The Role of Serine-Glyoxylate Aminotransferase and Malyl-CoA Lyase in the Metabolism of Methylococcus capsulatus Bath.

The genome of aerobic methanotroph Methylococcus capsulatus Bath possesses genes of three biochemical pathways of C1-carbon assimilation: the ribulose monophosphate cycle, the Calvin-Benson-Bassham cycle, and the partial serine cycle. Numerous studies have demonstrated that during methanotrophic growth cells of Methylococcus capsulatus Bath express key enzymes of these routes. In this study, the role of the serine cycle key enzymes, serine-glyoxylate aminotransferase (Sga) and malyl-CoA lyase (Mcl) in metabolism of Methylococcus capsulatus Bath was investigated by gene inactivation. The Δsga mutant obtained by double homologous recombination showed a prolonged lag phase, and after the lag period, the growth rate became similar to that of the wild type strain. The elevated intracellular levels of glutamate, serine, glycine, alanine, methionine, leucine, and succinate suggested significant metabolic changes in the mutant cells. Deletion of the mcl gene resulted in very poor growth and glycine only partially improved growth of the mutant strain. Cells of Δmcl mutant possess lower content of histidine, but enhanced level of alanine, leucine, and lysine than those of the wild type strain. Our data imply the importance of the serine cycle enzymes in metabolism of the methanotroph as well as relationships of the three C1 assimilation pathways in the gammaproteobacterial methanotrophs.

Concerning P450 Evolution: Structural Analyses Support Bacterial Origin of Sterol 14α-Demethylases.

Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbreviated pathway in the bacterium Methylococcus capsulatus. Sterol 14α-demethylation is an essential step in this pathway and is catalyzed by cytochrome P450 51 (CYP51). In M. capsulatus, the enzyme consists of the P450 domain naturally fused to a ferredoxin domain at the C-terminus (CYP51fx). The structure of M. capsulatus CYP51fx was solved to 2.7 Å resolution and is the first structure of a bacterial sterol biosynthetic enzyme. The structure contained one P450 molecule per asymmetric unit with no electron density seen for ferredoxin. We connect this with the requirement of P450 substrate binding in order to activate productive ferredoxin binding. Further, the structure of the P450 domain with bound detergent (which replaced the substrate upon crystallization) was solved to 2.4 Å resolution. Comparison of these two structures to the CYP51s from human, fungi, and protozoa reveals strict conservation of the overall protein architecture. However, the structure of an "orphan" P450 from nonsterol-producing Mycobacterium tuberculosis that also has CYP51 activity reveals marked differences, suggesting that loss of function in vivo might have led to alterations in the structural constraints. Our results are consistent with the idea that eukaryotic and bacterial CYP51s evolved from a common cenancestor and that early eukaryotes may have recruited CYP51 from a bacterial source. The idea is supported by bioinformatic analysis, revealing the presence of CYP51 genes in >1,000 bacteria from nine different phyla, >50 of them being natural CYP51fx fusion proteins.

Systems analysis of the effect of hydrogen sulfide on the growth of Methylococcus capsulatus Bath.

