mxaF Gene Research Articles

Methylocystis iwaonis sp. nov., a type II methane-oxidizing bacterium from surface soil of a rice paddy field in Japan, and emended description of the genus Methylocystis (ex Whittenbury et al. 1970) Bowman et al. 1993.

A novel methane-oxidizing bacterial strain SS37A-ReT was isolated from surface soil of a rice paddy field in Japan. Cells were Gram-stain-negative, motile rods with single polar flagellum and type II intracytoplasmic membrane arrangement. The strain grew on methane or methanol as the sole carbon and energy source. It grew at 15–37 °C (optimum 25–30 °C), pH 6.0–9.0 (optimum 7.0–8.0) and with 0–0.1 % (w/w) NaCl (no growth at 0.5 % or above). Cells formed cysts, but not exospores. The results of sequence analysis of the 16S rRNA gene indicated that SS37A-ReT represented a member of the family Methylocystaceae , with the highest similarity (98.9 %) to Methylocystis parva corrig. OBBPT. Phylogenetic analysis of pmoA and mxaF genes and core genes in the genome indicated that the strain was closely related to the members of the genus Methylocystis , while the analysis of the mmoX gene indicated the close relationships with the genus Methylosinus . The values of genome relatedness between SS37A-ReT and species of the genera Methylocystis and Methylosinus were 78.6–82.5% and 21.7–24.9 % estimated by the average nucleotide identity and digital DNA–DNA hybridisation, respectively, showing the highest values with Methylocystis echinoides LMG 27198T. The DNA G+C content was 63.2 mol% (genome). The major quinone and fatty acids were Q-8 and, C18 : 1 (C18 : 1ω8t and C18 : 1ω8c) and C18 : 2, respectively. On the basis of the phenotypic and phylogenetic features, the strain represents a novel species of the genus Methylocystis , for which the name Methylocystis iwaonis sp. nov. is proposed. The type strain is SS37A-ReT (=JCM 34278T =NBRC 114996T=KCTC 82710T).

Diversity of cultivated methylotrophs from the extremely oligotrophic system in the Cuatro Cienegas Basin, Mexico: An unexplored ecological guild

The simplest form of heterotrophy in the carbon cycle is to metabolize C1 compounds, this is a widely spread strategy that includes genus in different phyla inhabiting diverse environments that seem to have acquired the methanol dehydrogenase by horizontal gene transfer (HGT). The objective of this study was to isolate and explore the diversity of the ecological guild of methylotrophs in the water and riparian vegetation of the Churince system in the Cuatro Cienegas Basin (CCB), Coahuila, Mexico. Methylotrophy was verified by polymerase chain reaction (PCR) amplification of the mxaF gene that encodes the α-subunit of the enzyme methanol dehydrogenase (MDH), while phylogenetic affiliations were assigned following 16S rRNA phylogenetic analyses. Among the isolated strains we observed a phylogenetic association with a common species of Methylobacterium (M. radiotolerans). In addition, other methylotrophs were isolated, like Methylorubrum aminovorans, Methylorubrum extorquems and Methylophilus methylotrophus. Interestingly, we also isolated other strains able to grow in methanol and mxaF+, their 16S rRNA identified them as Jiella, Pseudomonas, Rhizobium, Serratia and Stenotrophomonas. This study addresses, for the first time, the diversity of cultivated methylotrophic bacteria within CCB and inserts this knowledge in the context of a total inventory of the microbiota in the site.

A Novel Moderately Thermophilic Facultative Methylotroph within the Class Alphaproteobacteria.

