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.

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.

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.

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.

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.

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.

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.

The ecology of pelagic freshwater methylotrophs assessed by a high-resolution monitoring and isolation campaign.

Methylotrophic planktonic bacteria fulfill a particular role in the carbon cycle of lakes via the turnover of single-carbon compounds. We studied two planktonic freshwater lineages (LD28 and PRD01a001B) affiliated with Methylophilaceae (Betaproteobacteria) in Lake Zurich, Switzerland, by a combination of molecular and cultivation-based approaches. Their spatio-temporal distribution was monitored at high resolution (n=992 samples) for 4 consecutive years. LD28 methylotrophs constituted up to 11 × 10(7) cells l(-1) with pronounced peaks in spring and autumn-winter, concomitant with blooms of primary producers. They were rare in the warm water layers during summer but abundant in the cold hypolimnion, hinting at psychrophilic growth. Members of the PRD01a001B lineage were generally less abundant but also had maxima in spring. More than 120 axenic strains from these so far uncultivated lineages were isolated from the pelagic zone by dilution to extinction. Phylogenetic analysis separated isolates into two distinct genotypes. Isolates grew slowly (µmax=0.4 d(-1)), were of conspicuously small size, and were indeed psychrophilic, with higher growth yield at low temperatures. Growth was enhanced upon addition of methanol and methylamine to sterile lake water. Genomic analyses of two strains confirmed a methylotrophic lifestyle with a reduced set of genes involved in C1 metabolism. The very small and streamlined genomes (1.36 and 1.75 Mb) shared several pathways with the marine OM43 lineage. As the closest described taxa (Methylotenera sp.) are only distantly related to either set of isolates, we propose a new genus with two species, that is, 'Candidatus Methylopumilus planktonicus' (LD28) and 'Candidatus Methylopumilus turicensis' (PRD01a001B).

SIP metagenomics identifies uncultivated Methylophilaceae as dimethylsulphide degrading bacteria in soil and lake sediment.

Dimethylsulphide (DMS) has an important role in the global sulphur cycle and atmospheric chemistry. Microorganisms using DMS as sole carbon, sulphur or energy source, contribute to the cycling of DMS in a wide variety of ecosystems. The diversity of microbial populations degrading DMS in terrestrial environments is poorly understood. Based on cultivation studies, a wide range of bacteria isolated from terrestrial ecosystems were shown to be able to degrade DMS, yet it remains unknown whether any of these have important roles in situ. In this study, we identified bacteria using DMS as a carbon and energy source in terrestrial environments, an agricultural soil and a lake sediment, by DNA stable isotope probing (SIP). Microbial communities involved in DMS degradation were analysed by denaturing gradient gel electrophoresis, high-throughput sequencing of SIP gradient fractions and metagenomic sequencing of phi29-amplified community DNA. Labelling patterns of time course SIP experiments identified members of the Methylophilaceae family, not previously implicated in DMS degradation, as dominant DMS-degrading populations in soil and lake sediment. Thiobacillus spp. were also detected in (13)C-DNA from SIP incubations. Metagenomic sequencing also suggested involvement of Methylophilaceae in DMS degradation and further indicated shifts in the functional profile of the DMS-assimilating communities in line with methylotrophy and oxidation of inorganic sulphur compounds. Overall, these data suggest that unlike in the marine environment where gammaproteobacterial populations were identified by SIP as DMS degraders, betaproteobacterial Methylophilaceae may have a key role in DMS cycling in terrestrial environments.

Bacterial metabolism of methylated amines and identification of novel methylotrophs in Movile Cave.

