Methylobacterium amylolyticum sp. nov. and Methylobacterium ligniniphilum sp. nov., isolated from soil.

Two pink-pigmented bacteria, designated strains NEAU-140T and NEAU-KT, were isolated from field soil collected from Linyi, Shandong Province, PR China. Both isolates were aerobic, Gram-stain-negative, rod-shaped, and facultatively methylotrophic. 16S rRNA gene sequences analysis showed that these two strains belong to the genus Methylobacterium. Strain NEAU-140T exhibited high 16S rRNA gene sequence similarities to Methylobacterium radiotolerans NBRC 15690T (97.43 %) and Methylobacterium phyllostachyos NBRC 105206T (97.36 %). Strain NEAU-KT exhibited high 16S rRNA gene sequence similarities to M. phyllostachyos NBRC 105206T (99.00 %) and Methylobacterium longum DSM 23933T (98.72 %). A phylogenetic tree based on 16S rRNA gene sequences showed that strain NEAU-140T formed a clade with Methylobacterium aerolatum (95.94 %), Methylobacterium persicinum (95.66 %) and Methylobacterium komagatae (96.87 %), and strain NEAU-KT formed a cluster with M. phyllostachyos and M. longum. The predominant fatty acid in both strains was C18 : 1 ω7c. Both strains contained ubiquinone Q-10 as the only respiratory quinone. The polar lipid profiles of both strains contained diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. Whole-genome phylogeny showed that strains NEAU-140T and NEAU-KT formed a phyletic line with M. aerolatum, M. persicinum, Methylobacterium radiotolerans, Methylobacterium fujisawaense, Methylobacterium oryzae, Methylobacterium tardum, M. longum and M. phyllostachyos. The orthologous average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain NEAU-140T and its closely related strains were lower than 82.62 and 25.90  %, respectively. The ANI and dDDH values between strain NEAU-KT and its closely related strains were lower than 86.29 and 31.7 %, respectively. The genomic DNA G+C contents were 71.63 mol% for strain NEAU-140T and 69.08 mol% for strain NEAU-KT. On the basis of their phenotypic and phylogenetic distinctiveness and the results of dDDH and ANI hybridization, these two isolates represent two novel species within the genus Methylobacterium, for which the names Methylobacterium amylolyticum sp. nov. (type strain NEAU-140T=MCCC 1K08801T=DSM 110568T) and Methylobacterium ligniniphilum sp. nov. (type strain NEAU-KT=MCCC 1K08800T=DSM 110567T) are proposed.

Methylobacterium nigriterrae sp. nov., isolated from black soil.

An aerobic, Gram-stain-negative, motile rod bacterium, designated as SYSU BS000021T, was isolated from a black soil sample in Harbin, Heilongjiang province, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the genus Methylobacterium, and showed the highest sequence similarity to Methylobacterium segetis KCTC 62267 T (98.51%) and Methylobacterium oxalidis DSM 24028 T (97.79%). Growth occurred at 20-37℃ (optimum, 28 °C), pH 6.0-8.0 (optimum, pH 7.0) and in the presence of 0% (w/v) NaCl. Polar lipids comprised of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminolipid and one unidentified polar lipid. The major cellular fatty acids (> 5%) were C18:0 and C18:1 ω7c and/or C18:1 ω6c. The predominant respiratory quinone was Q-10. The genomic G + C content was 68.36% based on the whole genome analysis. The average nucleotide identity (≤ 83.5%) and digital DNA-DNA hybridization (≤ 27.3%) values between strain SYSU BS000021T and other members of the genus Methylobacterium were all lower than the threshold values recommended for distinguishing novel prokaryotic species. Based on the results of phenotypic, chemotaxonomic and phylogenetic analyses, strain SYSU BS000021T represents a novel species of the genus Methylobacterium, for which the name Methylobacterium nigriterrae sp. nov. is proposed. The type strain of the proposed novel species is SYSU BS000021T (= GDMCC 1.3814 T = KCTC 8051 T).

Draft genome sequence of Methylobacterium sp. 37f isolated from forest soil.