Methanotrophs are bacteria capable on growing on methane as their sole carbon source. They may provide a promising route for upgrading natural gas into more valuable fuels and chemicals. However, natural gas may contain significant quantities of hydrogen sulfide. Little is known about how hydrogen sulfide affects the growth and physiology of methanotrophs aside from a few studies showing that it is inhibitory. This study investigated how hydrogen sulfide affects the growth and physiology of the model methanotroph, Methylococcus capsulatus Bath. Growth studies demonstrated that hydrogen sulfide inhibits the growth of M. capsulatus Bath when the concentration exceeds 0.5% (v/v). To better understand how hydrogen sulfide is inhibiting the growth of M. capsulatus Bath, transcription and metabolite concentrations were profiled using RNA sequencing and gas chromatography-mass spectrometry, respectively. Our analysis of the differentially expressed genes and changes in metabolite concentrations suggests that hydrogen sulfide inhibits cellular respiration. The cells respond to sulfide stress in part by increasing the rate of sulfide oxidation and by increasing the expression of sulfide quinone reductase and a putative persulfide dioxygenase. In addition, they reduce the expression of the native calcium-dependent methanol dehydrogenase and increase the expression of XoxF, a lanthanide-dependent methanol dehydrogenase. While the reason of this switch in unknown, XoxF has previously been shown to be induced by lanthanides or nitric oxide in methanotrophs. Collectively, these results further our understanding of how methanotrophs respond to sulfide stress and may aid in the engineering of strains resistant to hydrogen sulfide. KEY POINTS: • Hydrogen sulfide inhibits growth of Methylococcus capsulatus Bath • Sulfide stress inhibits cellular respiration • Sulfide stress induces XoxF, a lanthanide-dependent methanol dehydrogenase.

C-4 sterol demethylation enzymes distinguish bacterial and eukaryotic sterol synthesis.

Sterols are essential eukaryotic lipids that are required for a variety of physiological roles. The diagenetic products of sterol lipids, sterane hydrocarbons, are preserved in ancient sedimentary rocks and are utilized as geological biomarkers, indicating the presence of both eukaryotes and oxic environments throughout Earth's history. However, a few bacterial species are also known to produce sterols, bringing into question the significance of bacterial sterol synthesis for our interpretation of sterane biomarkers. Recent studies suggest that bacterial sterol synthesis may be distinct from what is observed in eukaryotes. In particular, phylogenomic analyses of sterol-producing bacteria have failed to identify homologs of several key eukaryotic sterol synthesis enzymes, most notably those required for demethylation at the C-4 position. In this study, we identified two genes of previously unknown function in the aerobic methanotrophic γ-Proteobacterium Methylococcus capsulatus that encode sterol demethylase proteins (Sdm). We show that a Rieske-type oxygenase (SdmA) and an NAD(P)-dependent reductase (SdmB) are responsible for converting 4,4-dimethylsterols to 4α-methylsterols. Identification of intermediate products synthesized during heterologous expression of SdmA-SdmB along with 13C-labeling studies support a sterol C-4 demethylation mechanism distinct from that of eukaryotes. SdmA-SdmB homologs were identified in several other sterol-producing bacterial genomes but not in any eukaryotic genomes, indicating that these proteins are unrelated to the eukaryotic C-4 sterol demethylase enzymes. These findings reveal a separate pathway for sterol synthesis exclusive to bacteria and show that demethylation of sterols evolved at least twice-once in bacteria and once in eukaryotes.

Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing.

Methanotrophic bacteria play a crucial role in the Earth's biogeochemical cycle and have the potential to be employed in industrial biomanufacturing processes due to their capacity to use natural gas- and biogas-derived methane as a sole carbon and energy source. Advanced gene-editing systems have the potential to enable rapid, high-throughput methanotrophic genetics and biocatalyst development. To this end, we employed a series of broad-host-range expression plasmids to construct a conjugatable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing system in Methylococcus capsulatus (Bath). Heterologous coexpression of the Streptococcus pyogenes Cas9 endonuclease and a synthetic single guide RNA (gRNA) showed efficient Cas9 DNA targeting and double-stranded DNA (dsDNA) cleavage that resulted in cell death. We demonstrated effective in vivo editing of plasmid DNA using both Cas9 and Cas9D10A nickase to convert green fluorescent protein (GFP)- to blue fluorescent protein (BFP)-expressing cells with 71% efficiency. Further, we successfully introduced a premature stop codon into the soluble methane monooxygenase (sMMO) hydroxylase component-encoding mmoX gene with the Cas9D10A nickase, disrupting sMMO function. These data provide proof of concept for CRISPR/Cas9-mediated gene editing in M. capsulatus Given the broad-host-range replicons and conjugation capability of these CRISPR/Cas9 tools, they have potential utility in other methanotrophs and a wide array of Gram-negative microorganisms.IMPORTANCE In this study, we targeted the development and evaluation of broad-host-range CRISPR/Cas9 gene-editing tools in order to enhance the genetic-engineering capabilities of an industrially relevant methanotrophic biocatalyst. The CRISPR/Cas9 system developed in this study expands the genetic tools available to define molecular mechanisms in methanotrophic bacteria and has the potential to foster advances in the generation of novel biocatalysts to produce biofuels, platform chemicals, and high-value products from natural gas- and biogas-derived methane. Further, due to the broad-host-range applicability, these genetic tools may also enable innovative approaches to overcome the barriers associated with genetically engineering diverse, industrially promising nonmodel microorganisms.

Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene.

Methylococcus capsulatus (Bath) is a representative gammaproteobacterial methanotroph that has been studied extensively in diverse research fields. The sacB gene, which encodes levansucrase, causing cell death in the presence of sucrose, is widely used as a counterselectable marker for disruption of a target gene in Gram-negative bacteria. However, sacB is not applicable to all Gram-negative bacteria, and its efficiency for the counterselection of M. capsulatus (Bath) is low. Here, we report the construction of an alternative counterselectable marker, pheS*, by introduction of two point mutations (A306G and T252A) into the pheS gene from M. capsulatus (Bath), which encodes the α-subunit of phenylalanyl-tRNA synthetase. The transformant harboring pheS* on an expression plasmid showed sensitivity to 10 mM p-chloro-phenylalanine, whereas the transformant harboring an empty plasmid showed no sensitivity, indicating the availability of pheS* as a counterselectable marker in M. capsulatus (Bath). To validate the utility of the pheS* marker in counterselection, we attempted to obtain an unmarked mutant of xoxF, a gene encoding the major subunit of Xox methanol dehydrogenase, which we failed to obtain by counterselection using the sacB marker. PCR, immunodetection using an anti-XoxF antiserum, and a cell growth assay in the absence of calcium demonstrated successful disruption of the xoxF gene in M. capsulatus (Bath). The difference in counterselection efficiencies of the markers indicated that pheS* is more suitable than sacB for counterselection in M. capsulatus (Bath). This study provides a new genetic tool enabling efficient counterselection in M. capsulatus (Bath).IMPORTANCE Methanotrophs have long been considered promising strains for biologically reducing methane from the environment and converting it into valuable products, because they can oxidize methane at ambient temperatures and pressures. Although several methodologies and tools for the genetic manipulation of methanotrophs have been developed, their mutagenic efficiency remains lower than that of tractable strains such as Escherichia coli Therefore, further improvements are still desired. The significance of our study is that we increased the efficiency of counterselection in M. capsulatus (Bath) by employing pheS*, which was newly constructed as a counterselectable marker. This will allow for the efficient production of gene-disrupted and gene-integrated mutants of M. capsulatus (Bath). We anticipate that this counterselection system will be utilized widely by the methanotroph research community, leading to improved productivity of methane-based bioproduction and new insights into methanotrophy.

Expression, purification and biochemical characterization of a family 6 carboxylesterase from Methylococcus capsulatus (bath).

