Methylotrophic Bacteria Research Articles

Impact of foliar fertilization of nutrients and biofertilizer on the growth and yield of hybrid cotton (Gossypium hirsutum L.)

The newly released high yielding cotton cultivars are higher boll retention rate than conventional cultivars that have higher overall nutrient demand during the flowering and boll development stages. Therefore, this field experiment was conducted at during summer, 2020 at Annamalai University experimental farm, Department of Agronomy, Faculty of Agriculture, Annamalai Nagar, Tamil Nadu, India, 608 002 to ascertain the impact of foliar application of essential nutrients and biofertilizers on growth and yield of cotton. The experiment was laid out in randomized block design with twelve treatments of foliar application macro and micro-nutrients with and without Pink pigmented facultative methylotrophic bacteria (PPFM) and replicated thrice. Foliar application of macro, micronutrients and PPFM registered the increased values of growth characters, yield attributes and yield of hybrid cotton. Among the foliar application practice tested, foliar application of 2% urea + 1% PPFM at flowering and boll development stages significantly resulted in the enhances values of growth characters viz., plant height (157.40 cm), Dry matter production (6003 kg/ha), number of monopodial branches (3.28/plant), growth analysis like Leaf area index (4.30), Crop growth rate (7.01 g/m2/day) and Relative growth rate (0.0144 g/g/day), yield attributes viz., number of sympodial branches (21.77/plant), number of squares (83.77/plant), number of bolls (45.04/plant), boll weight (5.58 g) and seed cotton yield (3185 kg/ha). This was statistically on par with the foliar application of 2% MgSO4 + 1% PPFM at flowering and boll development stages. The lower values of growth characters, yield attributes and yield were recorded under water spray treatment. Based on the results of field investigation it can be concluded that foliar application of 2% urea + 1% PPFM at flowering and boll development stages can possibly enhance the growth and yield of hybrid cotton grown under dryland condition.

The potential role of betaine in enhancement of microbial-assisted phytoremediation of benzophenone-3 contaminated soil

Benzophenone-3 (BP-3) is an emerging environmental pollutant used in personal care products, helping to reduce the risk of ultraviolet radiation to human skin. The BP-3 removal potential from soil by tobacco (Nicotiana tabacum) assisted with Methylophilus sp. FP-6 was explored in our previous study. However, the reduced BP-3 remediation efficiency by FP-6 in soil and the inhibited plant growth by BP-3 limited the application of this phytoremediation strategy. The aim of the present study was to reveal the potential roles of betaine, as the methyl donor of methylotrophic bacteria and plant regulator, in improving the strain FP-6-assisted phytoremediation capacity of BP-3 contaminated soil. The results revealed that strain FP-6 could use betaine as a co-metabolism substrate to enhance the BP-3 degradation activity. About 97.32% BP-3 in soil was effectively removed in the phytoremediation system using tobacco in combination with FP-6 and betaine for 40 d while the concentration of BP-3 in tobacco significantly reduced. Moreover, the biomass and photosynthetic efficiency of plants were remarkably improved through the combined treatment of betaine and strain FP-6. Simultaneously, inoculation of FP-6 in the presence of betaine stimulated the change of local microbial community structure, which might correlate with the production of a series of hydrolases and reductases involved in soil carbon, nitrogen and phosphorus cycling processes. Meantime, some of the dominant bacteria could secrete various multiple enzymes involved in degrading organic pollutants, such as laccase, to accelerate the demethylation and hydroxylation of BP-3. Overall, the results from this study proposed that the co-metabolic role of betaine could be utilized to strengthen microbial-assisted phytoremediation process by increasing the degradation ability of methylotrophic bacteria and enhancing plant tolerance to BP-3. The present results provide novel insights and perspectives for broadening the engineering application scope of microbial-assisted phytoremediation of organic pollutants without sacrificing economic crop safety.