In this study, we report the draft genome sequence data of Methylobacterium sp. 37f, isolated from soil beneath Quercus semiserrata Roxb. in Thailand. The genome consists of 5,305,449 base pairs, with a GC content of 67.5%.

Draft genome sequence of an ubiquinone-10 producing Methylobacterium durans LRY1-08 isolated from lichen in Thailand.

A ubiquitous and pink-pigmented facultatively methylotrophic bacterium, designated LRY1-08 (=JCM 33120), was isolated from a lichen in Thailand. Strain LRY1-08 and Methylobacterium durans NBRC 112876T shared 99.92 % similarity based on the 16S rRNA gene sequence. The draft genome of LRY1-08 was 5.26 Mbp with 4,952 protein-coding sequences and an average G + C content of 70.0 mol%. Comparing strain LRY1-08 to M. durans NBRC 112876T, the ANIb, ANIm, AAI, and digital DNA-DNA hybridization values were 96.29 %, 97.10 %, 96.7 %, and 82.29 %, respectively. Based on the phenotypic characteristics and genome analysis, it was identified as M. durans. Its genomic sequence data revealed the PHB and CoQ10 biosynthesis genes. Therefore, the results offer suggestions for further investigation into possible applications of this bacterium in biotechnology. The draft genome was deposited at DDBJ/EMBL/GenBank (DNA Databank of Japan/European Molecular Biology Laboratory/Genbank) (JAYEEX000000000).

Isolation and identification of Methylobacterium komagatae and its application in textile industries.

A light pink-coloured, rod-shaped, gram-negative bacterium isolated from an unproductive crude oil production area was considered as a sample for this study. The 16S rRNA gene sequence identified the isolate as Methylobacterium komagatae. Comparing the standard colour measurement values set by the International Commission on Illumination (CIE) method confirms the colourant produced by the biomass of this microorganism as a 'light pink' colouration. The energy-dispersive X-ray spectroscopy and High-Resolution Mass Spectroscopy process help in the structural elucidation of the sample. It indicates the presence of magnesium (Mg) as a central metal atom in the bacterial colourant, i.e. 'bacteriochlorophyll' (BChl) (MgC55H74N4O). The recovered bacterial colourant was applied to cotton fabric and cotton yarns to dye and examine their fastness quality. The result shows the cotton fabrics retained colourant in normal washing while it got reduced after detergent-based washing. Therefore, its fastness quality must be improved to equalise with current colourants.

Draft genome sequence of Methylobacterium sp. OT2 isolated from human skin and capable of growing on t-octylphenol polyethoxylate.

Here, we present the draft whole-genome sequence of Methylobacterium sp. OT2, isolated from human skin on a minimal medium containing t-octylphenol ethoxylate (Triton X-100). This genomic information contributes to understanding the niche adaptation on human skin and its catabolism of Triton X-100 in this strain.

Nitrogen removal intensification of biofilm through bioaugmentation with Methylobacterium gregans DC-1 during wastewater treatment.

The increasing concern for environmental remediation has led to a search for effective methods to remove eutrophic nutrients. In this study, Methylobacterium gregans DC-1 was utilized to improve nitrogen removal in a sequencing batch biofilm reactor (SBBR) via aerobic denitrification. This bacterium has the extraordinary characteristics of strong auto-aggregation and a high ability to remove nitrogen efficiently, making it an ideal candidate for enhanced treatment of nitrogen-rich wastewater. This strain was used for the bioassessment of a test reactor (SBBRbio), which showed a shorter biofilm formation time compared to a control reactor (SBBRcon) without this strain inoculation. Moreover, the enhanced biofilm was enriched in TB-EPS and had a wider variety of protein secondary structures than SBBRcon. During the stabilization phase of SBBRbio, the EPS molecules showed the highest proportion of intermolecular hydrogen bonding. It is possible that bioaugmentation with this strain positively affects the structural stability of biofilm. At influent ammonia loadings of 100 and 150 mg. L-1, the average reduction of ammonia and nitrate-nitrogen was higher in the experimental system compared to the control system. Additionally, nitrite-N accumulation was lower and N2O production decreased compared to the control. Analysis of the microbial community structure demonstrated successful colonization in the bioreactor by a highly nitrogen-tolerant strain that efficiently removed inorganic nitrogen. These results illustrate the great potential of this type of denitrifying bacteria in the application of bioaugmentation systems.

