Flexible genomic island conservation across freshwater and marine Methylophilaceae.

The evolutionary trajectory of Methylophilaceae includes habitat transitions from freshwater sediments to freshwater and marine pelagial that resulted in genome reduction (genome-streamlining) of the pelagic taxa. However, the extent of genetic similarities in the genomic structure and microdiversity of the two genome-streamlined pelagic lineages (freshwater "Ca. Methylopumilus" and the marine OM43 lineage) has so far never been compared. Here, we analyzed complete genomes of 91 "Ca. Methylopumilus" strains isolated from 14 lakes in Central Europe and 12 coastal marine OM43 strains. The two lineages showed a remarkable niche differentiation with clear species-specific differences in habitat preference and seasonal distribution. On the other hand, we observed a synteny preservation in their genomes by having similar locations and types of flexible genomic islands (fGIs). Three main fGIs were identified: a replacement fGI acting as phage defense, an additive fGI harboring metabolic and resistance-related functions, and a tycheposon containing nitrogen-, thiamine-, and heme-related functions. The fGIs differed in relative abundances in metagenomic datasets suggesting different levels of variability ranging from strain-specific to population-level adaptations. Moreover, variations in one gene seemed to be responsible for different growth at low substrate concentrations and a potential biogeographic separation within one species. Our study provides a first insight into genomic microdiversity of closely related taxa within the family Methylophilaceae and revealed remarkably similar dynamics involving mobile genetic elements and recombination between freshwater and marine family members.

Optimizing operational parameters for the enzymatic production of furandicarboxylic acid building block.

BACKGROUND: 2,5-Furandicarboxylic acid (FDCA) is a precursor for green plastics due to its structural similarity to terephthalic acid, a common precursor of oil-derived polymers, and its potential production from sugars obtained from plant biomass. Hydroxymethylfurfural oxidase (HMFO) has been reported as a promising biocatalyst for FDCA production since it can convert bio-based 5-hydroxymethylfurfural (HMF) into FDCA building block. This three-step oxidation reaction occurs through the diformylfuran and 2,5-formylfurancarboxylic acid (FFCA) intermediates. Several efforts have been made for the development of HMFO variants that increase FDCA yields by improving their activities over the reaction intermediates. However, there is still limited insight into how operational conditions can influence these enzymatic reactions. The setup of optimal reaction conditions would enable to understand potential problems hampering the effective industrial production of this bioplastic precursor using HMFO as biocatalyst. RESULTS: In this work, several parameters affecting the performance of Methylovorus sp HMFO oxidizing HMF have been analyzed for the wild-type enzyme, and its V367R and W466F single variants, V367R/W466F double variant, and I73V/H74Y/G356H/V367R/T414K/A419Y/A435E/W466F (8BxHMFO) octuple variant. Our results show how the oxidation of HMF by HMFO enzymes is highly influenced by pH, with different optimal pH values for the different improved variants. Moreover, the enzymes are not stable at high hydrogen peroxide concentrations and their activity is inhibited by the FFCA intermediate in a pH-dependent way. These limitations can be efficiently overcome with the addition of catalase to the reaction medium, which removes the hydrogen peroxide formed during the oxidations, and the controlled dosage of the substrate to limit the amount of FFCA accumulated in the reaction. The different behavior of wild-type HMFO and its variants against pH, hydrogen peroxide and FFCA highlights the importance of considering each variant as an individual enzyme with its own operational conditions for an eventual industrial FDCA production. CONCLUSIONS: This work provides information of those parameters that condition a high production of FDCA by HMFO. Unraveling these factors allowed to increase the FDCA yields by using the most stable enzymes at their optimal pH for HMF oxidation, removing the peroxide with catalase, and avoiding FFCA accumulation by controlling substrate and/or enzyme concentration. These above findings will be useful when planning a future scale-up of these conversions and will provide new viewpoints for the design of HMFO variants that render a more effective performance during HMF conversion into FDCA.

A Novel Freshwater to Marine Evolutionary Transition Revealed within Methylophilaceae Bacteria from the Arctic Ocean.