Role of EPS in mitigation of plant abiotic stress: The case of Methylobacterium extorquens PA1

Methylobacterium extorquens is a facultative methylotrophic Gram-negative bacterium, often associated with plants, that exhibits a unique ability to grow in the presence of high methanol concentrations, which serves as a single carbon energy source. We found that M. extorquens strain PA1 secretes a mixture of different exopolysaccharides (EPSs) when grown in reference medium or in presence of methanol, that induces the secretion of a peculiar and heterogenous mixture of EPSs, with different structure, composition, repeating units, bulk and a variable degree of methylation. These factors influenced 3D structure and supramolecular assets, diffusion properties and hydrodynamic radius, and likely contribute to increase methanol tolerance and cell stability. No direct methanol involvement in the EPSs solvation shell was detected, indicating that the polymer exposure to methanol is water mediated. The presence of methanol induces no changes in size and shape of the polymer chains, highlighting how water-methanol mixtures are a good solvent for refEPS and metEPS.

Siderophore for Lanthanide and Iron Uptake for Methylotrophy and Plant Growth Promotion in Methylobacterium aquaticum Strain 22A.

Methylobacterium and Methylorubrum species are facultative methylotrophic bacteria that are abundant in the plant phyllosphere. They have two methanol dehydrogenases, MxaF and XoxF, which are dependent on either calcium or lanthanides (Lns), respectively. Lns exist as insoluble minerals in nature, and their solubilization and uptake require a siderophore-like substance (lanthanophore). Methylobacterium species have also been identified as plant growth-promoting bacteria although the actual mechanism has not been well-investigated. This study aimed to reveal the roles of siderophore in Methylobacterium aquaticum strain 22A in Ln uptake, bacterial physiology, and plant growth promotion. The strain 22A genome contains an eight-gene cluster encoding the staphyloferrin B-like (sbn) siderophore. We demonstrate that the sbn siderophore gene cluster is necessary for growth under low iron conditions and was complemented by supplementation with citrate or spent medium of the wild type or other strains of the genera. The siderophore exhibited adaptive features, including tolerance to oxidative and nitrosative stress, biofilm formation, and heavy metal sequestration. The contribution of the siderophore to plant growth was shown by the repressive growth of duckweed treated with siderophore mutant under iron-limited conditions; however, the siderophore was dispensable for strain 22A to colonize the phyllosphere. Importantly, the siderophore mutant could not grow on methanol, but the siderophore could solubilize insoluble Ln oxide, suggesting its critical role in methylotrophy. We also identified TonB-dependent receptors (TBDRs) for the siderophore–iron complex, iron citrate, and Ln, among 12 TBDRs in strain 22A. Analysis of the siderophore synthesis gene clusters and TBDR genes in Methylobacterium genomes revealed the existence of diverse types of siderophores and TBDRs. Methylorubrum species have an exclusive TBDR for Ln uptake that has been identified as LutH. Collectively, the results of this study provide insight into the importance of the sbn siderophore in Ln chelation, bacterial physiology, and the diversity of siderophore and TBDRs in Methylobacterium species.

Genome-Wide Transcription Start Sites Mapping in Methylorubrum Grown with Dichloromethane and Methanol.

Dichloromethane (DCM, methylene chloride) is a toxic halogenated volatile organic compound massively used for industrial applications, and consequently often detected in the environment as a major pollutant. DCM biotransformation suggests a sustainable decontamination strategy of polluted sites. Among methylotrophic bacteria able to use DCM as a sole source of carbon and energy for growth, Methylorubrum extorquens DM4 is a longstanding reference strain. Here, the primary 5′-ends of transcripts were obtained using a differential RNA-seq (dRNA-seq) approach to provide the first transcription start site (TSS) genome-wide landscape of a methylotroph using DCM or methanol. In total, 7231 putative TSSs were annotated and classified with respect to their localization to coding sequences (CDSs). TSSs on the opposite strand of CDS (antisense TSS) account for 31% of all identified TSSs. One-third of the detected TSSs were located at a distance to the start codon inferior to 250 nt (average of 84 nt) with 7% of leaderless mRNA. Taken together, the global TSS map for bacterial growth using DCM or methanol will facilitate future studies in which transcriptional regulation is crucial, and efficient DCM removal at polluted sites is limited by regulatory processes.

