Methylotrophic Bacteria Research Articles

ASYMMETRIC, BIMODAL TRADE-OFFS DURING ADAPTATION OFMETHYLOBACTERIUMTO DISTINCT GROWTH SUBSTRATES

Trade-offs between selected and nonselected environments are often assumed to exist during adaptation. This phenomenon is prevalent in microbial metabolism, where many organisms have come to specialize on a narrow breadth of substrates. One well-studied example is methylotrophic bacteria that can use single-carbon (C(1)) compounds as their sole source of carbon and energy, but generally use few, if any, multi-C compounds. Here, we use adaptation of experimental populations of the model methylotroph, Methylobacterium extorquens AM1, to C(1) (methanol) or multi-C (succinate) compounds to investigate specialization and trade-offs between these two metabolic lifestyles. We found a general trend toward trade-offs during adaptation to succinate, but this was neither universal nor showed a quantitative relationship with the extent of adaptation. After 1500 generations, succinate-evolved strains had a remarkably bimodal distribution of fitness values on methanol: either an improvement comparable to the strains adapted on methanol or the complete loss of the ability to grow on C(1) compounds. In contrast, adaptation to methanol resulted in no such trade-offs. Based on the substantial, asymmetric loss of C(1) growth during growth on succinate, we suggest that the long-term maintenance of C(1) metabolism across the genus Methylobacterium requires relatively frequent use of C(1) compounds to prevent rapid loss.

Isolation and Characterization of Pink-Pigmented, Facultative Methylotrophic (PPFM) Bacteria from Leaves of Neem, <i>Azadirachta indica</i> A. Juss.

Isolation and Characterization of Pink-Pigmented, Facultative Methylotrophic (PPFM) Bacteria from Leaves of Neem, <i>Azadirachta indica</i> A. Juss.

Methylobacterium Genome Sequences: A Reference Blueprint to Investigate Microbial Metabolism of C1 Compounds from Natural and Industrial Sources

BackgroundMethylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared.Methodology/Principal FindingsThe 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid).Conclusion/SignificanceThese two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.

The aerobic degradation of dichloromethane: Structural-functional aspects (a review)

This review summarizes current notions on the mechanisms of transport and degradation of dichloromethane (DCM) by aerobic methylotrophic bacteria as well as enzymological and genetic aspects of DCM dehalogenation, including probable pathways used by cells to overcome accompanying stresses (acid, osmotic, and oxidative). The topicality of the problem of the degradation of this genotoxic solvent is associated with the search for and creation of new DCM-destroying strains, which would provide for more efficient bioremediation of industrial sewage and ecosystems with extreme pH and salinity and could be used for the development of modern techniques for DCM degradation on the basis of existing strains. Special attention is given to the consideration of methodological approaches to the interpretation of physiological—biochemical and molecular bases of adaptation of bacteria to the utilization of DCM and other halogenated pollutants.

Phenotypic Characterization of Pink Pigmented Facultative Methylotrophic Bacteria from Soil Exposed to Vehicular Soot

Phenotypic Characterization of Pink Pigmented Facultative Methylotrophic Bacteria from Soil Exposed to Vehicular Soot

Vertical diversity of bacteria in an oxygen-stratified humic lake, evaluated using DNA and phospholipid analyses

Microbes play a particularly important role in the food web in lakes with high dissolved organic carbon content. The bacterial community of a polyhumic lake, Mekkojarvi, was studied using DNA techniques and phospholipid fatty acid (PLFA) analysis during the mid-summer period of water column strati- fication. According to the 16S rRNA gene clone libraries and length heterogeneity analysis (LH-PCR), heterotrophic bacteria dominated only in the oxic epilimnion, in which various Actinobacteria (mostly cluster acI-B) and Betaproteobacteria (especially Poly- nucleobacter subcluster PnecC) were common. Se- quences assigned to heterotrophic, methylotrophic, photoautotrophic, and chemoautotrophic genera were all abundant in the oxic-anoxic boundary layer. Methylobacter and Methylophilus were dominant genera among methylotrophic bacteria. Sequences assigned to the photoautotrophic green sulfur bac- terium Chlorobium sp. dominated in the anoxic water column, in which the microbial PLFA biomass was 6 times higher than in the oxic surface layer. All PLFA- profiles were dominated by 16 monounsaturated fatty acids typical of Gram-negative bacteria, whereas iso- and anteiso-branched PLFAs typical of Actinobacteria were present only in minor proportions. The high bio- mass of the potentially autotrophic meta- and hypolim- netic bacteria may form an important carbon source for the whole lake after spring and autumn overturns; thus, the role of these bacteria in the seasonal energy mobilization deserves more study in oxygen-stratified humic lakes and ponds.