Bacteria inhabiting polar oceans, particularly the Arctic Ocean, are less studied than those at lower latitudes. Discovering bacterial adaptations to Arctic Ocean conditions is essential for understanding responses to the accelerated environmental changes occurring in the North. The Methylophilaceae are emerging as a model for investigating the genomic basis of habitat adaptation, because related lineages are widely distributed across both freshwater and marine ecosystems. Here, we investigated Methylophilaceae diversity in the salinity-stratified surface waters of the Canada Basin, Arctic Ocean. In addition to a diversity of marine OM43 lineages, we report on the genomic characteristics and evolution of a previously undescribed Methylophilaceae clade (BS01) common to polar surface waters yet related to freshwater sediment Methylotenera species. BS01 is restricted to the lower-salinity surface waters, while OM43 is found throughout the halocline. An acidic proteome supports a marine lifestyle for BS01, but gene content shows increased metabolic versatility compared to OM43 and evidence for ongoing genome-streamlining. Phylogenetic reconstruction shows that BS01 colonized the pelagic ocean independently of OM43 via convergent evolution. Salinity adaptation and differences in one-carbon and nitrogen metabolism may play a role in niche differentiation between BS01 and OM43. In particular, urea utilization by BS01 is predicted to provide an ecological advantage over OM43 given the limited amount of inorganic nitrogen in the Canada Basin. These observations provide further evidence that the Arctic Ocean is inhabited by distinct bacterial groups and that at least one group (BS01) evolved via a freshwater to marine environmental transition. IMPORTANCE Global warming is profoundly influencing the Arctic Ocean. Rapid ice melt and increased freshwater input is increasing ocean stratification, driving shifts in nutrient availability and the primary production that supports marine food webs. Determining bacterial responses to Arctic Ocean change is challenging because of limited knowledge on the specific adaptations of Arctic Ocean bacteria. In this study, we investigated the diversity and genomic adaptations of a globally distributed group of marine bacteria, the Methylophilaceae, in the surface waters of the Arctic Ocean. We discovered a novel lineage of marine Methylophilaceae inhabiting the Arctic Ocean whose evolutionary origin involved a freshwater to marine environmental transition. Crossing the salinity barrier is thought to rarely occur in bacterial evolution. However, given the ongoing freshening of the Arctic Ocean, our results suggest that these relative newcomers to the ocean microbiome increase in abundance and, therefore, ecological significance in a near-future Arctic Ocean.

Efficient synthesis of γ-glutamyl compounds by co-expression of γ-glutamylmethylamide synthetase and polyphosphate kinase in engineered Escherichia coli.

γ-Glutamyl compounds have unveiled their importance as active substances or precursors of pharmaceuticals. In this research, an approach for enzymatic synthesis of γ-glutamyl compounds was developed using γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays and polyphosphate kinase (PPK) from Corynebacterium glutamicum. GMAS and PPK were co-recombined in pETDuet-1 plasmid and co-expressed in E. coli BL21 (DE3), and the enzymatic properties of GMAS and PPK were investigated, respectively. Under the catalysis of the co-expression system, L-theanine was synthesized with 89.8% conversion when the substrate molar ratio of sodium glutamate and ethylamine (1:1.4) and only 2 mM ATP were used. A total of 14 γ-glutamyl compounds were synthesized by this one-pot method and purified by cation exchange resin and isoelectric point crystallization with a yield range from 22.3 to 72.7%. This study provided an efficient approach for the synthesis of γ-glutamyl compounds by GMAS and PPK co-expression system.

Production of L-Theanine Using Escherichia coli Whole-Cell Overexpressing γ-Glutamylmethylamide Synthetase with Bakers Yeast.