Stratification of microbial communities and their functions in mossy biofilms colonizing the giant monolithic statue of buddha

Moss-microbial biofilm is an important topic for the biodeterioration of sandstone heritages. However, the processes that biofilms colonize and erode sandstone heritages should be better defined. We divided mosses into three strata perpendicular to the sandstone face in the direction from the atmosphere to the sandstone surface (the upper layer, the middle layer, and the lower layer) to explore the mechanisms involved. The results showed that the bacteria in various layers of moss exhibited a distinct layered pattern of nutrient strategies. The bacteria in the upper layer were mostly photoautotrophic (e.g., Cyanobacteria, WPS-2, etc.) and methylotrophic bacteria (e.g., Methylobacterium-Methylorubrum, Sphingomonas, etc.). The bacteria in the lower layer were primarily heterotrophic as special Actinobacteria with the ability of weathering (e.g., Actinomycetospora, Nocardioides, etc.). Particularly, the potential acid condition in the lower layer would promote the growth of acidophilic bacteria (e.g., Acidipila, Acidiphilium, etc.) and acidophilic iron-reducing bacteria (e.g., Acidiphilium, Acidicaldus, etc.), which deteriorate the alkaline cement and ferric oxides. Furthermore, nucleotide sequences associated with the Calvin-Benson-Bassham cycle, methanol utilization (methanol dehydrogenase), nitrogen fixation (nitrogenase), and ammonia oxidation (ammonia monooxygenase) were more prevalent in the upper layer of bacterial communities. The nucleotide sequences related to the reductive tricarboxylic acid cycle, reduced acetyl-CoA pathway, decomposition of organic carbon (β-1,4-glucosidase), and organic nitrogen (β-N-acetyl glucosaminidase) were more abundant in the lower layer. Therefore, in addition to the damage to the moss itself, we should pay more attention to the microorganisms that live in symbiosis with it. Our study shows that the strategy of stratified colonization of microorganisms would supply the nutrients available for moss-microbial biofilm, the formation of which would in turn deteriorate sandstone heritages.

Role of methylotrophic bacteria in managing abiotic stresses for enhancing agricultural production

Abiotic stresses are a significant factor that considerably limits plant growth and productivity. Methylotrophs are an essential group of bacteria that utilize volatile carbon compounds, are prolific colonizers of different plant parts, and play a vital role in plant growth promotion (PGP) under stress conditions. Numerous rhizospheric and phyllosphere methylotrophs have been reported to exhibit PGP activities with superior stress-tolerating capacity against drought, heavy metal, salinity, high and low temperatures, solar ultraviolet radiation, and other harsh environmental conditions. Methylotrophs promote plant growth through N2 fixation, phosphate solubilization, production of phytohormones (i.e., auxins, gibberellins, and cytokinins), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, siderophore, ammonia production, and secondary metabolites. The production of these compounds by methylotrophs protects a plant against adverse environmental conditions and influences its productivity. This review discusses the role methylotrophs play in managing various abiotic stresses, how they help mitigate these stresses, and how they improve agricultural productivity.

Growth of Methylobacterium organophilum in Methanol for the Simultaneous Production of Single-Cell Protein and Metabolites of Interest.

This study aims to monitor the growth of the methylotrophic bacteria Methylobacterium organophilum in a culture medium with methanol as a carbon source and to verify the production of unicellular proteins and other biomolecules, such as carotenoids, exopolysaccharides and polyhydroxyalkanoates, making them more attractive as animal feed. Bacterial growth was studied in shake flasks using different carbon/nitrogen (C:N) ratios to determine their best ratio for achieving the highest volumetric productivity of cells and substrate consumption rate. This optimal parameter was further used in a fed-batch operating bioreactor system to define the kinetic profile of cell growth. Methanol consumption was measured by HPLC analysis and the extracted pigments were analyzed by liquid chromatography/mass spectrometry. Chemical composition and rheological properties of the produced exopolysaccharides were also determined. The best experimental parameters were verified using an initial methanol concentration of 7 g/L in the culture medium. The same initial substrate concentration was used in the fed-batch operation and after 60 h of cultivation 5 g/L of biomass were obtained. The accumulation of carotenoids associated with cell growth was monitored, reaching a concentration of 1.6 mg/L at the end of the process. These pigments were then analyzed and characterized as a set of xanthophylls (oxidized carotenoids). In addition, two other product types were identified during the fed-batch operation: exopolysaccharides, which reached a concentration of 8.9 g/L at the end of the cultivation, and an intracellular granular structure that was detected by transmission electron microscopy (TEM), suggesting the accumulation of polyhydroxyalkanoate (PHA), most likely polyhydroxybutyrate. Methylobacterium organophilum demonstrated a unique ability to produce compounds of commercial interest. The distinct metabolic diversity of this bacterium makes room for its use in biorefineries.