Identification and cloning of a gene encoding dichloromethane dehalogenase from a methylotrophic bacterium, Bacillus circulans WZ-12 CCTCC M 207006

The gene dehalA encoding a novel dichloromethane dehalogenases (DehalA), has been cloned from Bacillus circulans WZ-12 CCTCC M 207006. The open reading frame of dehalA, spanning 864 bp, encoded a 288-amino acid protein that showed 85.76% identity to the dichloromethane dehalogenases of Hyphomicrobium sp. GJ21 with several commonly conserved sequences. These sequences could not be found in putative dichloromethane (DCM) dehalogenases reported from other bacteria and fungi. DehalA was expressed in Escherichia coli BL21 (DE3) from a pET28b(+) expression system and purified. The subunit molecular mass of the recombinant DehalA as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 33 kDa. Subsequent enzymatic characterization revealed that DehalA was most active in a acidic pH range at 30 degrees , which was quite different from that observed from a facultative bacterium dichloromethane dehalogenases of Methylophilus sp. strain DM11. The Michaelis-Menten constant of DCM dehalogenase was markedly lower than that of standard DCM dehalogenases.

Enhancing Production of l-Serine by Increasing the glyA Gene Expression in Methylobacterium sp. MB200

Microbial fermentation using methylotrophic bacteria is one of the most promising methods for L-serine production. Here we describe the metabolic engineering of a Methylobacterium strain to increase the production of L-serine. The glyA gene, encoding serine hydroxymethyltransferase (SHMT), was isolated from the genomic DNA of Methylobacterium sp. MB200, using a DNA fragment encoding Methylobacterium extorquens AM1 SHMT as a probe, and inserted into the vector pLAFR3. The resulting construct was transformed into Methylobacterium sp. MB200 using triparental mating. The genetic-engineered strain, designated as Methylobacterium sp. MB202, was shown to produce 11.4 + or - 0.6 mg/ml serine in resting cell reactions from 30 mg/ml wet cells, 20 mg/ml glycine, and 70 mg/ml methanol in 2 days, representing a 4.4-fold increase from that of the wild strain. The results demonstrated the potential for improving L-serine production by manipulating the glyA in bacteria and should facilitate the production of L-serine using Methylobacterium sp. strains.

Thiosulfate Oxidation, Mixotrophic Growth of Methylobacterium goesingense and Methylobacterium fujisawaense

The mixotrophic growth with methanol plus thiosulfate was examined in nutrient-limited mixotrophic condition for Methylobacterium goesingense CBMB5 and Methylobacterium fujisawaense CBMB37. Thiosulfate oxidation increased the growth and protein yield in mixotrophic medium that contained 150 mM methanol and 20 mM sodium thiosulfate, at 144 h. Respirometric study revealed that thiosulfate was the most preferable reduced inorganic sulfur source, followed by sulfite and sulfur. M. goesingense CBMB5 and M. fujisawaense CBMB37 oxidized thiosulfate directly to sulfate, and intermediate products of thiosulfate oxidation such as polythionates, sulfite, and sulfur were not detected in spent medium and they did not yield positive amplification for tested soxB primers. Enzymes of thiosulfate oxidation such as rhodanese and sulfite oxidase activities were detected in cell-free extracts of M. goesingense CBMB5, and M. fujisawaense CBMB37, and thiosulfate oxidase (tetrathionate synthase) activity was not observed. It indicated that both the organisms use the "non-S4 intermediate" sulfur oxidation pathway for thiosulfate oxidation. It is concluded from this study that M. goesingense CBMB5, and M. fujisawaense CBMB37 exhibited mixotrophic metabolism in medium containing methanol plus thiosulfate and that thiosulfate oxidation and the presence of a "Paracoccus sulfur oxidation" (PSO) pathway in methylotrophic bacteria are species dependant.

Statistical media optimization for growth and PHB production from methanol by a methylotrophic bacterium

Media components were optimized by statistical design for cell growth and PHB production of Methylobacterium extorquens DSMZ 1340. Four important components of growth media were optimized by central composite design. The growth increased from an OD = 1.35 for Choi medium as control to an OD = 2.15 for optimal medium. Then media components for PHB production were optimized. Optimization of five important factors was conducted by response surface method. The optimal composition of PHB production medium was found to be at 7.8 (g/L) Na 2HPO 4 · 12H 2O, and surprisingly at zero concentration of (NH 4) 2SO 4, KH 2PO 4, MgSO 4 and MnSO 4. The PHB production was found to be 2.95 (g/L) at this medium. RSM results indicated that a deficiency of nitrogen and magnesium is crucial for PHB accumulation in this microorganism. Also, PHB production was carried out in a 5 L fermentor at the optimum condition which resulted in 9.5 g/L PHB and 15.4 g/L cell dry weight with 62.3% polymer content.

Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria

Methanol is one of the building blocks in the chemical industry and can be synthesized either from petrochemical or renewable resources, such as biogas. Bioprocess technology with methylotrophic bacteria is well established, as illustrated by large-scale single-cell protein production in the past. During recent years, the first genomes of methylotrophs have been sequenced and significant progress in elucidating their metabolism has been made. In addition, the tool set for genetic engineering of methylotrophic bacteria has expanded greatly and strategies to produce fine and bulk chemicals with methylotrophs have been described. This review highlights the potential of these bacteria for the development of economically competitive bioprocesses based on methanol as an alternative carbon source, bringing together biological, technical and economic considerations.

Atomic resolution structure of pseudoazurin from the methylotrophic denitrifying bacteriumHyphomicrobium denitrificans: structural insights into its spectroscopic properties

The crystal structure of native pseudoazurin (HdPAz) from the methylotrophic denitrifying bacterium Hyphomicrobium denitrificans has been determined at a resolution of 1.18 A. After refinement with SHELX employing anisotropic displacement parameters and riding H atoms, R(work) and R(free) were 0.135 and 0.169, respectively. Visualization of the anisotropic displacement parameters as thermal ellipsoids provided insight into the atomic motion within the perturbed type 1 Cu site. The asymmetric unit includes three HdPAz molecules which are tightly packed by head-to-head cupredoxin dimer formation. The shape of the Cu-atom ellipsoid implies significant vibrational motion diagonal to the equatorial xy plane defined by the three ligands (two His and one Cys). The geometric parameters of the type 1 Cu site in the HdPAz structure differ unambiguously from those of other pseudoazurins. It is demonstrated that their structural aspects are consistent with the unique visible absorption spectrum.

Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria

The goal of this study was to identify bacterial populations that assimilated methanol in a denitrifying sequencing batch reactor (SBR), using stable isotope probing (SIP) of (13)C labeled DNA and quantitatively track changes in these populations upon changing the electron donor from methanol to ethanol in the SBR feed. Based on SIP derived (13)C 16S rRNA gene clone libraries, dominant SBR methylotrophic bacteria were related to Methyloversatilis spp. and Hyphomicrobium spp. These methylotrophic populations were quantified via newly developed real-time PCR assays. Upon switching the electron donor from methanol to ethanol, Hyphomicrobium spp. concentrations decreased significantly in accordance with their obligately methylotrophic nutritional mode. In contrast, Methyloversatilis spp. concentrations were relatively unchanged, in accordance with their ability to assimilate both methanol and ethanol. Direct assimilation of ethanol by Methyloversatilis spp. but not Hyphomicrobium spp. was also confirmed via SIP. The reduction in methylotrophic bacterial concentration upon switching to ethanol was paralleled by a significant decrease in the methanol supported denitrification biokinetics of the SBR on nitrate. In sum, the results of this study demonstrate that the metabolic capabilities (methanol assimilation and metabolism) and substrate specificity (obligately or facultatively methylotrophic) of two distinct methylotrophic bacterial populations contributed to their survival or washout in denitrifying bioreactors.

Analysis of Pink Pigmented Facultative Methylotroph Bacteria from Human Environments

