophan in PC was an order of magnitude lower than in chicken egg protein; the content of this amino acid in PC is comparable with incomplete plant proteins (sunflower, flax, rapeseed). The results of the biological value evaluation of the Methylococcus capsulatus protein in the experiment on rats indicate a relatively low biological value of the microbial synthesis protein, that is caused, most likely, by tryptophan deficiency. Rats of the test group had a significant decrease in body weight gain, feed/protein intake, coefficient of protein efficiency ratio, coefficient of net protein ratio, true protein biological value, true net protein utilization. Conclusion. The results of a comparative evaluation of PC from methanotrophic bacteria Methylococcus capsulatus denucleinized biomass and basic food of animal and plant origin indicate its relatively high nutritional value. However, the characteristics of this PC sample were not optimal in regard of protein biological value by reason of tryptophan deficiency. A single amino acid deficiency is not a valid argument for not using microbially synthesized protein in human nutrition, considering the capabilities of the modern food industry, including ways to enrich foodstuffs with missing components. In addition, there is every cause to believe that adjusting the hydrolysis technology used in the production of PC will allow to eliminate the essential amino acid loss, thereby increasing the biological value of this product.

Oxidation of L-Ascorbic Acid in the Presence of the Copper-Binding Compound from Methanotrophic Bacteria Methylococcus capsulatus (M).

The oxidation of ascorbic acid by air oxygen and hydrogen peroxide in the presence of the copper-binding compound (cbc) from bacteria Methylococcus capsulatus (M) was studied. The rate constant of ascorbic acid oxidation by air oxygen in the presence of the copper complex with cbc from M. capsulatus (M) was shown to be 1.5 times higher than that of the noncatalytic reaction. The rate constant of ascorbic acid oxidation by hydrogen peroxide in the presence of the copper complex with cbc from M. capsulatus (M) decreased by almost one-third compared to the reaction in the absence of the copper complex with cbc. It was assumed that cbc can be involved in a multilevel system of antioxidant protection and can protect a bacterial cell from oxidation stress. Thus, the cbc is mimetic ascorbate oxidase in the oxidation of ascorbic acid by molecular oxygen.

Methanotroph (Methylococcus capsulatus, Bath) bacteria meal as an alternative protein source for Japanese yellowtail, Seriola quinqueradiata

Two trials were carried out to determine the optimal replacement level of fish meal (FM) protein by the bacterial protein meal FeedKind® (FK), in the diet of juvenile Japanese yellowtail, Seriola quinqueradiata. FK protein was produced from the methanotrophic bacteria Methylococcus capsulatus Bath, which utilizes methane as its sole carbon and energy source. In Trial 1, six diets were formulated: control diet (C) with FM as protein source; four experimental diets in which the FM of diet C was replaced at 25 (FK25), 50 (FK50), 75 (FK75) and 100% (FK100) by FK; and a final experimental diet in which 3% of FK in diet FK100 was replaced by enzyme-treated FM (EFK). In Trial 2, seven diets were prepared: control diet C similar to Trial 1; three experimental diets in which the FM of diet C was replaced at 20 (FK20), 25 (FK25) and 30% (FK30) by FK; two experimental diets in which FK from diet FK25 was replaced either by further grinding FK (FK25J) or lower digestible FK (FK25L); and experimental diet EFK in which 3% of the FK in diet FK25 was replaced by enzyme-treated FM. Fifteen (ca. 126g) or ten fish (ca. 80 g) were stocked into each of eighteen 500 L tanks in triplicates in Trial 1 and 2, respectively, fed two times daily until apparent satiation, and cultured for 8 weeks. At the end of both trials, feces were collected after feeding with chromic oxide (Cr2O3) mixed diets. In Trial 1, all growth parameters and nutrients retention efficiencies showed similar patterns and no significant differences were observed between diets C and FK25 (P > .05), though feed efficiency (FE) was reduced by 10% in the latter diet. However, other diets showed significantly lower growth performance compared to diet C (P < .05). When different lots of FK was used and feed formula was slightly adjusted in Trial 2, there were no significant differences in the growth performance, nutrients digestibility and retention efficiency, among the treatments even up to 30% FM protein replacement (P > .05). FE was also either equal to or higher in all FK-based diets compared with control group. Overall, results indicated that the FK can comprise up to 20% of total diet, replacing 30% of the FM protein in diet for yellowtail, without compromising growth performance or FE.