Engineering photo-methylotrophic Methylobacterium for enhanced 3-hydroxypropionic acid production during non-growth stage fermentation.

This study explored the potential of methanol as a sustainable feedstock for biomanufacturing, focusing on Methylobacterium extorquens, a well-established representative of methylotrophic cell factories. Despite this bacterium's long history, its untapped photosynthetic capabilities for production enhancement have remained unreported. Using genome-scale flux balance analysis, it was hypothesized that introducing photon fluxes could boost the yield of 3-hydroxypropionic acid (3-HP), an energy- and reducing equivalent-consuming chemicals. To realize this, M. extorquens was genetically modified by eliminating the negative regulator of photosynthesis, leading to improved ATP levels and metabolic activity in non-growth cells during a two-stage fermentation process. This modification resulted in a remarkable 3.0-fold increase in 3-HP titer and a 2.1-fold increase in its yield during stage (II). Transcriptomics revealed that enhanced light-driven methanol oxidation, NADH transhydrogenation, ATP generation, and fatty acid degradation were key factors. This development of photo-methylotrophy as a platform technology introduced novel opportunities for future production enhancements.

Application and effectiveness of Methylobacterium symbioticum as a biological inoculant in maize and strawberry crops.

The effectiveness of Methylobacterium symbioticum in maize and strawberry plants was measured under different doses of nitrogen fertilisation. The biostimulant effect of the bacteria was observed in maize and strawberry plants treated with the biological inoculant under different doses of nitrogen fertiliser compared to untreated plants (control). It was found that bacteria allowed a 50 and 25% decrease in the amount of nitrogen applied in maize and strawberry crops, respectively, and the photosynthetic capacity increased compared with the control plant under all nutritional conditions. A decrease in nitrate reductase activity in inoculated maize plants indicated that the bacteria affects the metabolism of the plant. In addition, inoculated strawberry plants grown with a 25% reduction in nitrogen had a higher concentration of nitrogen in leaves than control plants under optimal nutritional conditions. Again, this indicates that Methylobacterium symbioticum provide an additional supply of nitrogen.

Biodegradation of Petroleum Sludge by Methylobacterium sp. Strain ZASH.

A bacterium was isolated from sludge-contaminated soil in a petroleum refinery and tested for its ability to degrade aliphatic hydrocarbon compounds present in petroleum sludge. The isolate was grown on minimal salt media agar supplemented with 1% (w/v) petroleum sludge. The isolate was tentatively identified as Methylobacterium s p. s t rain ZASH based on the partial 16s rDNA molecular phylogeny. The bacterium grew optimally between the temperatures of 30°C and 35°C, pH 7 and 7.5, 0.5% and 1.5% (v/v) Tween 80 as the surfactant, and between 1% and 2% (w/v) peptone as the nitrogen source. The constants derived from the Haldane equation were µmax = 0.039 hr-1, Ks = 0.385% (w/v) total petroleum hydrocarbons (TPH) or 3,850 mg/L TPH, and Ki =1.12% (w/v) TPH or 11,200 mg/L. The maximum biodegradation rate exhibited by this strain was 19 mg/L/hr at an initial TPH concentration of 10,000 mg/L. Gas chromatography analysis revealed that after 15 days the strain was able to degrade all aliphatic n-alkanes investigated with different efficiencies. Shorter n-alkanes were generally degraded more rapidly than longer n-alkanes with 90% removal for C-12 compared to only 30% removal for C-36. The addition of sawdust did not improve bacterial degradation of petroleum hydrocarbons, but it assisted in the removal of remaining undegraded hydrocarbons through adsorption.

Diversity of Methylobacterium species associated with New Zealand native plants.