Movile Cave, Romania, is an unusual underground ecosystem that has been sealed off from the outside world for several million years and is sustained by non-phototrophic carbon fixation. Methane and sulfur-oxidising bacteria are the main primary producers, supporting a complex food web that includes bacteria, fungi and cave-adapted invertebrates. A range of methylotrophic bacteria in Movile Cave grow on one-carbon compounds including methylated amines, which are produced via decomposition of organic-rich microbial mats. The role of methylated amines as a carbon and nitrogen source for bacteria in Movile Cave was investigated using a combination of cultivation studies and DNA stable isotope probing (DNA-SIP) using (13)C-monomethylamine (MMA). Two newly developed primer sets targeting the gene for gamma-glutamylmethylamide synthetase (gmaS), the first enzyme of the recently-discovered indirect MMA-oxidation pathway, were applied in functional gene probing. SIP experiments revealed that the obligate methylotroph Methylotenera mobilis is one of the dominant MMA utilisers in the cave. DNA-SIP experiments also showed that a new facultative methylotroph isolated in this study, Catellibacterium sp. LW-1 is probably one of the most active MMA utilisers in Movile Cave. Methylated amines were also used as a nitrogen source by a wide range of non-methylotrophic bacteria in Movile Cave. PCR-based screening of bacterial isolates suggested that the indirect MMA-oxidation pathway involving GMA and N-methylglutamate is widespread among both methylotrophic and non-methylotrophic MMA utilisers from the cave.

Multiple pqqA genes respond differently to environment and one contributes dominantly to pyrroloquinoline quinone synthesis.

Pyrroloquinoline quinone is the third redox cofactor after nicotinamide and flavin in bacteria, and its biosynthesis pathway comprise five steps initiated from a precursor peptide PqqA coded by pqqA gene. Methylovorus sp. MP688 is equipped with five copies of pqqA genes. Herein, the transcription of pqqA genes under different conditions by real-time quantitative PCR and ß-galactosidase reporter genes are reported. Multiple pqqA genes were proved to play significant roles and contribute differently in PQQ synthesis. pqqA1, pqqA2, and pqqA4 were determined to be dominantly transcribed over the others, and correspondingly absence of any of the three genes caused a decrease in PQQ synthesis. Notably, pqqA was up-regulated in low pH and limited oxygen environment, and it is pqqA2 promoter that could be induced when bacteria were transferred from pH 7.0 to pH 5.5. Deletion analysis revealed a region within pqqA2 promoter inhibiting transcription. PQQ concentration was increased by overexpression of pqq genes under control of truncated pqqA2 promoter. The results not only imply there exist negative transcriptional regulators for pqqA2 but also provide us a new approach to achieve higher PQQ production by deleting the target binding sequence.

[Culturable psychrotolerant methanotrophic bacteria in landfill cover soil].

Methanotrophs closely related to psychrotolerant members of the genera Methylobacter and Methylocella were identified in cultures enriched at 10@C from landfill cover soil samples collected in the period from April to November. Mesophilic methanotrophs of the genera Methylobacter and Methylosinus were found in cultures enriched at 20 degrees C from the same cover soil samples. A thermotolerant methanotroph related to Methylocaldum gracile was identified in the culture enriched at 40 degrees C from a sample collected in May (the temperature of the cover soil was 11.5-12.5 degrees C). In addition to methanotrophs, methylobacteria of the genera Methylotenera and Methylovorus and members of the genera Verrucomicrobium, Pseudomonas, Pseudoxanthomonas, Dokdonella, Candidatus Protochlamydia, and Thiorhodospira were also identified in the enrichment cultures. A methanotroph closely related to the psychrotolerant species Methylobacter tundripaludum (98% sequence identity of 16S r-RNA genes with the type strain SV96(T)) was isolated in pure culture. The introduction of a mixture of the methanotrophic enrichments, grown at 15 degrees C, into the landfill cover soil resulted in a decrease in methane emission from the landfill surface in autumn (October, November). The inoculum used was demonstrated to contain methanotrophs closely related to Methylobacter tundripaludum SV96.

The expanded diversity of methylophilaceae from Lake Washington through cultivation and genomic sequencing of novel ecotypes.