Methanethiol Consumption and Hydrogen Sulfide Production by the Thermoacidophilic Methanotroph Methylacidiphilum fumariolicum SolV.

Methanotrophs aerobically oxidize methane to carbon dioxide to make a living and are known to degrade various other short chain carbon compounds as well. Volatile organic sulfur compounds such as methanethiol (CH3SH) are important intermediates in the sulfur cycle. Although volatile organic sulfur compounds co-occur with methane in various environments, little is known about how these compounds affect methanotrophy. The enzyme methanethiol oxidase catalyzing the oxidation of methanethiol has been known for decades, but only recently the mtoX gene encoding this enzyme was identified in a methylotrophic bacterium. The presence of a homologous gene in verrucomicrobial methanotrophs prompted us to examine how methanotrophs cope with methanethiol. Here, we show that the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV consumes methanethiol and produces H2S, which is concurrently oxidized. Consumption of methanethiol is required since methanethiol inhibits methane oxidation. Cells incubated with ∼15 μM methanethiol from the start clearly showed inhibition of growth. After depletion of methanethiol, growth resumed within 1 day. Genes encoding a putative methanethiol oxidase were found in a variety of methanotrophs. Therefore, we hypothesize that methanethiol degradation is a widespread detoxification mechanism in methanotrophs in a range of environments.

Hyperaccumulation of Gadolinium by Methylorubrum extorquens AM1 Reveals Impacts of Lanthanides on Cellular Processes Beyond Methylotrophy.

Lanthanides (Ln) are a new group of life metals, and many questions remain regarding how they are acquired and used in biology. Methylotrophic bacteria can acquire, transport, biomineralize, and use Ln as part of a cofactor complex with pyrroloquinoline quinone (PQQ) in alcohol dehydrogenases. For most methylotrophic bacteria use is restricted to the light Ln, which range from lanthanum to samarium (atomic numbers 57–62). Understanding how the cell differentiates between light and heavy Ln, and the impacts of these metals on the metabolic network, will advance the field of Ln biochemistry and give insights into enzyme catalysis, stress homeostasis, and metal biomineralization and compartmentalization. We report robust methanol growth with the heavy Ln gadolinium by a genetic variant of the model methylotrophic bacterium Methylorubrum extorquens AM1, named evo-HLn, for “evolved for Heavy Lanthanides.” A non-synonymous single nucleotide polymorphism in a cytosolic hybrid histidine kinase/response regulator allowed for sweeping transcriptional alterations to heavy metal stress response, methanol oxidation, and central metabolism. Increased expression of genes for Ln acquisition and uptake, production of the Ln-chelating lanthanophore, PQQ biosynthesis, and phosphate transport and metabolism resulted in gadolinium hyperaccumulation of 36-fold with a trade-off for light Ln accumulation. Gadolinium was hyperaccumulated in an enlarged acidocalcisome-like compartment. This is the first evidence of a bacterial intracellular Ln-containing compartment that we name the “lanthasome.” Carotenoid and toblerol biosynthesis were also upregulated. Due to its unique capabilities, evo-HLn can be used to further magnetic resonance imaging (MRI) and bioremediation technologies. In this regard, we show that gadolinium hyperaccumulation was sufficient to produce MRI contrast in whole cells, and that evo-HLn was able to readily acquire the metal from the MRI contrast agent gadopentetic acid. Finally, hyperaccumulation of gadolinium, differential uptake of light and heavy Ln, increased PQQ levels, and phosphate transport provide new insights into strategies for Ln recovery.

Stimulated Organic Carbon Cycling and Microbial Community Shift Driven by a Simulated Cold-Seep Eruption.