Methylobacterium species are abundant colonizers of the phyllosphere due to the availability of methanol, a waste product of pectin metabolism during plant cell division. The phyllosphere is an extreme environment, with a landscape that is heterogeneous and continuously changing as the plant grows and is exposed to high levels of ultraviolet irradiation. Geographically, New Zealand (NZ) has been isolated for over a million years, has a biologically diverse flora, and is considered a biodiversity hotspot, with most native plants being endemic. We therefore hypothesize that the phyllosphere of NZ native plants harbor diverse groups of Methylobacterium species. Leaf imprinting using methanol-supplemented agar medium was used to isolate bacteria, and diversity was determined using ARDRA and 16S rRNA gene sequencing. Methylobacterium species were successfully isolated from the phyllosphere of 18 of the 20 native NZ plant species in this study, and six different species were identified: M. marchantiae, M. mesophilicum, M. adhaesivum, M. komagatae, M. extorquens, and M. phyllosphaerae. Other α, ß, and γ-Proteobacteria, Actinomycetes, Bacteroidetes, and Firmicutes were also isolated, highlighting the presence of other potentially novel methanol utilizers within this ecosystem. This study identified that Methylobacterium are abundant members of the NZ phyllosphere, with species diversity and composition dependent on plant species.

Growth Suppression of a Robust Bacterium Methylobacterium extorquens by Porous Materials with Oxygen Functional Groups.

Suppressing the growth of Methylobacterium species without the use of toxic chemicals has been a challenging task owing to their robustness against previous antimicrobial techniques. In this work, we prepared porous materials with various numbers and types of oxygen functional groups and investigated their ability to suppress the growth of Methylobacterium extorquens. It turned out that the number and type of oxygen functional groups in the porous materials greatly affected the growth of the bacterium. Three porous materials (resorcinol-formaldehyde gel (RF), hydrothermally treated RF (RFH), and Wakkanai siliceous shale (WS)) were tested, and RF exhibited the best performance in suppressing the growth of the bacterium. This performance is possibly due to abundant phenolic groups in the porous material.

Five-year trends in bacterial contamination of dialysis equipment.

Bedside dialysis monitoring equipment for hemodialysis are located in the bioburden section upstream of the endotoxin-retentive filter for dialysis fluid sterilization. We observed 26 equipment at our institution for bacterial contamination at least once every 4 weeks for 5 years with another ultrafiltration membrane upstream to prevent bacterial contamination. Bacterial contamination levels were highest and most diverse at the time of the first flush. During subsequent initial cleanng, the contamination level decreased, and bacterial species converged almost exclusively to one genus, namely Methylobacterium spp. During clinical use, the equipment were cleaned and disinfected daily after dialysis, and daily operations and maintenance were performed using aseptic techniques. Although the frequency of bacterial detection decreased annually, the same bacterial genotypes observed at the first flush were isolated even after long time periods and were thought to persist in the equipment possibly by forming biofilm. Pseudomonas aeruginosa was newly detected after the replacement of parts during breakdown maintenance, indicating the need to sterilize replacement parts. Thus, the bioburden should be assessed regularly as part of the management of in-house-produced dialysis fluid.

Metabolism-linked methylotaxis sensors responsible for plant colonization in Methylobacterium aquaticum strain 22A.