Methylotrophic bacteria (non-methanotrophic methanol oxidizers) consuming reduced carbon compounds containing no carbon–carbon bonds as their sole carbon and energy source have been found in a great variety of environments. Here, we report a unique moderately thermophilic methanol-oxidising bacterium (strain LS7-MT) that grows optimally at 55 °C (with a growth range spanning 30 to 60 °C). The pure isolate was recovered from a methane-utilizing mixed culture enrichment from an alkaline thermal spring in the Ethiopia Rift Valley, and utilized methanol, methylamine, glucose and a variety of multi-carbon compounds. Phylogenetic analysis of the 16S rRNA gene sequences demonstrated that strain LS7-MT represented a new facultatively methylotrophic bacterium within the order Hyphomicrobiales of the class Alphaproteobacteria. This new strain showed 94 to 96% 16S rRNA gene identity to the two methylotroph genera, Methyloceanibacter and Methyloligella. Analysis of the draft genome of strain LS7-MT revealed genes for methanol dehydrogenase, essential for methanol oxidation. Functional and comparative genomics of this new isolate revealed genomic and physiological divergence from extant methylotrophs. Strain LS7-MT contained a complete mxaF gene cluster and xoxF1 encoding the lanthanide-dependent methanol dehydrogenase (XoxF). This is the first report of methanol oxidation at 55 °C by a moderately thermophilic bacterium within the class Alphaproteobacteria. These findings expand our knowledge of methylotrophy by the phylum Proteobacteria in thermal ecosystems and their contribution to global carbon and nitrogen cycles.

Methylotrophic bacteria from underarm of human: Isolation, molecular detection, antibiofilm activity, and toxicity property

Background: Methylotrophic bacteria are a group of bacteria that capable in utilize one-carbon and reduced carbon compounds as a source of carbon and energy for their growth. This ability was supported by the presence of mxaF gene that encodes methanol dehydrogenase (MDH) enzyme. Previous studies have reported the presence of these bacteria in the mouth and feet of human. However, research about the presence of methylotrophic bacteria in human underarm is still limited. Biofilm is known as one of the self-defense mechanisms against the antibacterial agents, possessed by several bacteria, resulting in resistance and many chronic infections. This research is aimed to isolate methylotrophic bacteria from underarm of human, detect the genes encode MDH, screen the antibiofilm activity of these bacteria, and continue to determine their toxicity property. Methods and materials: Methylotrophic bacteria were isolated from 80 participants. Subsequently, molecular detection of mxaF gene as marker of these bacteria was conducted by performing polymerase chain reaction (PCR) using specific primer, namely f1003 and r1561. Then, antibiofilm activity assay was performed in two different mechanisms (inhibition and destruction activity) at two different concetration treatment (5 and 10% of crude extract). Finally, toxicity assay was performed by using brine shrimp lethality assay (BSLA) to determine LC50 values of these isolates. Results: In total, 120 isolates were isolated from 80 participants, and mxaF genes of 54 isolates were successfully amplified. Most of the methylotrophic bacteria exhibited specific antibiofilm activity against specific pathogen at specific concentration. The highest antibiofilm activity was demonstrated by isolate B2.4, which was identified as Serratia marcescens through 16S rRNA gene sequencing method. This isolate showed the highest inhibition activity against ETEC and also the destruction activity against Staphylococcus aureus ATCC 25923 with the result up to 65.09% and 58.56%, respectively. Then, five selected isolates showed LC50 values greater than 1000 μg/mL, indicating that these isolates had no cytotoxic effect. Conclusion: Further study needs to be conducted to explore other molecular detection of methylotrophic bacteria, other than using mxaF gene as a molecular marker. Moreover, it is also important to characterize, purify, and do more advanced toxicity assay on the potential bioactive compound.

Methylobacterium nonmethylotrophicum sp. nov., isolated from tungsten mine tailing.

