Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase

Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining protein structure. Despite recent advances in instruments and algorithms, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air/water interface and the presence of preferred orientations and nonuniform ice layers. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently attracted attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Graphene minimizes background noise and provides a stable platform for specimens under a high-voltage electron beam and cryogenic conditions. Here, we introduce a reliable, easily implemented, and reproducible method of producing 36 graphene-coated grids at once within 1.5 days. The quality of the graphene grids was assessed using various tools such as scanning EM, Raman spectroscopy, and atomic force microscopy. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.4 angstrom using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages; for example, it requires less protein, enables easy control of the ice thickness, and prevents pro-tein denaturation at the air/water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we revealed subtle yet significant geometrical differences at the non-heme di-iron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.

Nitrogen (N2) fixation activity under different oxygen concentration of methanotrophic bacteria isolated from rice fields and their molecular identification

Rice fields are a significantly sources of atmospheric methane. Methanotrophic bacteria are unique in their ability to utilize methane as a sole carbon source and their ability to fix N2. This research successfully characterized N2 fixation activity under different oxygen concentrations of methanotrophic bacteria isolated from rice fields. From 19 tested isolates, four isolates performed activity to fix N2. They could fix N2 on different concentration of air saturation (10 % up to 100%). The growth of methanotrophs is not directly corelated with the N2 fixation activity, and their N2 fixation activities are affected by O2 concentrations. The BGM 3 and BGM 9 isolates had very good N2 fixation activity. Their activities were increased by increasing air saturation up to 50% (approximately 10% O2), but then decrease by increasing air saturation from 50% (approximately 10% O2) to 100% (approximately 20% O2). However, the highest N2 fixation activity was performed by the BGM 9 isolate at 30% air saturation (approximately 6% O2), and the isolate was identified as Methylococcus capsulatus. This information can support application of the isolates to achieve sustainable and environmentally friendly agricultural system.

Evaluation of Methanotroph (Methylococcus capsulatus, Bath) Bacteria Meal (FeedKind®) as an Alternative Protein Source for Juvenile Black Sea Bream, Acanthopagrus schlegelii

Single-cell proteins are attracting growing attention as viable alternatives for fishmeal (FM) in aquatic feed. Methanotroph (Methylococcus capsulatus, Bath) bacteria meal FeedKind® (FK) is a type of single cell protein with high protein content (75.14%) and desirable amino acids profile, produced by Methylococcus capsulatus (Bath) living on methane consumption. The present study evaluated the potential of replacing FM with FK in the diet of black sea bream (Acanthopagrus schlegelii). Five iso-energetic and iso-nitrogenous diets were designed with FK replacing 0, 4.13, 8.27, 16.53, and 24.80% FM protein in the basal diet (40% FM content), respectively. All the diets were fed to three replicates of fish (initial weight 6.56 ± 0.02 g) for 70 days. After the feeding trial, replacing dietary 8.27% FM protein with FK significantly improved the weight gain and specific growth rate of fish (P < 0.05), while other groups showed no significant difference in the growth performance (P > 0.05). The fish fed diets with 8.27 and 16.53% replacement levels exhibited significantly increased feeding rates. The 8.27% FK diet significantly increased the whole-body and muscle crude protein contents, apparent digestibility of crude lipid, foregut, and midgut amylase activities. The microvillus density in the midgut of fish fed the 24.80% FK diet significantly increased. The diet with 8.27% FK increased the serum triglyceride content of the fish, while the 24.80% FK diet reduced the serum triglyceride, total cholesterol, and low-density lipoprotein cholesterol contents of the fish. In conclusion, the results indicated that replacing dietary FM protein with up to 24.80% FK had no adverse effects on the growth of black sea bream, whilst replacing 8.27% FM protein with FK enhanced its growth performance and feed utilization.

Effects of a Single Cell Protein (Methylococcus capsulatus, Bath) in Pacific White Shrimp (Penaeus vannamei) Diet on Growth Performance, Survival Rate and Resistance to Vibrio parahaemolyticus, the Causative Agent of Acute Hepatopancreatic Necrosis Disease

The efficacy of a single cell protein (SCP) methanotroph (Methylococcus capsulatus, Bath) bacteria meal (FeedKind®, Calysta, Menlo Park, CA, United States), in Pacific white shrimp (Penaeus vannamei) diets was studied to determine growth performance, survival rate and disease resistance against Vibrio parahaemolyticus causing Acute Hepatopancreatic Necrosis Disease (AHPND). The growth trial was assigned in a completely randomized design (CRD) with four treatments and 5 replicates of each, T1: a fishmeal-based control containing 15% fish meal and 3 diets with graded levels of methanotroph bacteria meal, namely T2: 5% methanotroph bacteria meal, T3: 10% methanotroph bacteria meal, and T4: 15% methanotroph bacteria meal. Shrimp were fed ad libitum for 6 weeks on trial diets to assess growth. Subsequent to the growth trial, three replicates of the same groups were exposed to V. parahaemolyticus by a single bath challenge and held for a further 15 days on the same diets as the growth study to assess survival and resistance. No significant differences (p > 0.05) in survival or in growth performance, including final weight, weight gain, specific growth rate, feed consumption or feed conversion ratio of white shrimp fed feeds containing methanotroph bacteria meal or control diets for 6 weeks. Immune markers such as hemocyte counts, phenoloxidase, superoxide dismutase and lysozyme activity were similar across all groups after the 6-week feeding trial. In a V. parahaemolyticus challenge, methanotroph bacteria meal in the diet significantly promoted the survival rate, and the reduction of Vibrio sp. in the hepatopancreas of white shrimp. Hemocyte count and phenoloxidase activity showed no significant differences (p > 0.05) between diet treatment groups, but hemolymph protein was significantly higher (p < 0.05) in shrimp fed diets containing 15% methanotroph bacteria meal after challenge. The Vibrio colony counts from hepatopancreas in the treatment groups were all significantly lower than the control (p < 0.05). The findings show that methanotroph bacteria meal can entirely replace fishmeal in white shrimp diets and the 15% inclusion of methanotroph bacteria meal in shrimp diet shows no adverse effects on growth performance, feed utilization and survival rate. In addition, shrimp fed methanotroph bacteria meal diets exhibited improved survival rates to an AHPND challenge.