The genome of Methylococcus capsulatus (bath) encodes a protein R-est6 that is annotated as a lipase family 3 protein. The phylogenetic and the sequence analyses linked this protein to the family 6 carboxylesterase. The gene encoding R-est6 was cloned and overexpressed in Escherichia coli and the recombinant 6x-His tagged protein was purified by Ni-NTA affinity chromatography. The buffers used in the purification were modified by adding 1% glycerol instead of the salt to prevent the protein aggregation. Far UV-CD spectrum and gel filtration chromatography of the purified R-est6 confirmed that the protein was well folded like a typical α/ß hydrolase and had the quaternary structure of a tetramer, in addition to a compact monomer. The optimum pH was in the range of 7.0-9.0 and the optimum temperature was at 55 °C for the hydrolysis of pNP-butyrate. As expected, being a member of the family 6 carboxylesterase, R-est6 hydrolyzed triglycerides, pNP esters of the small and the medium fatty acid chain esters and an aryl ester-phenyl acetate. However, R-est6 was also found to hydrolyze the long-chain fatty acid ester which had never been reported for the family 6 carboxylesterase. Additionally, R-est6 was stable and active in the different water-miscible organic solvents. Therefore, the broad substrate range and the structural stability of R-est6 would be advantageous for its application in industrial processes.

Discovery, taxonomic distribution, and phenotypic characterization of a gene required for 3-methylhopanoid production.

Hopanoids methylated at the C-3 position are a subset of bacterial triterpenoids that are readily preserved in modern and ancient sediments and in petroleum. The production of 3-methylhopanoids by extant aerobic methanotrophs and their common occurrence in modern and fossil methane seep communities, in conjunction with carbon isotope analysis, has led to their use as biomarker proxies for aerobic methanotrophy. In addition, these lipids are also produced by aerobic acetic acid bacteria and, lacking carbon isotope analysis, are more generally used as indicators for aerobiosis in ancient ecosystems. However, recent genetic studies have brought into question our current understanding of the taxonomic diversity of methylhopanoid-producing bacteria and have highlighted that a proper interpretation of methylhopanes in the rock record requires a deeper understanding of their cellular function. In this study, we identified and deleted a gene, hpnR, required for methylation of hopanoids at the C-3 position in the obligate methanotroph Methylococcus capsulatus strain Bath. Bioinformatics analysis revealed that the taxonomic distribution of HpnR extends beyond methanotrophic and acetic acid bacteria. Phenotypic analysis of the M. capsulatus hpnR deletion mutant demonstrated a potential physiological role for 3-methylhopanoids; they appear to be required for the maintenance of intracytoplasmic membranes and cell survival in late stationary phase. Therefore, 3-methylhopanoids may prove more useful as proxies for specific environmental conditions encountered during stationary phase rather than a particular bacterial group.

Engineered Methylococcus capsulatus Bath for efficient methane conversion to isoprene.

Isoprene has numerous industrial applications, including rubber polymer and potential biofuel. Microbial methane-based isoprene production could be a cost-effective and environmentally benign process, owing to a reduced carbon footprint and economical utilization of methane. In this study, Methylococcus capsulatus Bath was engineered to produce isoprene from methane by introducing the exogenous mevalonate (MVA) pathway. Overexpression of MVA pathway enzymes and isoprene synthase from Populus trichocarpa under the control of a phenol-inducible promoter substantially improved isoprene production. M. capsulatus Bath was further engineered using a CRISPR-base editor to disrupt the expression of soluble methane monooxygenase (sMMO), which oxidizes isoprene to cause toxicity. Additionally, optimization of the metabolic flux in the MVA pathway and culture conditions increased isoprene production to 228.1 mg/L, the highest known titer for methanotroph-based isoprene production. The developed methanotroph could facilitate the efficient conversion of methane to isoprene, resulting in the sustainable production of value-added chemicals.

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.

Draft genome sequence of the methane-oxidizing bacterium Methylococcus capsulatus (Texas).

Methanotrophic bacteria perform major roles in global carbon cycles via their unique enzymatic activities that enable the oxidation of one-carbon compounds, most notably methane. Here we describe the annotated draft genome sequence of the aerobic methanotroph Methylococcus capsulatus (Texas), a type strain originally isolated from sewer sludge.

Structure of the redox sensor domain of Methylococcus capsulatus (Bath) MmoS.