Dynamic investigation and modeling of the nitrogen cometabolism in Methylococcus capsulatus ( Bath).

The methanotrophic bacterium Methylococcus capsulatus is capable of assimilating methane and oxygen into protein-rich biomass, however, the diverse metabolism of the microorganism also allows for several undesired cometabolic side-reactions to occur. In this study, the ammonia cometabolism in Methylococcus capsulatus is investigated using pulse experiments. Surprisingly Methylococcus capsulatus oxidizes ammonia to nitrate through a yet unknown mechanism and fixes molecular nitrogen even at a high dissolved oxygen tension. The observed phenomena can be modeled using 14 ordinary differential equations and 18 kinetic parameters, of which 6 were revealed by Morris screening to be identifiable from the experimental data. Monte Carlo simulations showed that the model was robust and accurate even with uncertainty in the parameter values as confirmed by statistical error analysis.

Extracellular Electron Transfer via Outer Membrane Cytochromes in a Methanotrophic Bacterium Methylococcus capsulatus (Bath).

Electron exchange reactions between microbial cells and solid materials, referred to as extracellular electron transfer (EET), have attracted attention in the fields of microbial physiology, microbial ecology, and biotechnology. Studies of model species of iron-reducing, or equivalently, current-generating bacteria such as Geobacter spp. and Shewanella spp. have revealed that redox-active proteins, especially outer membrane c-type cytochromes (OMCs), play a pivotal role in the EET process. Recent (meta)genomic analyses have revealed that diverse microorganisms that have not been demonstrated to have EET ability also harbor OMC-like proteins, indicating that EET via OMCs could be more widely preserved in microorganisms than originally thought. A methanotrophic bacterium Methylococcus capsulatus (Bath) was reported to harbor multiple OMC genes whose expression is elevated by Cu starvation. However, the physiological role of these genes is unknown. Therefore, in this study, we explored whether M. capsulatus (Bath) displays EET abilities via OMCs. In electrochemical analysis, M. capsulatus (Bath) generated anodic current only when electron donors such as formate were available, and could reduce insoluble iron oxides in the presence of electron donor compounds. Furthermore, the current-generating and iron-reducing activities of M. capsulatus (Bath) cells that were cultured in a Cu-deficient medium, which promotes high levels of OMC expression, were higher than those cultured in a Cu-supplemented medium. Anodic current production by the Cu-deficient cells was significantly suppressed by disruption of MCA0421, a highly expressed OMC gene, and by treatment with carbon monoxide (CO) gas (an inhibitor of c-type cytochromes). Our results provide evidence of EET in M. capsulatus (Bath) and demonstrate the pivotal role of OMCs in this process. This study raises the possibility that EET to solid compounds is a novel survival strategy of methanotrophic bacteria.

Effects of the non-commensal Methylococcus capsulatus Bath on mammalian immune cells

Dietary inclusions of a bacterial meal consisting mainly of the non-commensal, methanotrophic bacteria Methylococcus capsulatus Bath have been shown to ameliorate symptoms of intestinal inflammation in different animal models. In order to investigate the molecular mechanisms causing these effects, we have studied the influence of this strain on different immune cells central for the regulation of inflammatory responses. Effects were compared to those induced by the closely related strain M. capsulatus Texas and the well-described probiotic strain Escherichia coli Nissle 1917.M. capsulatus Bath induced macrophage polarization toward a pro-inflammatory phenotype, but not to the extent observed after exposure to E. coli Nissle 1917. Likewise, dose-dependent abilities to activate NF-κB transcription in U937 cells were observed, with E. coli Nissle 1917 being most potent. High levels of CD141 on human primary monocyte-derived dendritic cells (moDCs) were only detected after exposure to E. coli Nissle 1917, which collectively indicate a superior capacity to induce Th1 cell responses for this strain. On the other hand, the M. capsulatus strains were more potent in increasing the expression of the maturation markers CD80, CD83 and CD86 than E. coli Nissle 1917. M. capsulatus Bath induced the highest levels of IL-6, IL-10 and IL-12 secretion from dendritic cells, suggesting that this strain generally the post potent inducer of cytokine secretion.These results show that M. capsulatus Bath exhibit immunogenic properties in mammalian in vitro systems which diverge from that of E. coli Nissle 1917. This may provide clues to how M. capsulatus Bath influence the adaptive immune system in vivo. However, further in vivo experiments are required for a complete understanding of how this strain ameliorates intestinal inflammation in animal models.