ABSTRACTCold seeps are a major methane source in marine systems, and microbe-mediated anaerobic oxidation of methane (AOM) serves as an effective barrier for preventing methane emissions from sediment to water. However, how the periodic eruption of cold seeps drives the microbial community shift and further affects carbon cycling has been largely neglected, mainly due to the technical challenge of analyzing the in situ communities undergoing such geological events. Using a continuously running high-pressure bioreactor to simulate these events, we found that under the condition of simulated eruptions, the abundance of AOM-related species decreased, and some methane was oxidized to methyl compounds to feed heterotrophs. The methanogenic archaeon Methanolobus replaced ANME-2a as the dominant archaeal group; moreover, the levels of methylotrophic bacteria, such as Pseudomonas, Halomonas, and Methylobacter, quickly increased, while those of sulfate-reducing bacteria decreased. According to the genomic analysis, Methylobacter played an important role in incomplete methane oxidation during eruptions; this process was catalyzed by the genes pmoABC under anaerobic conditions when the methane pressure was high, possibly generating organic carbon. Additionally, the findings showed that methyl compounds can also be released to the environment during methanogenesis and AOM under eruption conditions when the methane pressure is high.

Hydrous ferric oxides (HFO's) precipitated from contaminated waters at several abandoned Sb deposits – Interdisciplinary assessment

The presented paper represents a comprehensive analysis of ochre sediments precipitated from Fe rich drainage waters contaminated by arsenic and antimony. Ochre samples from three abandoned Sb deposits were collected in three different seasons and were characterized from the mineralogical, geochemical, and microbiological point of view. They were formed mainly by poorly crystallized 2-line ferrihydrite, with the content of arsenic in samples ranging from 7 g·kg−1 to 130 g·kg−1 and content of antimony ranging from 0.25 g·kg−1 up to 12 g·kg−1. Next-generation sequencing approach with 16S RNA, 18S RNA and ITS markers was used to characterize bacterial, fungal, algal, metazoal and protozoal communities occurring in the HFOs. In the 16S RNA, the analysis dominated bacteria (96.2%) were mainly Proteobacteria (68.8%) and Bacteroidetes (10.2%) and to less extent also Acidobacteria, Actinobacteria, Cyanobacteria, Firmicutes, Nitrosprae and Chloroflexi. Alpha and beta diversity analysis revealed that the bacterial communities of individual sites do not differ significantly, and only subtle seasonal changes were observed. In this As and Sb rich, circumneutral microenvironment, rich in iron, sulfates and carbonates, methylotrophic bacteria (Methylobacter, Methylotenera), metal/reducing bacteria (Geobacter, Rhodoferax), metal-oxidizing and denitrifying bacteria (Gallionella, Azospira, Sphingopyxis, Leptothrix and Dechloromonas), sulfur-oxidizing bacteria (Sulfuricurvum, Desulphobulbaceae) and nitrifying bacteria (Nitrospira, Nitrosospira) accounted for the most dominant ecological groups and their impact over Fe, As, Sb, sulfur and nitrogen geocycles is discussed. This study provides evidence of diverse microbial communities that exist in drainage waters and are highly important in the process of mobilization or immobilization of the potentially toxic elements.

Methanol Dehydrogenases as a Key Biocatalysts for Synthetic Methylotrophy.

One-carbon (C1) chemicals are potential building blocks for cheap and sustainable re-sources such as methane, methanol, formaldehyde, formate, carbon monoxide, and more. These resources have the potential to be made into raw materials for various products used in our daily life or precursors for pharmaceuticals through biological and chemical processes. Among the soluble C1 substrates, methanol is regarded as a biorenewable platform feedstock because nearly all bioresources can be converted into methanol through syngas. Synthetic methylotrophy can be exploited to produce fuels and chemicals using methanol as a feedstock that integrates natural or artificial methanol assimilation pathways in platform microorganisms. In the methanol utilization in methylotrophy, methanol dehydrogenase (Mdh) is a primary enzyme that converts methanol to formaldehyde. The discovery of new Mdhs and engineering of present Mdhs have been attempted to develop synthetic methylotrophic bacteria. In this review, we describe Mdhs, including in terms of their enzyme properties and engineering for desired activity. In addition, we specifically focus on the application of various Mdhs for synthetic methylotrophy.