L-Theanine, found in green tea leaves has been shown to positively affect immunity and relaxation in humans. There have been many attempts to produce L-theanine through enzymatic synthesis to overcome the limitations of traditional methods. Among the many genes coding for enzymes in the L-theanine biosynthesis, glutamylmethylamide synthetase (GMAS) exhibits the greatest possibility of producing large amounts of production. Thus, GMAS from Methylovorus mays No. 9 was overexpressed in several strains including vectors with different copy numbers. BW25113(DE3) cells containing the pET24ma::gmas was selected for strains. The optimal temperature, pH, and metal ion concentration were 50°C, 7, and 5 mM MnCl2, respectively. Additionally, ATP was found to be an important factor for producing high concentration of L-theanine so several strains were tested during the reaction for ATP regeneration. Bakers yeast was found to decrease the demand for ATP most effectively. Addition of potassium phosphate source was demonstrated by producing 4-fold higher L-theanine. To enhance the conversion yield, GMAS was additionally overexpressed in the system. A maximum of 198 mM L-theanine was produced with 16.5 mmol/l/h productivity. The whole-cell reaction involving GMAS has greatest potential for scale-up production of L-theanine.

Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production.

The enzymatic production of 2,5-furandicarboxylic acid (FDCA) from 5-hydroxymethylfurfural (HMF) has gained interest in recent years, as FDCA is a renewable precursor of poly(ethylene-2,5-furandicarboxylate) (PEF). 5-Hydroxymethylfurfural oxidases (HMFOs) form a flavoenzyme family with genes annotated in a dozen bacterial species but only one enzyme purified and characterized to date (after heterologous expression of a Methylovorus sp. HMFO gene). This oxidase acts on both furfuryl alcohols and aldehydes and, therefore, is able to catalyze the conversion of HMF into FDCA through 2,5-diformylfuran (DFF) and 2,5-formylfurancarboxylic acid (FFCA), with only the need of oxygen as a cosubstrate. To enlarge the repertoire of HMFO enzymes available, genetic databases were screened for putative HMFO genes, followed by heterologous expression in Escherichia coli After unsuccessful trials with other bacterial HMFO genes, HMFOs from two Pseudomonas species were produced as active soluble enzymes, purified, and characterized. The Methylovorus sp. enzyme was also produced and purified in parallel for comparison. Enzyme stability against temperature, pH, and hydrogen peroxide, three key aspects for application, were evaluated (together with optimal conditions for activity), revealing differences between the three HMFOs. Also, the kinetic parameters for HMF, DFF, and FFCA oxidation were determined, the new HMFOs having higher efficiencies for the oxidation of FFCA, which constitutes the bottleneck in the enzymatic route for FDCA production. These results were used to set up the best conditions for FDCA production by each enzyme, attaining a compromise between optimal activity and half-life under different conditions of operation.IMPORTANCE HMFO is the only enzyme described to date that can catalyze by itself the three consecutive oxidation steps to produce FDCA from HMF. Unfortunately, only one HMFO enzyme is currently available for biotechnological application. This availability is enlarged here by the identification, heterologous production, purification, and characterization of two new HMFOs, one from Pseudomonas nitroreducens and one from an unidentified Pseudomonas species. Compared to the previously known Methylovorus HMFO, the new enzyme from P. nitroreducens exhibits better performance for FDCA production in wider pH and temperature ranges, with higher tolerance for the hydrogen peroxide formed, longer half-life during oxidation, and higher yield and total turnover numbers in long-term conversions under optimized conditions. All these features are relevant properties for the industrial production of FDCA. In summary, gene screening and heterologous expression can facilitate the selection and improvement of HMFO enzymes as biocatalysts for the enzymatic synthesis of renewable building blocks in the production of bioplastics.

A Novel 2,5-Furandicarboxylic Acid Biosynthesis Route from Biomass-Derived 5-Hydroxymethylfurfural Based on the Consecutive Enzyme Reactions.

2,5-Furandicarboxylic acid (FDCA) is a promising bio-based building block as a green alternative to petroleum-based terephthalate in polymer production. Most of FDCA is produced by the oxidation of 5-hydroxymethylfurfural (HMF), which is derived from hexose. Although the chemical conversion is widely applied, the biocatalytic conversion is expected due to the relatively mild condition and fewer toxic chemicals consumption. However, it's difficult to catalyze the conversion of HMF to FDCA by a single enzyme. Here, a newly enzymatic cascade reaction process was introduced with a yield of 94.0% by the combination of 5-hydroxymethylfurfural oxidase (HMFO) and lipase. Briefly, a flavine adenosine dinucleotide independent (FAD-independent) HMFO of Methylovorus sp. MP688 was used to convert HMF to 2,5-diformylfuran (DFF) and 5-formylfuroic acid (FFA), which consecutively transformed to FDCA by a lipase Novozym 435. To facilitate the purification, a coupled alkali precipitation was developed to recover FDCA from organic solvent with an improved purity from 84.4 to 99.0% and recovery of 78.1%. This work will help to construct the green biorefinery route for the bulk FDCA from biomass by enzymes.