Expanding Characterized Diversity and the Pool of Complete Genome Sequences of Methylococcus Species, the Bacteria of High Environmental and Biotechnological Relevance

The bacterial genus Methylococcus, which comprises aerobic thermotolerant methanotrophic cocci, was described half-a-century ago. Over the years, a member of this genus, Methylococcus capsulatus Bath, has become a major model organism to study genomic and metabolic basis of obligate methanotrophy. High biotechnological potential of fast-growing Methylococcus species, mainly as a promising source of feed protein, has also been recognized. Despite this big research attention, the currently cultured Methylococcus diversity is represented by members of the two species, M. capsulatus and M. geothermalis, while finished genome sequences are available only for two strains of these methanotrophs. This study extends the pool of phenotypically characterized Methylococcus strains with good-quality genome sequences by contributing four novel isolates of these bacteria from activated sludge, landfill cover soil, and freshwater sediments. The determined genome sizes of novel isolates varied between 3.2 and 4.0Mb. As revealed by the phylogenomic analysis, strains IO1, BH, and KN2 affiliate with M. capsulatus, while strain Mc7 may potentially represent a novel species. Highest temperature optima (45–50°C) and highest growth rates in bioreactor cultures (up to 0.3h−1) were recorded for strains obtained from activated sludge. The comparative analysis of all complete genomes of Methylococcus species revealed 4,485 gene clusters. Of these, pan-genome core comprised 2,331 genes (on average 51.9% of each genome), with the accessory genome containing 846 and 1,308 genes in the shell and the cloud, respectively. Independently of the isolation source, all strains of M. capsulatus displayed surprisingly high genome synteny and a striking similarity in gene content. Strain Mc7 from a landfill cover soil differed from other isolates by the high content of mobile genetic elements in the genome and a number of genome-encoded features missing in M. capsulatus, such as sucrose biosynthesis and the ability to scavenge phosphorus and sulfur from the environment.

Design of a Microbial Remediation Inoculation Program for Petroleum Hydrocarbon Contaminated Sites Based on Degradation Pathways

This paper analyzed the degradation pathways of petroleum hydrocarbon degradation bacteria, screened the main degradation pathways, and found the petroleum hydrocarbon degradation enzymes corresponding to each step of the degradation pathway. Through the Copeland method, the best inoculation program of petroleum hydrocarbon degradation bacteria in a polluted site was selected as follows: single oxygenation path was dominated by Streptomyces avermitilis, hydroxylation path was dominated by Methylosinus trichosporium OB3b, secondary oxygenation path was dominated by Pseudomonas aeruginosa, secondary hydroxylation path was dominated by Methylococcus capsulatus, double oxygenation path was dominated by Acinetobacter baylyi ADP1, hydrolysis path was dominated by Rhodococcus erythropolis, and CoA path was dominated by Geobacter metallireducens GS-15 to repair petroleum hydrocarbon contaminated sites. The Copeland method score for this solution is 22, which is the highest among the 375 solutions designed in this paper, indicating that it has the best degradation effect. Meanwhile, we verified its effect by the Cdocker method, and the Cdocker energy of this solution is −285.811 kcal/mol, which has the highest absolute value. Among the inoculation programs of the top 13 petroleum hydrocarbon degradation bacteria, the effect of the best inoculation program of petroleum hydrocarbon degradation bacteria was 18% higher than that of the 13th group, verifying that this solution has the best overall degradation effect. The inoculation program of petroleum hydrocarbon degradation bacteria designed in this paper considered the main pathways of petroleum hydrocarbon pollutant degradation, especially highlighting the degradability of petroleum hydrocarbon intermediate degradation products, and enriching the theoretical program of microbial remediation of petroleum hydrocarbon contaminated sites.

Expression of soluble methane monooxygenase in Escherichia coli enables methane conversion

Natural gas and biogas provide an opportunity to harness methane as an industrial feedstock. Bioconversion is a promising alternative to chemical catalysis, which requires extreme operating conditions and exhibits poor specificities. Though methanotrophs natively utilize methane, efforts have been focused on engineering platform organisms like Escherichia coli for synthetic methanotrophy. Here, a synthetic E. coli methanotroph was developed by engineering functional expression of the Methylococcus capsulatus soluble methane monooxygenase in vivo via expression of its cognate GroESL chaperone. Additional overexpression of E. coli GroESL further improved activity. Incorporation of an acetone formation pathway then enabled the conversion of methane to acetone in vivo, as validated via 13C tracing. This work provides the first reported demonstration of methane bioconversion to liquid chemicals in a synthetic methanotroph.