Development of deficit irrigation program for improving the water use efficiency, growth, and yield of groundnut in the semiarid tropical region of India

In this study, we assessed the efficiency of a deficit irrigation program (DIP) on groundnut (<i>Arachis hypogaea</i> L.) yield and economic profits. The DIP was designed to combine frequency and volume of irrigation to develop an appropriate irrigation water management (IWM) strategy for groundnut. Two field experiments were conducted at the Agricultural College and Research Institute, Madurai, Tamil Nadu, India, with a bunch variety of groundnut, VRI 2, under a randomized block design (RBD) with three replications. The treatments included DIP in combination with a foliar application of pink-pigmented facultative methylotrophic bacteria (PPFM). The irrigation frequencies adopted for the first set of treatments were high frequency (5/5), medium frequency (7/5), and lower frequency (7/7) starting from 14 days after sowing (DAS). The effects of early water stress on groundnut pod yield and water use efficiency were investigated in a second experiment by starting irrigation at 21 DAS. The number of irrigation frequencies and irrigation volume was fixed to develop an easily readable irrigation chart for end-users. The number of irrigation frequencies was fixed as three, and the volume of irrigation was fixed as a depth of 3 cm. In the first experiment, a mini mobile pumping unit was used for scheduling irrigation frequencies; in the second experiment, the surface drip method was used. In both experiments, we also assessed the effects of foliar application of PPFM, widely used as a growth stimulant for several crops, especially under drought conditions. Overall, we observed that DIP led to up to 25.6% water saving compared to surface irrigation. Use of the DIP at the medium frequency with PPFM spray had a significantly positive influence on water use efficiency (WUE) and irrigation water use efficiency (IWUE) compared to conventional drip irrigation (CDI). Compared to surface irrigation (SI), DIP had a positive impact on growth parameters, yield attributes, and economic profitability with a higher benefit-cost ratio. Additionally, there were only minor differences between deficit irrigation (DI) results starting from 21 DAS and 14 DAS. Irrespective of the irrigation techniques, PPFM application had a positive effect on the yield attributes and WUE. Our results indicated that DI at a medium frequency (7/5) with PPFM spray, starting from 21 DAS under drip fertigation, is the best irrigation management innovation (IMI) strategy for optimum utilization of irrigation water without significant reduction in pod yield. This management had a positive influence on WUE and IWUE of summer groundnut in sandy clay loam soil. However, the exact scheduling of the irrigation may differ in different environments as well as soil types. Nevertheless, we propose that the application and further optimization of the DIP devised in this study would help the farmers attain a similar yield using a lower amount of irrigation water and lead to significant economic profits for the farmers.

Potential of pink pigmented methylotrophic bacteria on growth and physiology of cluster bean and soil microbial community

Methylotrophs are one of the prominent microbial community which are known for utilizing methanol as well as carbon sources from various plant metabolic activities and in turn facilitates the host plant growth by synthesizing phytohormones. A pot study was conducted to examine the bio-fertilizing potential of pink pigmented methylotrophic bacteria (PPFM) on cluster bean ( Cyamopsis tetragonolaba (L.) Taub and its impact on soil microbial communities . Foliar and soil application modes of treatment was employed for the study. Our results showed that isolated strain from Quisqualis indica leaf was identified as Methylobacterium tardum (strain IHBB) by 16s rRNA gene sequence and phylogenetic analysis confirmed its relationship with other Methylobacterium spp. Soil microbes such as bacterial population (9.25 CFU x 10- 6 /g), fungal population (70.05 CFU x 10 −3 /g) and microalgae (9.65 CFU x 10 −4 /g) were found to be enhanced in 3% of M. tardum (T2) when compared to T1, T3 and control. Soil application of M. tardum exhibited better response on growth, biochemical and yield parameters of cluster bean than foliar application . This study confirms that M. tardum could be a promising candidate as microbial inoculant for plant growth and development as well as a positive driver for enriching the soil microbial community. •Isolated strain from Quisqualis indica leaf was identified as Methylobacterium tardum (strain IHBB) by 16s rRNA gene sequence and phylogenetic analysis confirmed its relationship with other Methylobacterium spp. • Soil application of M. tardum exhibited better response on growth, biochemical and yield parameters of cluster bean than foliar application. • Soil microbes such as bacterial population, fungal population and microalgae were found to be enhanced in 3% of M. tardum (T2). • M. tardum could be a suitable candidate as microbial inoculant for plant growth and development has the potential of enriching the soil microbiota.