Motile bacteria take a competitive advantage in colonization of plant surfaces to establish beneficial associations that eventually support plant health. Plant exudates serve not only as primary growth substrates for bacteria but also as bacterial chemotaxis attractants. A number of plant-derived compounds and corresponding chemotaxis sensors have been documented, however, the sensors for methanol, one of the major volatile compounds released by plants, have not been identified. Methylobacterium species are ubiquitous plant surface-symbiotic, methylotrophic bacteria. A plant-growth promoting bacterium, M. aquaticum strain 22A exhibits chemotaxis toward methanol (methylotaxis). Its genome encodes 52 methyl-accepting chemotaxis proteins (MCPs), among which we identified three MCPs (methylotaxis proteins, MtpA, MtpB, and MtpC) responsible for methylotaxis. The triple gene mutant of the MCPs exhibited no methylotaxis, slower gathering to plant tissues, and less efficient colonization on plants than the wild type, suggesting that the methylotaxis mediates initiation of plant-Methylobacterium symbiosis and engages in proliferation on plants. To examine how these MCPs are operating methylotaxis, we generated multiple gene knockouts of the MCPs, and Ca2+-dependent MxaFI and lanthanide (Ln3+)-dependent XoxF methanol dehydrogenases (MDHs), whose expression is regulated by the presence of Ln3+. MtpA was found to be a cytosolic sensor that conducts formaldehyde taxis (formtaxis), as well as methylotaxis when MDHs generate formaldehyde. MtpB contained a dCache domain and exhibited differential cellular localization in response to La3+. MtpB expression was induced by La3+, and its activity required XoxF1. MtpC exhibited typical cell pole localization, required MxaFI activity, and was regulated under MxbDM that is also required for MxaF expression. Strain 22A methylotaxis is realized by three independent MCPs, two of which monitor methanol oxidation by Ln3+-regulated MDHs, and one of which monitors the common methanol oxidation product, formaldehyde. We propose that methanol metabolism-linked chemotaxis is the key factor for the efficient colonization of Methylobacterium on plants.

Methylobacterium spp. mitigation of UV stress in mung bean (Vigna radiata L.).

Methylotrophs are a diverse group of bacteria that abundantly colonize the phyllosphere and have great potential to withstand UV irradiation because of their pigmented nature and ability to promote plant growth through various mechanisms. The present study investigated the effects of UVB radiation on plant growth-promoting (PGP) properties of methylotrophic bacteria and the growth of Vigna radiata L. A total of 55 methylotrophic bacteria were isolated from desert plants, and 15 methylotrophs were resistant to UVB radiation for 4 h. All UVB-resistant methylotrophs possess a methyldehydrogenase gene. Identification based on 16S rRNA gene sequencing revealed that all 15 UVB-resistant methylotrophs belonged to the genera Methylorubrum (07), Methylobacterium (07), and Rhodococcus (01). Screening of methylotrophs for PGP activity in the presence and absence of UVB radiation revealed that all isolates showed ACC deaminase activity and growth on a nitrogen-free medium. Furthermore, the production of IAA-like substances ranged from 8.62 to 85.76 µg/mL, siderophore production increased from 3.47 to 65.75% compared to the control. Seed germination assay with V. radiata L. (mung bean) exposed to UVB radiation revealed that methylotrophs improved seed germination, root length, and shoot length compared to the control. The present findings revealed that the isolates SD3, SD2, KD1, KD5, UK1, and UK3 reduced the deleterious effects of UVB radiation on mung bean plants and can be used to protect seedlings from UVB radiation for sustainable agriculture.

Draft genome of methanol-oxidizing Methylobacterium fujisawaense strain LAC1.

We report the draft genome of Methylobacterium fujisawaense LAC1 isolated from an acidic aquifer in Indian Head, MD, USA. The genome contains 5,883,000 bp and has a GC content of 70% with 5,434 protein-encoding genes with functional assignments. This strain can grow on methanol with lanthanum, a rare earth element.

Draft genome sequence of the highly copper-tolerant Methylobacterium radiotolerans MLP1 isolated from the rhizosphere of grasses adjacent to mine tailings.

We present the genome of a highly copper-tolerant pink-pigmented facultative methylotroph isolated from the rhizosphere of grasses growing close to mine tailings. Based on whole-genome taxonomic analyses, this isolate was named Methylobacterium radiotolerans MLP1. Studies are in progress to infer its genome-based copper resistome.

Characterization of C30 carotenoid and identification of its biosynthetic gene cluster in Methylobacterium extorquens AM1.