Methylotenera oryzisoli sp. nov., a lanthanide-dependent methylotrophic bacteria isolated from rice field soil.

A new lanthanide (Ln3+)-dependent methanol-utilizing bacterial strain, La3113T, was isolated from rice field soil and its taxonomic position was investigated using polyphasic approaches. The strain was aerobic, Gram-stain-negative, strongly motile, catalase-positive and cytochrome oxidase-positive. It could neither catalyse the hydrolysis of urea nor reduce nitrate to nitrite. Growth was observed within a temperature range of 10-40 °C and a pH range of 6-8, with optimum growth at 28 °C and pH 7. Methylamine was utilized as the single source of energy, carbon and nitrogen, and it was oxidized by methylamine dehydrogenase. C16 : 1 ω7c, C16 : 1 ω6c and C16 : 0 were the dominant cellular fatty acids. Its draft genome (2.67 Mbp and 44.9 mol% G+C content) encodes genes including three Ln3+-dependent methanol dehydrogenase (XoxF-type MDH) genes, those for formaldehyde assimilation (ribulose monophosphate pathway), formate dehydrogenases and methylamine dehydrogenases, but not Ca2+-dependent MDH (MxaFI-MDH), which characterizes the species as a Ln3+-dependent methylotroph. The 16S rRNA gene sequence showed that strain La3113T belongs to the genus Methylotenera and is closely related to Methylotenera mobilis JLW8T (98.29 % identity). The digital DNA-DNA hybridization (dDDH) values (less than 30 %) and average nucleotide identity (ANI) values (less than 85 %) between genomes of strain La3113T and related type strains were lower than the thresholds for species delineation (70 % for dDDH and 95-96 % for ANI). On the basis of these polyphasic approaches, we propose a novel Methylotenera species, Methylotenera oryzisoli sp. nov. (type strain La3113T=NBRC 111954T=DSM 103219T).

Pseudomethylobacillus aquaticus gen. nov., sp. nov., a new member of the family Methylophilaceae isolated from an artificial reservoir.

A bacterial strain, designated H-5T, was isolated from an artificial reservoir in Taiwan and characterized using a polyphasic taxonomic approach. Cells of strain H-5T were Gram-stain-negative, aerobic, motile by means of a single polar flagellum, rod-shaped, covered by large capsules and formed white colonies. Growth occurred at 15-30 °C (optimum, 25 °C), at pH 6-8 (optimum, pH 7) and with 0-0.5 % NaCl (optimum, 0 %). Phylogenetic analyses based on the 16S rRNA gene, the methanol dehydrogenase gene and the coding sequences of 92 protein clusters indicated that strain H-5T was affiliated with genera in the family Methylophilaceae in the class Betaproteobacteria. Strain H-5T was most closely related to Methylobacillus methanolivorans ZT with a 95.0 % 16S rRNA gene sequence similarity. Strain H-5T showed less than 73.7 % average nucleotide identity and less than 23.6 % digital DNA-DNA hybridization identity compared to the strains of related genera within the family Methylophilaceae. The predominant fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The major isoprenoid quinone was Q-8 and the DNA G+C content was 58.3 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, one uncharacterized aminophospholipid, one uncharacterized phospholipid and one uncharacterized lipid. On the basis of the genotypic and phenotypic data presented here, strain H-5T represents a novel species of a new genus in the family Methylophilaceae, for which the name Pseudomethylobacillus aquaticus gen. nov., sp. nov. is proposed. The type strain is H-5T (=BCRC 81154T=KCTC 62865T).

Communal metabolism by Methylococcaceae and Methylophilaceae is driving rapid aerobic methane oxidation in sediments of a shallow seep near Elba, Italy.