Characterization of Americium and Curium Complexes with the Protein Lanmodulin: A Potential Macromolecular Mechanism for Actinide Mobility in the Environment.

Anthropogenic radionuclides, including long-lived heavy actinides such as americium and curium, represent the primary long-term challenge for management of nuclear waste. The potential release of these wastes into the environment necessitates understanding their interactions with biogeochemical compounds present in nature. Here, we characterize the interactions between the heavy actinides, Am3+ and Cm3+, and the natural lanthanide-binding protein, lanmodulin (LanM). LanM is produced abundantly by methylotrophic bacteria, including Methylorubrum extorquens, that are widespread in the environment. We determine the first stability constant for an Am3+-protein complex (Am3LanM) and confirm the results with Cm3LanM, indicating a ∼5-fold higher affinity than that for lanthanides with most similar ionic radius, Nd3+ and Sm3+, and making LanM the strongest known heavy actinide-binding protein. The protein's high selectivity over 243Am's daughter nuclide 239Np enables lab-scale actinide-actinide separations as well as provides insight into potential protein-driven mobilization for these actinides in the environment. The luminescence properties of the Cm3+-LanM complex, and NMR studies of Gd3+-LanM, reveal that lanmodulin-bound f-elements possess two coordinated solvent molecules across a range of metal ionic radii. Finally, we show under a wide range of environmentally relevant conditions that lanmodulin effectively outcompetes desferrioxamine B, a hydroxamate siderophore previously proposed to be important in trivalent actinide mobility. These results suggest that natural lanthanide-binding proteins such as lanmodulin may play important roles in speciation and mobility of actinides in the environment; it also suggests that protein-based biotechnologies may provide a new frontier in actinide remediation, detection, and separations.

Characterization of two 3-deoxy-d-Arabino-Heptulosonate 7-phosphate synthases from Bacillusmethanolicus

3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with d-erythrose 4-phosphate (E4P) and plays an important role in regulating carbon flux toward aromatic amino acid biosynthesis in bacteria and plants. Sequence analysis of the DAHP synthases AroG1 and AroG2 from Bacillus methanolicus MGA3 suggested this thermophilic, methylotrophic bacterium possesses two type Iβ DAHP synthases. This study describes production of AroG1 and AroG2 in Escherichia coli as hexa-histidine fused proteins, which were purified by affinity chromatography. Treatment with TEV protease afforded native proteins for characterization and kinetic analysis. AroG1 and AroG2 are, respectively, 30.1 kDa and 40.0 kDa proteins. Both enzymes have maximal activity over a pH range of 6.3–7.2. The apparent kinetic parameters at 50 °C and pH 7.2 for AroG1 are KmPEP 1100 ± 100 μM, KmE4P 530 ± 100 μM, and kcat 10.3 ± 1.2 s−1. The kinetic parameters for AroG2 are KmPEP 90 ± 20 μM, KmE4P 130 ± 40 μM, and kcat 2.0 ± 0.2 s−1. At 50 °C AroG2 retains 50% of its activity after 96 min whereas AroG1 retains less than 5% of its activity after 10 min. AroG2, which contains an N-terminal regulatory domain, is inhibited by chorismate and prephenate but not l-phenylalanine, l-tyrosine, or l-tryptophan. AroG1 is not inhibited by any of the molecules examined. Understanding DAHP synthase regulation in B. methanolicus is a first step toward generating biocatalysts that exploit the target-rich aromatic amino acid biosynthetic pathway for synthesis of chemicals from methanol.

Methyloradius palustris gen. nov., sp. nov., a methanol-oxidizing bacterium isolated from snow.

A novel methylotrophic bacterium, strain Zm11T, was isolated from reddish brown snow collected in a moor in Japan. Cells of the isolate were Gram-stain-negative, motile, and rod-shaped (0.6-0.7 × 1.2-2.7μm). Growth was observed at 5-32°C with an optimum growth temperature of 25-28°C. The pH range for growth was 5.4-7.8 with an optimum pH of 6.8. The strain utilized only methanol as carbon and energy sources for aerobic growth. The major cellular fatty acids (> 40% of total) were summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C16: 0. The predominant quinone was Q-8, and major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The complete genome of strain Zm11T is composed of a circular chromosome (2,800,413bp), with G + C content of 46.4mol%. Phylogenetic analyses were conducted based on the 16S rRNA gene sequence and conserved proteins encoded in the genome. The results of analyses indicate that strain Zm11T is a member of the family Methylophilaceae but does not belong to any existing genus. On the basis of its genomic and phenotypic properties, strain Zm11T (= DSM111909T = NBRC114766T) is proposed as the type strain of a new species in a new genus, Methyloradius palustris gen. nov., sp. nov.