MmoS from Methylococcus capsulatus (Bath) is the multidomain sensor protein of a two-component signaling system proposed to play a role in the copper-mediated regulation of soluble methane monooxygenase (sMMO). MmoS binds an FAD cofactor within its N-terminal tandem Per-Arnt-Sim (PAS) domains, suggesting that it functions as a redox sensor. The crystal structure of the MmoS tandem PAS domains, designated PAS-A and PAS-B, has been determined to 2.34 A resolution. Both domains adopt the typical PAS domain alpha/beta topology and are structurally similar. The two domains are linked by a long alpha helix and do not interact with one another. The FAD cofactor is housed solely within PAS-A and is stabilized by an extended hydrogen bonding network. The overall fold of PAS-A is similar to those of other flavin-containing PAS domains, but homodimeric interactions in other structures are not observed in the MmoS sensor, which crystallized as a monomer. The structure both provides new insight into the architecture of tandem PAS domains and suggests specific residues that may play a role in MmoS FAD redox chemistry and subsequent signal transduction.

Revisiting the mechanism of dioxygen activation in soluble methane monooxygenase from M. capsulatus (Bath): evidence for a multi-step, proton-dependent reaction pathway.

Stopped-flow kinetic investigations of soluble methane monooxygenase (sMMO) from M. capsulatus (Bath) have clarified discrepancies that exist in the literature regarding several aspects of catalysis by this enzyme. The development of thorough kinetic analytical techniques has led to the discovery of two novel oxygenated iron species that accumulate in addition to the well-established intermediates H(peroxo) and Q. The first intermediate, P*, is a precursor to H(peroxo) and was identified when the reaction of reduced MMOH and MMOB with O(2) was carried out in the presence of >or=540 microM methane to suppress the dominating absorbance signal due to Q. The optical properties of P* are similar to those of H(peroxo), with epsilon(420) = 3500 M(-1) cm(-1) and epsilon(720) = 1250 M(-1) cm(-1). These values are suggestive of a peroxo-to-iron(III) charge-transfer transition and resemble those of peroxodiiron(III) intermediates characterized in other carboxylate-bridged diiron proteins and synthetic model complexes. The second identified intermediate, Q*, forms on the pathway of Q decay when reactions are performed in the absence of hydrocarbon substrate. Q* does not react with methane, forms independently of buffer composition, and displays a unique shoulder at 455 nm in its optical spectrum. Studies conducted at different pH values reveal that rate constants corresponding to P* decay/H(peroxo) formation and H(peroxo) decay/Q formation are both significantly retarded at high pH and indicate that both events require proton transfer. The processes exhibit normal kinetic solvent isotope effects (KSIEs) of 2.0 and 1.8, respectively, when the reactions are performed in D(2)O. Mechanisms are proposed to account for the observations of these novel intermediates and the proton dependencies of P* to H(peroxo) and H(peroxo) to Q conversion.

The presence of multiple c-type cytochromes at the surface of the methanotrophic bacterium Methylococcus capsulatus (Bath) is regulated by copper.

Identification of surface proteins is essential to understand bacterial communication with its environment. Analysis of the surface-associated proteins of Methylococcus capsulatus (Bath) revealed a highly dynamic structure responding closely to the availability of copper in the medium in the range from approximately 0 to 10 microM. Several c-type cytochromes, including three novel multihaem proteins, are present at the cellular surface, a feature that is otherwise a peculiarity of dissimilatory metal-reducing bacteria. At low copper concentrations, the cytochrome c(553o) and the cytochrome c(553o) family protein, encoded by the MCA0421 and MCA0423 genes, respectively, are major constituents of the surfaceome and show a fine-tuned copper-dependent regulation of expression. Two novel members of the cytochrome c(553o) family were identified: MCA0338 was abundant between 5 and 10 microM copper, while MCA2259 was detected only in the surface fraction obtained from approximately 0 microM copper cultures. The presence at the bacterial surface of several c-type cytochromes, generally involved in energy transduction, indicates strongly that redox processes take place at the bacterial surface. Due to the unique role of copper in the biology of M. capsulatus (Bath), it appears that c-type cytochromes have essential functions in copper homeostasis allowing the cells to adapt to varying copper exposure.