We describe five novel Methylophilaceae ecotypes from a single ecological niche in Lake Washington, USA, and compare them to three previously described ecotypes, in terms of their phenotype and genome sequence divergence. Two of the ecotypes appear to represent novel genera within the Methylophilaceae. Genome-based metabolic reconstruction highlights metabolic versatility of Methylophilaceae with respect to methylotrophy and nitrogen metabolism, different ecotypes possessing different combinations of primary substrate oxidation systems (MxaFI-type methanol dehydrogenase versus XoxF-type methanol dehydrogenase; methylamine dehydrogenase versus N-methylglutamate pathway) and different potentials for denitrification (assimilatory versus respiratory nitrate reduction). By comparing pairs of closely related genomes, we uncover that site-specific recombination is the main means of genomic evolution and strain divergence, including lateral transfers of genes from both closely- and distantly related taxa. The new ecotypes and the new genomes contribute significantly to our understanding of the extent of genomic and metabolic diversity among organisms of the same family inhabiting the same ecological niche. These organisms also provide novel experimental models for studying the complexity and the function of the microbial communities active in methylotrophy.

Discovery and characterization of a 5-hydroxymethylfurfural oxidase from Methylovorus sp. strain MP688.

In the search for useful and renewable chemical building blocks, 5-hydroxymethylfurfural (HMF) has emerged as a very promising candidate, as it can be prepared from sugars. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a substitute for petroleum-based terephthalate in polymer production. On the basis of a recently identified bacterial degradation pathway for HMF, candidate genes responsible for selective HMF oxidation have been identified. Heterologous expression of a protein from Methylovorus sp. strain MP688 in Escherichia coli and subsequent enzyme characterization showed that the respective gene indeed encodes an efficient HMF oxidase (HMFO). HMFO is a flavin adenine dinucleotide-containing oxidase and belongs to the glucose-methanol-choline-type flavoprotein oxidase family. Intriguingly, the activity of HMFO is not restricted to HMF, as it is active with a wide range of aromatic primary alcohols and aldehydes. The enzyme was shown to be relatively thermostable and active over a broad pH range. This makes HMFO a promising oxidative biocatalyst that can be used for the production of FDCA from HMF, a reaction involving both alcohol and aldehyde oxidations.

Methylovorus sp. MP688 exopolysaccharides contribute to oxidative defense and bacterial survival under adverse condition.

Methylovorus sp. MP688 is an aerobic bacterium that can grow on reduced C1 compounds such as methanol, being regarded as an attractive producer for many commercial materials including polysaccharides. The aim of the study was to learn more information about the biochemical and physiological functions of extracellular polysaccharides (EPS) produced by Methylovorus sp. MP688. Firstly, gene clusters involved in EPS synthesis were identified by whole genome sequence analysis. Then EPS produced by Methylovorus sp. MP688 were isolated and purified by centrifugation, precipitation and deproteinization. Purified EPS displayed antioxidant activity towards DPPH free radical, hydroxyl radical and superoxide anion radical. Glucose, galactose and mannose were identified to be main component monosaccharides in EPS. One mutant with defect in EPS production was obtained by knocking out epsA gene within EPS synthesis cluster. Strain with deletion of epsA exhibited compromised growth ability in the presence of oxidative stress due to the sharp reduction in EPS synthesis. Meanwhile, the intracellular antioxidant scavengers were activated to a higher level in order to counteract with the excess harmful radicals. In addition, EPS were assimilated by Methylovorus sp. MP688 to survive under disadvantage condition when the preferred carbon source was exhausted. It was reasonable to conclude that EPS produced by Methylovorus sp. MP688 contributed to oxidative defense and bacterial survival under adverse condition.

Novel methylotrophic isolates from lake sediment, description of Methylotenera versatilis sp. nov. and emended description of the genus Methylotenera.