The release of abiotic methane from marine seeps into the atmosphere is a major source of this potent greenhouse gas. Methanotrophic microorganisms in methane seeps use methane as carbon and energy source, thus significantly mitigating global methane emissions. Here, we investigated microbial methane oxidation at the sediment-water interface of a shallow marine methane seep. Metagenomics and metaproteomics, combined with 13 C-methane stable isotope probing, demonstrated that various members of the gammaproteobacterial family Methylococcaceae were the key players for methane oxidation, catalysing the first reaction step to methanol. We observed a transfer of carbon to methanol-oxidizing methylotrophs of the betaproteobacterial family Methylophilaceae, suggesting an interaction between methanotrophic and methylotrophic microorganisms that allowed for rapid methane oxidation. From our microcosms, we estimated methane oxidation rates of up to 871 nmol of methane per gram sediment per day. This implies that more than 50% of methane at the seep is removed by microbial oxidation at the sediment-water interface, based on previously reported in situ methane fluxes. The organic carbon produced was further assimilated by different heterotrophic microbes, demonstrating that the methane-oxidizing community supported a complex trophic network. Our results provide valuable eco-physiological insights into this specialized microbial community performing an ecosystem function of global relevance.

Evolution in action: habitat transition from sediment to the pelagial leads to genome streamlining in Methylophilaceae.

The most abundant aquatic microbes are small in cell and genome size. Genome-streamlining theory predicts gene loss caused by evolutionary selection driven by environmental factors, favouring superior competitors for limiting resources. However, evolutionary histories of such abundant, genome-streamlined microbes remain largely unknown. Here we reconstruct the series of steps in the evolution of some of the most abundant genome-streamlined microbes in freshwaters ("Ca. Methylopumilus") and oceans (marine lineage OM43). A broad genomic spectrum is visible in the family Methylophilaceae (Betaproteobacteria), from sediment microbes with medium-sized genomes (2-3 Mbp genome size), an occasionally blooming pelagic intermediate (1.7 Mbp), and the most reduced pelagic forms (1.3 Mbp). We show that a habitat transition from freshwater sediment to the relatively oligotrophic pelagial was accompanied by progressive gene loss and adaptive gains. Gene loss has mainly affected functions not necessarily required or advantageous in the pelagial or is encoded by redundant pathways. Likewise, we identified genes providing adaptations to oligotrophic conditions that have been transmitted horizontally from pelagic freshwater microbes. Remarkably, the secondary transition from the pelagial of lakes to the oceans required only slight modifications, i.e., adaptations to higher salinity, gained via horizontal gene transfer from indigenous microbes. Our study provides first genomic evidence of genome reduction taking place during habitat transitions. In this regard, the family Methylophilaceae is an exceptional model for tracing the evolutionary history of genome streamlining as such a collection of evolutionarily related microbes from different habitats is rare in the microbial world.

Methylotrophs and Methylotroph Populations for Chloromethane Degradation.

Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of chloromethane by methylotrophic communities in terrestrial environments is a major underestimated chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using chloromethane as the sole carbon source. In addition to anaerobes that degrade chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterisized 'chloromethane utilization' (cmu) pathway, so far. This pathway is not representative of chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined 'omics' biological approaches with chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated chloromethane-degraders lacking the cmu genes display a singular isotope fractionation signature of chloromethane. Moreover, 13CH3Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from the taxa known for chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle.

Reconstruction and analysis of a genome-scale metabolic model of Methylovorus sp. MP688, a high-level pyrroloquinolone quinone producer.

Methylovorus sp. MP688 is a methylotrophic bacterium that can be used as a pyrroloquinolone quinone (PQQ) producer. To obtain a comprehensive understanding of its metabolic capabilities, we constructed a genome-scale metabolic model (iWZ583) of Methylovorus sp. MP688, based on its genome annotations, data from public metabolic databases, and literature mining. The model includes 772 reactions, 764 metabolites, and 583 genes. Growth of Methylovorus sp. MP688 was simulated using different carbon and nitrogen sources, and the results were consistent with experimental data. A core metabolic essential gene set of 218 genes was predicted by gene essentiality analysis on minimal medium containing methanol. Based on in silico predictions, the addition of aspartate to the medium increased PQQ production by 4.6- fold. Deletion of three reactions associated with four genes (MPQ_1150, MPQ_1560, MPQ_1561, MPQ_1562) was predicted to yield a PQQ production rate of 0.123 mmol/gDW/h, while cell growth decreased by 2.5%. Here, model iWZ583 represents a useful platform for understanding the phenotype of Methylovorus sp. MP688 and improving PQQ production.