A novel pink-pigmented strain, designated 6HR-1T, was isolated from tungsten mine tailings in Jiangxi Province, PR China. Cells were Gram-stain-negative, aerobic, non-spore-forming, rod-shaped and motile with a polar flagellum (monotrichous). It could not utilize methanol, methylamine, formaldehyde or formate as a sole carbon source. The methanol dehydrogenase mxaF gene was absent but the xoxF gene was present. Phylogenomic and 16S rRNA gene phylogenetic analyses clearly showed that strain 6HR-1T was affiliated to the genus Methylobacterium and closely related to 'Methylobacterium terrae' 17Sr1-28T (98.6 %), Methylobacterium platani JCM 14648T (97.7 %), Methylobacterium variabile DSM 16961T (97.7 %) and Methylobacterium currus KACC 19662T (97.4 %). The average nucleotide identity and digital DNA-DNA hybridization values between strain 6HR-1T and its closely related type species were 87.4-88.7 and 33.2-36.3 %, respectively. It had summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) as the major fatty acid and ubiquinone 10 as the predominant respiratory quinone. Polyphasic characterization supported that strain 6HR-1T represents a novel species of the genus Methylobacterium, for which the name Methylobacterium nonmethylotrophicum sp. nov. is proposed with the type strain 6HR-1T (=GDMCC 1.662T=KCTC 42760T).

Phyllosphere-associated Methylobacterium: a potential biostimulant for ginger (Zingiber officinale Rosc.) cultivation.

Methanol, a by-product associated with plant metabolism, is a substrate for pink pigmented facultative methylotrophs (PPFMs) of phyllosphere. The symbiotic interaction of PPFMs has many desirable effects on plant growth and disease resistance. The present study investigated the potential of native PPFMs for mitigating biotic stress and plant growth promotion in ginger. PPFMs were isolated from ginger phyllosphere by leaf imprint technique and screened against major fungal phytopathogens of ginger viz. Macrophomina phaseolina, Sclerotium rolfsii, Pythium myriotylum, Colletotrichum gloeosporioides and Fusarium oxysporum. Among the 60 PPFMs, IISRGPPFM13 was selected for its highly inhibitory activity against the target pathogens. The isolate was useful for mineral solubility, production of IAA, siderophores and hydrolytic enzymes like cellulase, pectinase, lipase, amylase and chitinase. On in planta experiments revealed that IISRGPPFM13 considerably increased plant growth parameters when the bacterium was applied as soil drenching cum foliar spraying. Methanol utilization potential of the isolate was confirmed by mxaF gene analysis where the sequence showing 95.51% identity towards Methylobacterium platani and M. iners. Further, 16S rRNA gene sequence showing 98.73% identity with M. komagatae 002-079T (AB252201). This is the first report of its kind that a genus of Methylobacterium with biostimulant potential isolated from the phyllosphere of ginger.

Bacterial Communities in Areas of Oil and Methane Seeps in Pelagic of Lake Baikal.

We have assessed the diversity of bacteria near oil-methane (area I) and methane (area II) seeps in the pelagic zone of Lake Baikal using massive parallel sequencing of 16S rRNA, pmoA, and mxaF gene fragments amplified from total DNA. At depths from the surface to 100m, sequences belonging to Cyanobacteria dominated. In the communities to a depth of 200m of the studied areas, Proteobacteria dominated the deeper layers of the water column. Alphaproteobacteria sequences were predominant in the community near the oil-methane seep, while the community near the methane seep was characterized by the prevalence of Alpha- and Gammaproteobacteria. Among representatives of these classes, type I methanotrophs prevailed in the 16S rRNA gene libraries from the near-bottom area, and type II methanotrophs were detected in minor quantities at different depths. In the analysis of the libraries of the pmoA and mxaF functional genes, we observed the different taxonomic composition of methanotrophic bacteria in the surface and deep layers of the water column. All pmoA sequences from area I were type II methanotrophs and were detected at a depth of 300m, while sequences of type I methanotrophs were the most abundant in deep layers of the water column of area II. All mxaF gene sequences belonged to Methylobacterium representatives. Based on comparative analyses of 16S rRNA, pmoA, and mxaF gene fragment libraries, we suggest that there must be a wider spectrum of functional genes facilitating methane oxidation that were not detected with the primers used.