Methylobacterium species, the representative bacteria distributed in phyllosphere region of plants, often synthesize carotenoids to resist harmful UV radiations. Methylobacterium extorquens is known to produce a carotenoid pigment and recent research revealed that this carotenoid has a C30 backbone. However, its exact structure remains unknown. In the present study, the carotenoid produced by M. extorquens AM1 was isolated and its structure was determined as 4-[2-O-11Z-octadecenoyl-ß-glucopyranosyl]-4,4'-diapolycopenedioc acid (1), a glycosylated C30 carotenoid. Furthermore, the genes related to the C30 carotenoid synthesis were investigated. Squalene, the precursor of the C30 carotenoid, is synthesized by the co-occurrence of META1p1815, META1p1816 and META1p1817. Further overexpression of the genes related to squalene synthesis improved the titer of carotenoid 1. By using gene deletion and gene complementation experiments, the glycosyltransferase META1p3663 and acyltransferase META1p3664 were firstly confirmed to catalyze the tailoring steps from 4,4'-diapolycopene-4,4'-dioic acid to carotenoid 1. In conclusion, the structure and biosynthetic genes of carotenoid 1 produced by M. extorquens AM1 were firstly characterized in this work, which shed lights on engineering M. extorquens AM1 for producing carotenoid 1 in high yield.

Methanol-based biomanufacturing of fuels and chemicals using native and synthetic methylotrophs.

Methanol has recently gained significant attention as a potential carbon substrate for the production of fuels and chemicals, owing to its high degree of reduction, abundance, and low price. Native methylotrophic yeasts and bacteria have been investigated for the production of fuels and chemicals. Alternatively, synthetic methylotrophic strains are also being developed by reconstructing methanol utilization pathways in model microorganisms, such as Escherichia coli. Owing to the complex metabolic pathways, limited availability of genetic tools, and methanol/formaldehyde toxicity, the high-level production of target products for industrial applications are still under development to satisfy commercial feasibility. This article reviews the production of biofuels and chemicals by native and synthetic methylotrophic microorganisms. It also highlights the advantages and limitations of both types of methylotrophs and provides an overview of ways to improve their efficiency for the production of fuels and chemicals from methanol.

Microbe-Responsive Proteomes During Plant-Microbe Interactions Between Rice Genotypes and the Multifunctional Methylobacterium oryzae CBMB20.

BACKGROUND: Rice is colonized by plant growth promoting bacteria such as Methylobacterium leading to mutually beneficial plant-microbe interactions. As modulators of the rice developmental process, Methylobacterium influences seed germination, growth, health, and development. However, little is known about the complex molecular responsive mechanisms modulating microbe-driven rice development. The application of proteomics to rice-microbe interactions helps us elucidate dynamic proteomic responses mediating this association. RESULTS: In this study, a total of 3908 proteins were detected across all treatments of which the non-inoculated IR29 and FL478 share up to 88% similar proteins. However, intrinsic differences appear in IR29 and FL478 as evident in the differentially abundant proteins (DAPs) and their associated gene ontology terms (GO). Successful colonization of M. oryzae CBMB20 in rice resulted to dynamic shifts in proteomes of both IR29 and FL478. The GO terms of DAPs for biological process in IR29 shifts in abundance from response to stimulus, cellular amino acid metabolic process, regulation of biological process and translation to cofactor metabolic process (6.31%), translation (5.41%) and photosynthesis (5.41%). FL478 showed a different shift from translation-related to response to stimulus (9%) and organic acid metabolic acid (8%). Both rice genotypes also showed a diversification of GO terms due to the inoculation of M. oryzae CBMB20. Specific proteins such as peptidyl-prolyl cis-trans isomerase (A2WJU9), thiamine thiazole synthase (A2YM28), and alanine-tRNA ligase (B8B4H5) upregulated in IR29 and FL478 indicate key mechanisms of M. oryzae CBMB20 mediated plant growth promotion in rice. CONCLUSIONS: Interaction of Methylobacterium oryzae CBMB20 to rice results in a dynamic, similar, and plant genotype-specific proteomic changes supporting associated growth and development. The multifaceted CBMB20 expands the gene ontology terms and increases the abundance of proteins associated with photosynthesis, diverse metabolic processes, protein synthesis and cell differentiation and fate potentially attributed to the growth and development of the host plant. The specific proteins and their functional relevance help us understand how CBMB20 mediate growth and development in their host under normal conditions and potentially link subsequent responses when the host plants are exposed to biotic and abiotic stresses.