Xanthobacter oligotrophicus sp.nov., isolated from paper mill sewage.

A novel, aerobic nitrogen-fixing methylotrophic bacterium, strain 29kT, was enriched and isolated from sludge generated during wastewater treatment at a paper mill in Baikal, Russian Federation. Cells were Gram-stain-variable. The cell wall was of the negative Gram-type. Cells were curved oval rod-shaped, 0.5-0.7×1.7-3.4 µm and formed yellow-coloured colonies. Cells tended to be pleomorphic if grown on media containing succinate or coccoid if grown in the presence of methyl alcohol as the sole carbon source. Cells were non-motile, non-spore-forming and contained retractile (polyphosphate) and lipid (poly-β-hydroxybutyrate) bodies. The major respiratory quinone was ubiquinone Q-10 and the predominant cellular fatty acids were C18:1 ω7, C19:0 cyclo and C16:0. The genomic DNA G+C content was 67.95 mol%. Strain 29kT was able to grow at 4-37 °C (optimum, 30 °C), at pH 6.0-8.5 (optimum, pH 6.5-7.0) and at salinities of 0-0.5% (w/v) NaCl (optimum, 0% NaCl). Catalase and oxidase were positive. Strain 29kT could grow chemolithoautotrophically in mineral media under an atmosphere of H2, O2 and CO2 as well as chemoorganoheterotrophically on methanol, ethanol, n-propanol, n-butanol and various organic acids. The carbohydrate utilization spectrum is limited by glucose and raffinose. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the newly isolated strain was a member of the genus Xanthobacter with Xanthobacter autotrophicus 7cT (99.9% similarity) and Xanthobacter viscosus 7dT (99.4 % similarity) as closest relatives among species with validly published names. The average nucleotide identity and digital DNA-DNA hybridization values of 92.7 and 44.9%, respectively, of the 29kT to the genome of the most closely related species, X. autotrophicus 7cT, were below the species cutoffs. Based on genotypic, phenotypic and chemotaxonomic characteristics, it is proposed that the isolate represents a novel species, Xanthobacter oligotrophicus sp. nov. The type strain is 29kT (=KCTC 72777T=VKM B-3453T).

Evaluation of Heterologous Biosynthetic Pathways for Methanol-Based 5-Aminovalerate Production by Thermophilic Bacillus methanolicus.

The use of methanol as carbon source for biotechnological processes has recently attracted great interest due to its relatively low price, high abundance, high purity, and the fact that it is a non-food raw material. In this study, methanol-based production of 5-aminovalerate (5AVA) was established using recombinant Bacillus methanolicus strains. 5AVA is a building block of polyamides and a candidate to become the C5 platform chemical for the production of, among others, δ-valerolactam, 5-hydroxy-valerate, glutarate, and 1,5-pentanediol. In this study, we test five different 5AVA biosynthesis pathways, whereof two directly convert L-lysine to 5AVA and three use cadaverine as an intermediate. The conversion of L-lysine to 5AVA employs lysine 2-monooxygenase (DavB) and 5-aminovaleramidase (DavA), encoded by the well-known Pseudomonas putida cluster davBA, among others, or lysine α-oxidase (RaiP) in the presence of hydrogen peroxide. Cadaverine is converted either to γ-glutamine-cadaverine by glutamine synthetase (SpuI) or to 5-aminopentanal through activity of putrescine oxidase (Puo) or putrescine transaminase (PatA). Our efforts resulted in proof-of-concept 5AVA production from methanol at 50°C, enabled by two pathways out of the five tested with the highest titer of 0.02 g l–1. To our knowledge, this is the first report of 5AVA production from methanol in methylotrophic bacteria, and the recombinant strains and knowledge generated should represent a valuable basis for further improved 5AVA production from methanol.