Indole Acetic Acid Production and Growth-Promoting Activity of Methylobacterium extorquens MP1 and Methylobacterium zatmanii MS4 in Tomato

ABSTRACTPink-pigmented facultative methylotrophs (PPFMs) are prevalent aerobic bacteria colonizing the phyllosphere of various plant species. PPFMs have the ability to utilize plant-derived methanol as an energy substrate when plants are being colonized under stress. The study was undertaken to explore the metabolic potential of methylotrophs isolated from diverse sources and their role in growth and development of Tomato (Solanum lycopersicum L.) plants, because it is easily grown under laboratory conditions. PPFMs were isolated from the phyllosphere of Peach (Prunus persica L.) and Strawberry (Fragaria ananassa L.) by the leaf imprint method. The bacterial isolates MP1 and MS4 are rod shaped as confirmed by scanning electron microscopy (SEM) analysis. Methylotrophy of the isolates was authenticated by polymerase chain reaction (PCR) amplification of the highly conserved mxaF gene. The 16S rRNA gene sequences demonstrated that the isolates MP1 and MS4 were Methylobacterium extorquens and Methylobacterium zatmanii, respectively. High-performance thin-layer chromatographic analysis indicated production of indole acetic acid (IAA) by M. extorquens MP1 and M. zatmanii MS4. The amount of IAA produced was 10.353 and 8.473 μg·mL−1 for M. extorquens MP1 and M. zatmanii MS4, respectively. Tomato seed were treated with M. extorquens and M. zatmanii and had seed vigor indices (SVIs) of 1383.33 ± 90.26 and 1240.79 ± 244.09, respectively, compared to untreated control seed, which had an SVI of 1031.68 ± 83.96. Foliar application of M. extorquens MP1 and M. zatmanii MS4 augmented its impact on plant growth promotion in tomato compared to the control. The increased production of IAA and subsequent enhancement in growth-promoting traits indicates that methylotrophs from diverse plant species can be used to improve early plant development in tomato under controlled conditions.

Plant Growth Promoting Activity of Pink Pigmented Facultative Methylotroph - Methylobacterium extorquens MM2 on Lycopersicon esculentum L.

Pink pigmented facultative methylotrophs (PPFMs) are diversified group of microorganisms that promote plant growth by producing indole acetic acid (IAA) and cytokinins. The objective of the present study is to isolate and characterize methylotrophic bacteria from the phyllosphere of mustard plant (Brassica niagra) and to study their plant growth promotion in tomato (Lycopersicon esculentum L.). Bacteria were isolated from the phyllosphere and the conserved mxaF gene sequence was amplified. Production of IAA was determined by High Performance Thin Layer Chromatography (HPTLC) and further quantified by spectrophotometric analysis. Effect of seed bacterization with the bacterial isolate MM2 was studied for percentage seed germination, root & shoot length. Based on NCBI BLAST search of the mxaF sequence obtained, the bacterial isolate was identified as Methylobacterium extorquens MM2. The amount of IAA produced by M. extorquens MM2 was 6.16 μg/ml. M. extorquens MM2 significantly increased seed vigour index (SVI) in tomato plants. The SVI of the treated plant was 1462.56 ± 83.96 as compared to the control, 1019.94 ± 113.61. Foliar application of M. extorquens MM2 also contributed a substantial impact on growth of tomato plant. The bacterial isolate M. extorquens MM2 thus can be employed in the crops for sustainable agriculture.