Phylogenetic positions, and genotypic and phenotypic characteristics of three novel methylotrophic isolates, strains 301(T), 30S and SIP3-4, from sediment of Lake Washington, Seattle, USA, are described. The strains were restricted facultative methylotrophs capable of growth on single carbon compounds (methylamine and methanol) in addition to a limited range of multicarbon compounds. All strains used the N-methylglutamate pathway for methylamine oxidation. Strain SIP3-4 possessed the canonical (MxaFI) methanol dehydrogenase, but strains 301(T) and 30S did not. All three strains used the ribulose monophosphate pathway for C1 assimilation. The major fatty acids in the three strains were C(16:0) and C(16:1)ω7c. The DNA G+C contents of strains 301(T) and SIP3-4 were 42.6 and 54.6 mol%, respectively. Based on 16S rRNA gene sequence phylogeny and the relevant phenotypic characteristics, strain SIP3-4 was assigned to the previously defined species Methylovorus glucosotrophus. Strains 301(T) and 30S were closely related to each other (100% 16S rRNA gene sequence similarity) and shared 96.6% 16S rRNA gene sequence similarity with a previously described isolate, Methylotenera mobilis JLW8(T). Based on significant genomic and phenotypic divergence with the latter, strains 301(T) and 30S represent a novel species within the genus Methylotenera, for which the name Methylotenera versatilis sp. nov. is proposed; the type strain is 301(T) (=VKM B-2679(T)=JCM 17579(T)). An emended description of the genus Methylotenera is provided.

An integrated proteomics/transcriptomics approach points to oxygen as the main electron sink for methanol metabolism in Methylotenera mobilis.

Methylotenera species, unlike their close relatives in the genera Methylophilus, Methylobacillus, and Methylovorus, neither exhibit the activity of methanol dehydrogenase nor possess mxaFI genes encoding this enzyme, yet they are able to grow on methanol. In this work, we integrated a genome-wide proteomics approach, shotgun proteomics, and a genome-wide transcriptomics approach, shotgun transcriptome sequencing (RNA-seq), of Methylotenera mobilis JLW8 to identify genes and enzymes potentially involved in methanol oxidation, with special attention to alternative nitrogen sources, to address the question of whether nitrate could play a role as an electron acceptor in place of oxygen. Both proteomics and transcriptomics identified a limited number of genes and enzymes specifically responding to methanol. This set includes genes involved in oxidative stress response systems, a number of oxidoreductases, including XoxF-type alcohol dehydrogenases, a type II secretion system, and proteins without a predicted function. Nitrate stimulated expression of some genes in assimilatory nitrate reduction and denitrification pathways, while ammonium downregulated some of the nitrogen metabolism genes. However, none of these genes appeared to respond to methanol, which suggests that oxygen may be the main electron sink during growth on methanol. This study identifies initial targets for future focused physiological studies, including mutant analysis, which will provide further details into this novel process.

Genomes of three methylotrophs from a single niche reveal the genetic and metabolic divergence of the methylophilaceae.

The genomes of three representatives of the family Methylophilaceae, Methylotenera mobilis JLW8, Methylotenera versatilis 301, and Methylovorus glucosetrophus SIP3-4, all isolated from a single study site, Lake Washington in Seattle, WA, were completely sequenced. These were compared to each other and to the previously published genomes of Methylobacillus flagellatus KT and an unclassified Methylophilales strain, HTCC2181. Comparative analysis revealed that the core genome of Methylophilaceae may be as small as approximately 600 genes, while the pangenome may be as large as approximately 6,000 genes. Significant divergence between the genomes in terms of both gene content and gene and protein conservation was uncovered, including the varied presence of certain genes involved in methylotrophy. Overall, our data demonstrate that metabolic potentials can vary significantly between different species of Methylophilaceae, including organisms inhabiting the very same environment. These data suggest that genetic divergence among the members of this family may be responsible for their specialized and nonredundant functions in C1 cycling, which in turn suggests means for their successful coexistence in their specific ecological niches.