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.

Cytochromes P460 and c'-beta; a new family of high-spin cytochromes c.

Cytochromes-P460 of Nitrosomonas europaea and Methylococcus capsulatus (Bath), and the cytochrome c' of M. capsulatus, believed to be involved in binding or transformation of N-oxides, are shown to represent an evolutionarily related new family of monoheme, approximately 17kDa, cytochromes c found in the genomes of diverse Proteobacteria. All members of this family have a predicted secondary structure predominantly of beta-sheets in contrast to the predominantly alpha-helical cytochromes c' found in photoheterotrophic and denitrifying Proteobacteria.

Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath).

Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.

Heterologous expression and biochemical studies of a thermostable glucose tolerant ß-glucosidase from Methylococcus capsulatus (bath strain).

Glucose inhibition of ß-glucosidase (BG) is a bottleneck in biomass hydrolysis. In this study, a glucose resistant GH1 ß-glucosidase gene- Mbgl from Methylococcus capsulatus (bath strain) was cloned and overexpressed in E.coli. The Ni-NTA affinity purified Mbgl displayed an optimum temperature of 70°C and optimum pH was 6.0. The calculated KM of the enzyme was 48.6mM and 0.12mM for cellobiose and 4-Nitrophenyl ß-d-glucopyranoside (PNPG) respectively. PNPG hydrolysis in presence of various glucose concentrations showed that the enzyme was stimulated by ∼2.2 fold at 50mM glucose and was not inhibited up to 450-500mM glucose. Homology modeling and structural comparisons of Mbgl with a glucose tolerant ß-glucosidase of Humicola insolens (HiBG) revealed that the Mbgl has a much broader active site unlike to a deep and narrow active site pocket of HiBG. The difference in active site shape reflects on an alternative mechanism of glucose tolerance in Mbgl. Supplementing a commercial cellulase enzyme mixture CTec with Mbgl in the hydrolysis of the pretreated rice straw enhanced the glucose yield by 10-15%. In addition, Mbgl was also stable in organic solvents, detergents and oxidative conditions which would be advantageous for biotechnological applications.

Insights into the obligate methanotroph Methylococcus capsulatus.

Completion of the genome sequence of Methylococcus capsulatus Bath is an important event in molecular microbiology, and an achievement for which the authors deserve congratulation. M. capsulatus, along with other methanotrophs, has been the subject of intense biochemical and molecular study because of its role in the global carbon cycle: the conversion of biogenic methane to carbon dioxide. The methane monooxygenase enzymes that are central to this process also have high biotechnological potential. Analysis of the genome sequence will potentially accelerate elucidation of the regulation of methane-dependent metabolism in obligate methanotrophs, and help explain the cause of obligate methanotrophy, the phenomenon making most methanotrophs unable to grow on any substrates other than methane and a very small number of other one-carbon compounds.

The RING-finger protein haprin: domains and function in the acrosome reaction.

The RBCC (RING finger, B-box type zinc finger, coiled-coil domain) motif family contains a large number of proteins implicated in many cellular processes, including vesicle exocytosis. The acrosome reaction, the sperm exocytotic event that is required for fertilization, involves essentially the same process of intracellular membrane fusions as vesicular exocytosis in somatic cells. We have previously isolated a haploid-germ-cell-specific gene designated haprin, which encodes a RBCC motif protein that plays a role in the acrosome reaction of sperm by mediating protein complex formation via the RBCC motif. In this review, we describe the potential role of Haprin in the molecular mechanisms of acrosome reaction, as compared with some other RBCC proteins. The conserved structure and localization of the Haprin protein in human and mouse suggest an indispensable role for Haprin in the functioning of mammalian sperm.