A novel Delftia plant symbiont capable of autotrophic methylotrophy

A facultative methylotrophic bacterium, strain Lp-1, which was isolated from root nodules of lupine (Lupinus polyphyllus L.) on the medium with methanol as a carbon and energy source, exhibited high similarity of the 16S rRNA gene sequences to Delftia strains (94‒99.9%). The cells of Delftia sp. Lp-1 were motile gram-negative rods dividing by binary fission. Predominant fatty acids were C16:0 (34.2%), C16:1ω9 (14.5%), and C18:1ω7c (17.3%). Phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol were the dominant phospholipids. Q8 was the major ubiquinone. Optimal growth occurred at 24‒26°C and pH 7.1‒7.3; growth was inhibited by 1% NaCl. The organism oxidized methanol with the classical methanol dehydrogenase and used the ribulose bisphosphate pathway of C1 metabolism. Analysis of translated amino acid sequence of the large subunit of the MxaF methanol dehydrogenase revealed 85.5‒94% similarity to the sequences of such autotrophic methylotrophs of the class Alphaproteobacteria as Angulomicrobium, Starkeya, and Ancylobacter, indicating the possible acquisition of the mxaF gene via horizontal gene transfer. Delftia sp. Lp-1 (VKM B-3039, DSM 24446), the first methylotrophic member of the genus Delftia, was shown to be a plant symbiont, stimulating plant growth and morphogenesis, increasing the level of photosynthetic pigments and specific leaf weight. It possesses the nifH gene of nitrogen fixation, is capable of phosphate solubilization, synthesis of auxins and siderophores, and is antagonistic to plant pathogenic fungi and bacilli.

Characterization of endophytic plant growth promoting traits of Methylobacterium sp. isolated from banana (Musa sp.)

A total of 26 endophytic Methylobacterium sp. strains were obtained from surface sterilized leaves of two banana cultivar (Robusta and Nattu poovan) and the strains were tested for its ability to fix atmospheric nitrogen, EPS and IAA production for promoting banana tree growth. The result of the present study demonstrated that endophytic population ranged from 4.03 and 4.14 log cfu per gram of leaf tissue. Among these four isolates were chosen based on colony morphology and their distinct pigmentation. All the selected four isolates were able to grow in nitrogen free methanol mineral salt medium. The synthesis of indole-3-acetic acid (IAA) in the presence of L-tryptophan was detected in all the isolates tested. The isolate FM2 ( Methylobacterium sp.) produced the highest amount of IAA (13.01 μgml -1 ) in medium supplemented with L-tryptophan and was able to synthesize IAA in the absence of L-tryptophan. The maximum amount of ASP and WSP was recorded in FM3 (21.73 μg ml -1 ) and FM1 (157.79 μg ml -1 ), respectively. They were tentatively identified at species level based on carbon utilization test. The classified strains were also screened for methanol dehydrogenase ( mxaF gene sequencing) using specific primers and obtained 555 bp PCR product. So the Methylobacterium sp. strains analyzed here had a promising potential for developing as a plant growth promoting bacteria for sustainable agriculture.

Overexpression of the methanol dehydrogenase gene mxaF in Methylobacterium sp. MB200 enhances L-serine production.

Increase in L-serine production is of interest for industry. Here, we describe ametabolic engineering approach to increase the production of L-serine in a methylotrophic bacterium. mxaF, the gene encoding the large subunit of a methanol dehydrogenase, was cloned from Methylobacterium sp. MB200 through transposon mutagenesis. Deletion of mxaF gene prevented the strain to grow on methanol, suggesting that mxaF is involved in methanol metabolism. Overexpression of mxaF gene in the strain MB200 resulted in a fivefold increase in methanol dehydrogenase activity compared to the wild-type. Resting cell assays showed that the recombinant strain accumulated 6·6mgml(-1) L-serine in 72h with 30mgml(-1) wet cells from 50mgml(-1) glycine and 50mgml(-1) methanol, representing a 1·5-fold increment for L-serine production in contrast to the wild-type strain. These results demonstrate that the potential for improving the production of L-serine can be achieved by overexpressing mxaF gene in methylotrophic bacteria. The amount of L-serine produced each year worldwide is relatively small compared with the amounts of the other amino acids and hence it is in great demand. Here, we describe a metabolic engineering approach to increase the production of L-serine in a methylotrophic bacterium Methylobacterium sp. MB200. The result demonstrates that raising the output of L-serine can be achieved by overexpressing mxaF gene in methylotrophic bacteria.