The Methylococcus capsulatus (Bath) Secreted Protein, MopE*, Binds Both Reduced and Oxidized Copper

Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (A|| = 20 mT). Immobilized metal affinity chromatography binding studies suggests that residues in the N-terminal part of MopE* are involved in forming binding site(s) for Cu(II) ions. Our results support the hypothesis that MopE plays an important role in copper uptake, possibly making use of both its high (Cu(I) and low Cu(II) affinity properties.

Fatty acid composition, oxidative stability and sensory quality of meat from broiler chicken fed autolysate from bacteria grown on natural gas

Bacterial autolysate, a down stream product of bacterial biomass grown on natural gas by mainly the methanotrophic bacteria Methylococcus capsulatus, was fed at 8% as is to broiler chickens from 1 to 35 days of age for studies of fatty acid composition, lipid oxidation and sensory quality of thigh meat stored frozen for 6 month at -18 °C or -80 °C. Lipid oxidation was measured by thiobarbituric acid reactive substances (TBARS) and volatile profile by dynamic headspace gas chromatography. Adding bacterial autolysate to diets did not affect the total content of saturated, monounsaturated or polyunsaturated fatty acids in thigh meat, but increased the levels of C14:0, C16:0, C18:0 and C16:1n-7 and reduced the levels of C18:1n-7, C18:2n-6 and C18:3n-3 fatty acids. Feeding of bacterial autolysate tended (p < 0.08) to reduce TBARS of meat samples. Contents of volatiles were generally low, but feeding of bacterial autolysate significantly reduced levels of butanal (p < 0.04) and tended to reduce levels of hexanal (p < 0.11), pentanal (p < 0.09), 1-penten-3-ol (p < 0.08) and butanone (p < 0.08). Bacterial autolysate had no effects on sensory quality parameters of meat related to odour and flavour. To conclude, adding bacterial autolysate to diets did not affect the relative proportion of saturated, monounsaturated or polyunsaturated fatty acids, but reduced content of volatiles in frozen-stored broiler meat. The reduced susceptibility to lipid oxidation in broiler meat may be related to antioxidant properties of the bacterial autolysate.

Protective properties of gold in the composition of nanoparticles and Au—rutin complexes

Protective properties of gold nanoparticles and gold—rutin complexes were studied. Aurophilic bacteria Micrococcus luteus and methanotrophic bacteria Methylococcus capsulatus were studied. Gold—rutin nanoparticles and complexes protect the respiratory activity of the bacteria against toxins. Pretreatment of the cells with gold is more efficient than the treatment after the action of toxin.

Physiological evidence for the presence of a cis-trans isomerase of unsaturated fatty acids in Methylococcus capsulatus Bath to adapt to the presence of toxic organic compounds

The physiology of the response in the methanotrophic bacterium Methylococcus capsulatus Bath towards thermal and solvent stress was studied. A systematic investigation of the toxic effects of organic compounds (chlorinated phenols and alkanols) on the growth of this bacterium was carried out. The sensitivity to the tested alkanols correlated with their chain length and hydrophobicity; methanol was shown to be an exception to which the cells showed a very high tolerance. This can be explained by the adaptation of these bacteria to growth on C1 compounds. On the other hand, M. capsulatus Bath was very sensitive towards the tested chlorinated phenols. The high toxic effect of phenolic compounds on methanotrophic bacteria might be explained by the occurrence of toxic reactive oxygen species. In addition, a physiological proof of the presence of cis-trans isomerization as a membrane-adaptive response mechanism in M. capsulatus was provided. This is the first report on physiological evidence for the presence of the unique postsynthetic membrane-adaptive response mechanism of the cis-trans isomerization of unsaturated fatty acids in a bacterium that does not belong to the genera Pseudomonas and Vibrio where this mechanism was already reported and described extensively.

Sterol Biosynthesis by a Prokaryote: Firstin VitroIdentification of the Genes Encoding Squalene Epoxidase and Lanosterol Synthase fromMethylococcus capsulatus

Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)-2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)- and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed.

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.

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.

Copper-dependent reciprocal transcriptional regulation of methane monooxygenase genes in Methylococcus capsulatus and Methylosinus trichosporium.