The formation of pink biofilm in wet places are usually correlated with chlorine-resistant pink pigmented facultative methylotrophs (PPFM). In this study we investigated the presence of PPFM bacteria through bacterial isolation and detection of mxaF gene from wet places of human-made environments. A total of eighteen PPFM bacterial isolates were recovered from the formation of biofilm bacterial of four test places such as washstands, bathrooms, and potable water supplies. Confirmation of the isolates through biochemical analysis were done using catalase, oxidase and urease tests. Chlorine-resistance-activity was assayed for all of the isolates. Antibiotic resistance were examined for ampicillin (25 μg), tetracycline (30 μg), kanamycin (30 μg), trimethoprim (1.25 μg), and streptomycin (10 μg) using the agar diffusion method. Genomic DNA was subjected to PCR analysis with primers corresponding to the 5’- and 3’- end conserved segments of the mxaF gene. PCR amplification followed by DNA sequencing of 16S rRNA gene were done for some isolates. We recovered 18 isolates of PPFM bacteria. Biochemical analysis indicated that the isolates were positive for catalase, oxidase, and urease activities. Chlorine-resistance-analysis showed the majority of the isolates were resistant to chlorine. Antibiotic resistance assays showed all of the isolates exhibited resistance to trimethoprim but were sensitive to streptomycin, kanamycin, and tetracycline but were variably resistant to ampicilin. PCR detection using specific primers for the mxaF gene gave a positive result for all of the isolates. DNA sequencing of the 16S rRNA gene of two isolates showed that isolate WD10 had a 98% similarity with the mxaF gene from Methylobacterium lusitanum strain MP2 and isolate WK2 had a 98% similarity to the mxaF gene from Afipia felis strain RD1. The formation of pink biofilm of four wet areas in this study were correlated with the presence of chlorine-resistant PPFM bacteria and we confirmed with the presence of the mxaF gene in all of the isolates. This finding needs to be widely publicized since some PPFM bacteria were known as opportunistic pathogens.

Novel methylotrophic bacteria isolated from the River Thames (London, UK)

Enrichment and elective culture for methylotrophs from sediment of the River Thames in central London yielded a diversity of pure cultures representing several genera of Gram-negative and Gram-positive bacteria, which were mainly of organisms not generally regarded as typically methylotrophic. Substrates leading to successful isolations included methanol, monomethylamine, dimethylamine, trimethylamine, methanesulfonate and dimethylsulfone. Several isolates were studied in detail and shown by their biochemical and morphological properties and 16S rRNA gene sequencing to be Sphingomonas melonis strain ET35, Mycobacterium fluoranthenivorans strain DSQ3, Rhodococcus erythropolis strain DSQ4, Brevibacterium casei strain MSQ5, Klebsiella oxytoca strains MMA/F and MMA/1, Pseudomonas mendocina strain TSQ4, and Flavobacterium sp. strains MSA/1 and MMA/2. The results show that facultative methylotrophy is present across a wide range of Bacteria, suggesting that turnover of diverse C(1)-compounds is of much greater microbiological and environmental significance than is generally thought. The origins of the genes encoding the enzymes of methylotrophy in diverse heterotrophs need further study, and could further our understanding of the phylogeny and antiquity of methylotrophic systems.

Activities of 6-phosphofructokinases and inorganic pyrophosphatase in aerobic methylotrophic bacteria

The classical radioisotopic and enzymological studies of J.R. Quayle led to the discovery of the ribulosemonophosphate (RuMP) cycle of formaldehyde assimilation in methanotrophs and methylobacteria [1, 2]. The key reactions of the RuMP cycle catalyzed, by 3-hexulosephosphate synthase (HPS) and phospho-3hexuloisomerase, form fructose-6-phosphate from formaldehyde and ribulose-5-phosphate. It was believed that further phosphorylation of fructose-6phosphate to fructose-1,6-bisphosphate is carried out by ATP-dependent 6-phosphofructokinase (ATP-PFK), although its low activity (2‐8 mU/mg protein) in methanotrophs questioned the significance of the glycolytic pathway of phosphosugar transformation in the RuMP cycle. The discovery of the activity of pyrophosphatedependent 6-phosphofructokinase (PP i -PFK) and a high intracellular concentration of inorganic pyrophosphate in three species of methanotrophs employing different pathways of e 1 compound assimilation stimulated the study of the role of PP i and reappraisal of the concepts of the organization and regulation of the RuMP cycle in aerobic methylotrophic bacteria of various taxonomic positions [3]. Along these lines, in this work, the activities of PP i - and ATP-dependent 6-phosphofructokinases and pyrophosphatase (PP i -ase) were determined in a broad spectrum of aerobic methanotrophs and methylobacteria employing various pathways of C 1 assimilation. The work used strains of obligate methanotrophs and methylobacteria from the collection of the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences. Cells were harvested in the early stationary phase, as described earlier [3, 4]. A 12-h Escherichia coli K12 culture grown in liquid medium with glucose was also used. Cells were separated from the medium by centrifugation, washed with 50 mM phosphate buffer (pH 7), and destroyed with an IBPM-press in a frozen state. To obtain cell-free extracts, cell homogenates were centrifuged (12000 g , 20 min) and dialyzed against the same buffer. The activities of PP i -PFK (EC 2.7.1.90), ATP-PFK (EC 2.7.1.11), and inorganic pyrophosphatase (EC 3.6.1.1) were determined as described earlier [4]. The data presented are the means of three measurements. The results of determination of the activities of PP i PFK, ATP-PFK, and PP i -ase in obligate and facultative methylobacteria are shown in the table. Since the detected activity of ATP-PFK could in fact be the activity of PP i -PFK at the expense of PP i admixtures, a preparation of yeast inorganic pyrophosphatase (0.2 U/ml) was introduced into the incubation mixture for ATPPFK determination in further experiments. Under these conditions, the ATP-PFK activity was not revealed. The highest PP i -PFK activity was revealed in type I methanotrophs, which assimilate formaldehyde via the RuMP cycle. In type II methanotrophs with the serine pathway, the activity of PP i -PFK was lower than in type I methanotrophs. The reversibility of this reaction and the necessity of the synthesis of hexose phosphates from triose phosphates in type II methanotrophs suggests the involvement of PP i -PFK in the gluconeogenesis of these bacteria. The lowest values of the PP i -PFK activity were recorded in type X methanotrophs of the genera Methylococcus and Methylocaldum , which simultaneously employ three pathways of C 1 assimilation. The activity of PP i -ase in cell-free extracts of methanotrophs was considerably lower than in cell-free extracts of most of the methylobacteria studied and E. coli K12.