Investigation of XoxF methanol dehydrogenases reveals new methylotrophic bacteria in pelagic marine and freshwater ecosystems.

The diversity and distribution of methylotrophic bacteria have been investigated in the oceans and lakes using the methanol dehydrogenase mxaF gene as a functional marker. However, pelagic marine (OM43) and freshwater (LD28 and PRD01a001B) methylotrophs within the Betaproteobacteria lack mxaF, instead possessing a related xoxF4-encoded methanol dehydrogenase. Here, we developed and employed xoxF4 as a complementary functional gene marker to mxaF for studying methylotrophs in aquatic environment. Using xoxF4, we detected OM43-related and LD28-related methylotrophs in the ocean and freshwaters of North America, respectively, and showed the coexistence of these two lineages in a large estuarine system (St Lawrence Estuary). Gene expression patterns of xoxF4 supported a positive relationship between xoxF4-containing methylotroph activity and spring time productivity, suggesting phytoplankton blooms are a source of methylotrophic substrates. Further investigation of methanol dehydrogenase diversity in pelagic ecosystems using comparative metagenomics provided strong support for a widespread distribution of xoxF4 (as well as several distinct xoxF5) containing methylotrophs in marine and freshwater surface waters. In total, these results demonstrate a geographical distribution of OM43/LD28-related methylotrophs that includes marine and freshwaters and suggest that methylotrophy occurring in the water column is an important component of lake and estuary carbon cycling and biogeochemistry.

Phylogenetic study of methanol oxidizers from chilika-lake sediments using genomic and metagenomic approaches.

Group-wise diversity of sediment methylotrophs of Chilika lake (Lat. 19°28'-19°54'N; Long. 85°06'-85°35'E) Odisha, India at various identified sites was studied. Both the culturable and unculturable (metagenome) methylotrophs were investigated in the lake sediments employing both mxaF and 16S rRNA genes as markers. ARDRA profiling, 16S rRNA gene sequencing, PAGE profiling of HaeIII, EcoRI restricted mxaF gene and the mxaF gene sequences using culture-dependent approach revealed the relatedness of α-proteobacteria and Methylobacterium, Hyphomicrobium and Ancyclobacter sp. The total viable counts of the culturable aerobic methylotrophs were relatively higher in sediments near the sea mouth (S3; Panaspada), also demonstrated relatively high salinity (0.1M NaCl) tolerance. Metagenomic DNA from the sediments, amplified using GC clamp mxaF primers and resolved through DGGE, revealed the diversity within the unculturable methylotrophic bacterium Methylobacterium organophilum, Ancyclobacter aquaticus, Burkholderiales and Hyphomicrobium sp. Culture-independent analyses revealed that up to 90% of the methylotrophs were unculturable. The study enhances the general understandings of the metagenomic methylotrophs from such a special ecological niche.

Screening and characterization of alkaline protease produced by a pink pigmented facultative methylotrophic (PPFM) strain, MSF 46

Abstract Among the various bacterial isolates, the strain MSF 46 isolated from thorn forest soil samples, Tamil Nadu, India, was screened and characterized for its proteolytic activity. While the 16S rRNA sequencing and biochemical characterization revealed that the strain closely resembles Methylobacterium sp., methylotrophy of the strain was confirmed by the sequence homology of mxaF gene with other relative Methylobacterium sp. The alkaline protease was purified to homogeneity using DEAE cellulose ion exchange chromatography, with a 5.2-fold increase in specific activity and 34% recovery. The apparent molecular weight of the enzyme was determined as 40 kDa by SDS–PAGE study. The pH and temperature optima were 9.0 and 50 °C respectively with maximum protease activity of 1164 U/ml. Protease of MSF 46 was active in a broad pH range 7.0–11.0 with a maximum at pH 8.5 and exhibited thermostability at 50 °C. The enzyme activity was inhibited by PMSF but showed stability with Tween 20, Triton X-100 and hydrogen peroxide. Nearly 30% reduction in enzyme activity was observed in the presence of EDTA and DTT. The enzyme was effective in hydrolyzing gelatin, skimmed milk and blood clots and exhibited the potency for dehairing of goat skin and removing blood stain from cotton fabric. Significant morphological changes were observed under scanning electron microscope between cells grown in normal and casein amended medium. This first detailed report on the production of alkaline protease by a PPFM strain appears promising toward development of protocols for mass production, study of the molecular mechanism and other applications.