The methanotrophic bacteria Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b convert methane to methanol using the enzyme, methane monooxygenase (MMO). These bacteria are able to express two distinct MMOs: a cytoplasmic or soluble form (sMMO) and a membrane-bound or particulate form (pMMO). Differential expression of sMMO and pMMO is regulated by the amount of copper ions available to the cells; sMMO is expressed at low copper-biomass ratios, whereas pMMO is expressed at high copper-biomass ratios. In both methanotrophs, transcription of the sMMO gene cluster is negatively regulated by copper ions. Data suggest that transcription of the M. trichosporium OB3b sMMO gene cluster is directed from a sigma54-like and a sigma70-like promoter. The pMMO (pmo) genes of M. capsulatus (Bath) are transcribed into a polycistronic mRNA of 3.3 kb. The synthesis of this mRNA was activated by copper ions. Activation of pmo transcription by copper ions was concomitant with repression of sMMO gene transcription in both methanotrophs. This suggests that a common regulatory pathway may be involved in the transcriptional switch between sMMO and pMMO gene expression.

Regulation of bacterial methane oxidation: transcription of the soluble methane mono-oxygenase operon of Methylococcus capsulatus (Bath) is repressed by copper ions.

Methane is oxidized to methanol by the enzyme methane mono-oxygenase (MMO) in methanotrophic bacteria. In previous work, this multicomponent enzyme system has been extensively characterized at the biochemical and molecular level. Copper ions have been shown to irreversibly inhibit MMO activity in vivo and in vitro, but the effect of copper ions on transcription of the genes encoding the soluble (cytoplasmic) MMO (sMMO) has not previously been investigated. To examine more closely the regulation of bacterial methane oxidation and to determine the role of copper in this process, we have investigated transcriptional regulation of the sMMO gene cluster in the methanotrophic bacterium Methylococcus capsulatus (Bath). Using Northern blot analysis and primer extension experiments, it was shown that the six ORFs of the sMMO gene cluster are organized as an operon and the transcripts produced upon expression of this operon have been identified. The synthesis of these transcripts was under control of a single copper-regulated promoter, which is as yet not precisely defined.

Online Monitoring of Biological Reactions at Low Parts-Per-Trillion Levels by Membrane Inlet Mass Spectrometry

A flow-through membrane inlet combined with a single quadrupole mass spectrometer has been used for the direct on-line monitoring of biological reactions at the extremely low parts-per-trillion level. Linear signals over at least 4 orders of magnitude were obtained. As an example, on-line monitoring data from the microbial oxygenation of chloroethylenes in pure cultures of the methanotrophic bacteria Methylococcus capsulatus (Bath) are presented

Carbon isotopic fractionation in lipids from methanotrophic bacteria: Relevance for interpretation of the geochemical record of biomarkers

Experiments with cultured aerobic methane oxidising bacteria confirm that their biomarker lipids will be significantly depleted in 13C compared to the substrate. The methanotrophic bacteria Methylococcus capsulatus and Methylomonas methanica, grown on methane and using the RuMP cycle for carbon assimilation, show maximum 13C fractionation of approximately 30%. in the resultant biomass. In M. capsulatus, the maximum fractionation is observed in the earliest part of the exponential growth stage and decreases to approximately 16%. as cells approach stationary phase. This change may be associated with a shift from the particulate form to the soluble form of the methane monooxygenase enzyme. Less than maximum fractionation is observed when cells are grown with reduced methane availability. Biomass of M. capsulatus grown on methanol was depleted by 9%. compared to the substrate. Additional strong 13C fractionation takes place during polyisoprenoid biosynthesis in methanotrophs. The δ 13 C values of individual hopanoid and steroid biomarkers produced by these organisms were as much as 10%. more negative than total biomass. In individual cultures, squalene was 13C-enriched by as much as 14%. compared to the triterpane skeleton of bacteriohopaneaminopentol. Much of the isotopic dispersion in lipid metabolites could be attributed to shifts in their relative abundances, combined with an overall reduction in fractionation during the growth cycle. In cells grown on methanol, where there was no apparent effect of growth stage on overall fractionation there were still significant isotopic differences between closely related lipids including a 5.3%. difference between the hopane and 3 β-methylhopane skeletons. Hopane and sterane polyisoprenoids were also 13C-depleted compared to fatty acids. These observations have significant implications for the interpretation of specific compound isotopic signatures now being measured for hydrocarbons and other lipids present in sediments and petroleum. In particular, biomarker lipids produced by a single organism do not necessarily have the same carbon isotopic composition.