Isolation and genomic characterization of Novimethylophilus kurashikiensis gen. nov. sp. nov., a new lanthanide-dependent methylotrophic species of Methylophilaceae.

Recently, it has been found that two types of methanol dehydrogenases (MDHs) exist in Gram-negative bacterial methylotrophs, calcium-dependent MxaFI-MDH and lanthanide-dependent XoxF-MDH and the latter is more widespread in bacterial genomes. We aimed to isolate and characterize lanthanide-dependent methylotrophs. The growth of strain La2-4T on methanol, which was isolated from rice rhizosphere soil, was strictly lanthanide dependent. Its 16S rRNA gene sequence showed only 93.4% identity to that of Methylophilus luteus MimT , and the name Novimethylophilus kurashikiensis gen. nov. sp. nov. is proposed. Its draft genome (ca. 3.69 Mbp, G + C content 56.1 mol%) encodes 3579 putative CDSs and 84 tRNAs. The genome harbors five xoxFs but no mxaFI. XoxF4 was the major MDH in the cells grown on methanol and methylamine, evidenced by protein identification and quantitative PCR analysis. Methylamine dehydrogenase gene was absent in the La2-4T genome, while genes for the glutamate-mediated methylamine utilization pathway were detected. The genome also harbors those for the tetrahydromethanopterin and ribulose monophosphate pathways. Additionally, as known species, isolates of Burkholderia ambifaria, Cupriavidus necator and Dyadobacter endophyticus exhibited lanthanide dependent growth on methanol. Thus, lanthanide can be used as an essential growth factor for methylotrophic bacteria that do not harbor MxaFI-MDH.

Microbial megacities fueled by methane oxidation in a mineral spring cave.

Massive biofilms have been discovered in the cave of an iodine-rich former medicinal spring in southern Germany. The biofilms completely cover the walls and ceilings of the cave, giving rise to speculations about their metabolism. Here we report on first insights into the structure and function of the biofilm microbiota, combining geochemical, imaging and molecular analytics. Stable isotope analysis indicated that thermogenic methane emerging into the cave served as an important driver of biofilm formation. The undisturbed cavern atmosphere contained up to 3000 p.p.m. methane and was microoxic. A high abundance and diversity of aerobic methanotrophs primarily within the Methylococcales (Gammaproteobacteria) and methylotrophic Methylophilaceae (Betaproteobacteria) were found in the biofilms, along with a surprising diversity of associated heterotrophic bacteria. The highest methane oxidation potentials were measured for submerged biofilms on the cavern wall. Highly organized globular structures of the biofilm matrix were revealed by fluorescent lectin staining. We propose that the extracellular matrix served not only as an electron sink for nutrient-limited biofilm methylotrophs but potentially also as a diffusive barrier against volatilized iodine species. Possible links between carbon and iodine cycling in this peculiar habitat are discussed.

Biochemical Properties and Phylogeny of Hydroxypyruvate Reductases from Methanotrophic Bacteria with Different C1-Assimilation Pathways.

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C1-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C1-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.

Methanethiol-dependent dimethylsulfide production in soil environments.

Dimethylsulfide (DMS) is an environmentally important trace gas with roles in sulfur cycling, signalling to higher organisms and in atmospheric chemistry. DMS is believed to be predominantly produced in marine environments via microbial degradation of the osmolyte dimethylsulfoniopropionate (DMSP). However, significant amounts of DMS are also generated from terrestrial environments, for example, peat bogs can emit ~6 µmol DMS m-2 per day, likely via the methylation of methanethiol (MeSH). A methyltransferase enzyme termed 'MddA', which catalyses the methylation of MeSH, generating DMS, in a wide range of bacteria and some cyanobacteria, may mediate this process, as the mddA gene is abundant in terrestrial metagenomes. This is the first study investigating the functionality of MeSH-dependent DMS production (Mdd) in a wide range of aerobic environments. All soils and marine sediment samples tested produced DMS when incubated with MeSH. Cultivation-dependent and cultivation-independent methods were used to assess microbial community changes in response to MeSH addition in a grassland soil where 35.9% of the bacteria were predicted to contain mddA. Bacteria of the genus Methylotenera were enriched in the presence of MeSH. Furthermore, many novel Mdd+ bacterial strains were isolated. Despite the abundance of mddA in the grassland soil, the Mdd pathway may not be a significant source of DMS in this environment as MeSH addition was required to detect DMS at only very low conversion rates.