Isolation and characterization of methane utilizing bacteria from wetland paddy ecosystem

Methylotrophic bacteria which are known to utilize C1 compounds including methane. Research during past few decades increased the interest in finding out novel genera of methane degrading bacteria to efficiently utilize methane to decrease global warming effect. Moreover, evaluation of certain known plant growth promoting strains for their methane degrading potential may open up a new direction for multiple utility of such cultures. In this study, efficient methylotrophic cultures were isolated from wetland paddy fields of Gujarat. From the overall morphological, biochemical and molecular characterization studies, the isolates were identified and designated as Bacillus aerius AAU M 8; Rhizobium sp. AAU M 10; B. subtilis AAU M 14; Paenibacillus illinoisensis AAU M 17 and B. megaterium AAU M 29. Gene specific PCR analysis of the isolates, P. illinoisensis, B. aerius, Rhizobium sp. and B. subtilis showed presence of pmoA gene encoding α subunit particulate methane monooxygenase cluster. B. megaterium, P. illinoisensis, Rhizobium sp. and Methylobacterium extrorquens showed presence of mmoX gene encoding α subunit of the hydroxylase component of the soluble methane monooxygenase cluster. P. illinoisensis and Rhizobium sp. showed presence mxaF gene encoding α subunit region of methanol dehydrogenase gene cluster showing that both isolates are efficient utilizers of methane. To the best of our knowledge, this is the first time report showing presence of methane degradation enzymes and genes within the known PGPB group of organisms from wet land paddy agro-ecosystem, which is considered as one of the leading methane producer.

Methylobacterium pseudosasae sp. nov., a pink-pigmented, facultatively methylotrophic bacterium isolated from the bamboo phyllosphere

A pink-pigmented, Gram negative, aerobic, facultatively methylotrophic bacterium, strain BL44(T), was isolated from bamboo leaves and identified as a member of the genus Methylobacterium. Phylogenetic analysis based on 16S rRNA gene sequences showed similarity values of 98.7-97.0 % with closely related type strains and showed highest similarity to Methylobacterium zatmanii DSM 5688(T) (98.7 %) and Methylobacterium thiocyanatum DSM 11490(T) (98.7 %). Methylotrophic metabolism in this strain was confirmed by PCR amplification and sequencing of the mxaF gene coding for the α-subunit of methanol dehydrogenase. Strain BL44(T) produced three known quorum sensing signal molecules with similar retention time to C8, C10 and C12-HSLs when characterized by GC-MS. The fatty acid profiles contained major amounts of C18:1 ω7c, iso-3OH C17:0 and summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH), which supported the grouping of the isolate in the genus Methylobacterium. The DNA G+C content was 66.9 mol%. DNA relatedness of the strain BL44(T) to its most closely related strains ranged from 12-43.3 %. On the basis of the phenotypic, phylogenetic and DNA-DNA hybridization data, strain BL44(T) is assigned to a novel species of the genus Methylobacterium for which the name Methylobacterium pseudosasae sp. nov. is proposed (type strain BL44(T) = NBRC 105205(T) = ICMP 17622(T)).

Methylobacterium-plant interaction genes regulated by plant exudate and quorum sensing molecules

Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction.

Vertical distribution of methane oxidation and methanotrophic response to elevated methane concentrations in stratified waters of the Arctic fjord Storfjorden (Svalbard, Norway)

Abstract. The bacterially mediated aerobic methane oxidation (MOx) is a key mechanism in controlling methane (CH4) emissions from the world's oceans to the atmosphere. In this study, we investigated MOx in the Arctic fjord Storfjorden (Svalbard) by applying a combination of radio-tracer-based incubation assays (3H-CH4 and 14C-CH4), stable C-CH4 isotope measurements, and molecular tools (16S rRNA gene Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, pmoA- and mxaF gene analyses). Storfjorden is stratified in the summertime with melt water (MW) in the upper 60 m of the water column, Arctic water (ArW) between 60 and 100 m, and brine-enriched shelf water (BSW) down to 140 m. CH4 concentrations were supersaturated with respect to the atmospheric equilibrium (about 3–4 nM) throughout the water column, increasing from ∼20 nM at the surface to a maximum of 72 nM at 60 m and decreasing below. MOx rate measurements at near in situ CH4 concentrations (here measured with 3H-CH4 raising the ambient CH4 pool by <2 nM) showed a similar trend: low rates at the sea surface, increasing to a maximum of ∼2.3 nM day−1 at 60 m, followed by a decrease in the deeper ArW/BSW. In contrast, rate measurements with 14C-CH4 (incubations were spiked with ∼450 nM of 14C-CH4, providing an estimate of the CH4 oxidation at elevated concentration) showed comparably low turnover rates (<1 nM day−1) at 60 m, and peak rates were found in ArW/BSW at ∼100 m water depth, concomitant with increasing 13C values in the residual CH4 pool. Our results indicate that the MOx community in the surface MW is adapted to relatively low CH4 concentrations. In contrast, the activity of the deep-water MOx community is relatively low at the ambient, summertime CH4 concentrations but has the potential to increase rapidly in response to CH4 availability. A similar distinction between surface and deep-water MOx is also suggested by our molecular analyses. The DGGE banding patterns of 16S rRNA gene fragments of the surface MW and deep water were clearly different. A DGGE band related to the known type I MOx bacterium Methylosphaera was observed in deep BWS, but absent in surface MW. Furthermore, the Polymerase Chain Reaction (PCR) amplicons of the deep water with the two functional primers sets pmoA and mxaF showed, in contrast to those of the surface MW, additional products besides the expected one of 530 base pairs (bp). Apparently, different MOx communities have developed in the stratified water masses in Storfjorden, which is possibly related to the spatiotemporal variability in CH4 supply to the distinct water masses.

The methanol dehydrogenase gene, mxaF, as a functional and phylogenetic marker for proteobacterial methanotrophs in natural environments.

The mxaF gene, coding for the large (α) subunit of methanol dehydrogenase, is highly conserved among distantly related methylotrophic species in the Alpha-, Beta- and Gammaproteobacteria. It is ubiquitous in methanotrophs, in contrast to other methanotroph-specific genes such as the pmoA and mmoX genes, which are absent in some methanotrophic proteobacterial genera. This study examined the potential for using the mxaF gene as a functional and phylogenetic marker for methanotrophs. mxaF and 16S rRNA gene phylogenies were constructed based on over 100 database sequences of known proteobacterial methanotrophs and other methylotrophs to assess their evolutionary histories. Topology tests revealed that mxaF and 16S rDNA genes of methanotrophs do not show congruent evolutionary histories, with incongruencies in methanotrophic taxa in the Methylococcaceae, Methylocystaceae, and Beijerinckiacea. However, known methanotrophs generally formed coherent clades based on mxaF gene sequences, allowing for phylogenetic discrimination of major taxa. This feature highlights the mxaF gene’s usefulness as a biomarker in studying the molecular diversity of proteobacterial methanotrophs in nature. To verify this, PCR-directed assays targeting this gene were used to detect novel methanotrophs from diverse environments including soil, peatland, hydrothermal vent mussel tissues, and methanotroph isolates. The placement of the majority of environmental mxaF gene sequences in distinct methanotroph-specific clades (Methylocystaceae and Methylococcaceae) detected in this study supports the use of mxaF as a biomarker for methanotrophic proteobacteria.