Methylophilaceae and Hyphomicrobium as target taxonomic groups in monitoring the function of methanol-fed denitrification biofilters in municipal wastewater treatment plants.

Molecular monitoring of bacterial communities can explain and predict the stability of bioprocesses in varying physicochemical conditions. To study methanol-fed denitrification biofilters of municipal wastewater treatment plants, bacterial communities of two full-scale biofilters were compared through fingerprinting and sequencing of the 16S rRNA genes. Additionally, 16S rRNA gene fingerprinting was used for 10-week temporal monitoring of the bacterial community in one of the biofilters. Combining the data with previous study results, the family Methylophilaceae and genus Hyphomicrobium were determined as suitable target groups for monitoring. An increase in the relative abundance of Hyphomicrobium-related biomarkers occurred simultaneously with increases in water flow, NO x- load, and methanol addition, as well as a higher denitrification rate, although the dominating biomarkers linked to Methylophilaceae showed an opposite pattern. The results indicate that during increased loading, stability of the bioprocess is maintained by selection of more efficient denitrifier populations, and this progress can be analyzed using simple molecular fingerprinting.

Emended Description of Methylovorus glucosotrophus Govorukhina and Trotsenko 1991.

Phylogeneticanalysis based,on comparison of the 16S rRNA gene sequences in combination with comparative analysis of physiological, biochemical, and chemotaxonomic characteristics and DNA-DNA hybridization revealed that "Methylobacillusfructoseoxidans" 34 (VKM B-1609 = DSM 5897 and-Methylov- orus glucosotrophus 6B 1T (ATCC 49758T = DSM 6874T = VKM B- 1745T = NCIMB 13222 ) belong to the same Methylovorus species. Extended description of the limited facultative methylotroph Methylovorus gluco- sotrophus is proposed, which includes the fructose-utilizing strain 34. Emended description of Methylovorus glucosotrophus is provided.

Comprehensive Genomic Analyses of the OM43 Clade, Including a Novel Species from the Red Sea, Indicate Ecotype Differentiation among Marine Methylotrophs.

The OM43 clade within the family Methylophilaceae of Betaproteobacteria represents a group of methylotrophs that play important roles in the metabolism of C1 compounds in marine environments and other aquatic environments around the globe. Using dilution-to-extinction cultivation techniques, we successfully isolated a novel species of this clade (here designated MBRS-H7) from the ultraoligotrophic open ocean waters of the central Red Sea. Phylogenomic analyses indicate that MBRS-H7 is a novel species that forms a distinct cluster together with isolate KB13 from Hawaii (Hawaii-Red Sea [H-RS] cluster) that is separate from the cluster represented by strain HTCC2181 (from the Oregon coast). Phylogenetic analyses using the robust 16S-23S internal transcribed spacer revealed a potential ecotype separation of the marine OM43 clade members, which was further confirmed by metagenomic fragment recruitment analyses that showed trends of higher abundance in low-chlorophyll and/or high-temperature provinces for the H-RS cluster but a preference for colder, highly productive waters for the HTCC2181 cluster. This potential environmentally driven niche differentiation is also reflected in the metabolic gene inventories, which in the case of the H-RS cluster include those conferring resistance to high levels of UV irradiation, temperature, and salinity. Interestingly, we also found different energy conservation modules between these OM43 subclades, namely, the existence of the NADH:quinone oxidoreductase complex I (NUO) system in the H-RS cluster and the nonhomologous NADH:quinone oxidoreductase (NQR) system in the HTCC2181 cluster, which might have implications for their overall energetic yields.