Comprehensive proteomics of Methylobacterium extorquens AM1 metabolism under single carbon and nonmethylotrophic conditions

In order to validate a gel free quantitative proteomics assay for the model methylotrophic bacterium Methylobacterium extorquens AM1, we examined the M. extorquens AM1 proteome under single carbon (methanol) and multicarbon (succinate) growth, conditions that have been studied for decades and for which extensive corroborative data have been compiled. In total, 4447 proteins from a database containing 7556 putative ORFs from M. extorquens AM1 could be identified with two or more peptide sequences, corresponding to a qualitative proteome coverage of 58%. Statistically significant nonzero (log(2) scale) differential abundance ratios of methanol/succinate could be detected for 317 proteins using summed ion intensity measurements and 585 proteins using spectral counting, at a q-value cut-off of 0.01, a measure of false discovery rate. The results were compared to recent microarray studies performed under equivalent chemostat conditions. The M. extorquens AM1 studies demonstrated the feasibility of scaling up the multidimensional capillary HPLC MS/MS approach to a prokaryotic organism with a proteome more than three times the size of microbes we have investigated previously, while maintaining a high degree of proteome coverage and reliable quantitative abundance ratios.

Molecular and biokinetic characterization of methylotrophic denitrification using nitrate and nitrite as terminal electron acceptors

Although methanol is a widely employed carbon source for denitrification, relatively little is known on the abundance and diversity of methylotrophic bacteria in activated sludge. The primary aim of this study was to specifically identify bacteria that metabolized methanol in a sequencing batch denitrifying reactor (SBDR), using a novel technique, stable isotope probing (SIP) of 13C labeled DNA. A secondary aim was to quantitatively track dominant methylotrophic bacteria in the SBDR exposed to different terminal electron acceptors. SIP enabled 13C 16S rDNA clone libraries revealed that SBDR methylotrophic populations were related to Methyloversatilis spp. and Hyphomicrobium spp. Based on newly developed quantitative polymerase chain reaction (qPCR) assays, Hyphomicrobium spp. were more abundant than Methyloversatilis spp. throughout the period of SBDR operation. The relative population abundance was stable despite a shift in electron acceptor from nitrate to nitrite (keeping the same methanol dose). However, the shift to nitrite resulted in a significant decrease in denitrification biokinetics on both nitrate and nitrite.

Phenotypic characterization of air-borne pink pigmented facultative methylotrophic bacteria from high vehicular traffic density environment in Manila, Philippines

Phenotypic characterization of air-borne pink pigmented facultative methylotrophic bacteria from high vehicular traffic density environment in Manila, Philippines

Genetic diversity and phylogeny of pink-pigmented facultative methylotrophic bacteria isolated from the phyllosphere of tropical crop plants

Genetic diversity of pink-pigmented facultative methylotrophic bacteria belonging to the genus, Methylobacterium, was assessed using 16S ribosomal RNA (rRNA) gene sequencing, amplified ribosomal DNA restriction analysis (ARDRA), and differential carbon-substrate utilization profile in the phyllosphere of cotton, maize, sunflower, soybean, and mentha plants. Methylobacterium populi, Methylobacterium thiocyanatum, Methylobacterium suomiense, M. aminovorans, and Methylobacterium fujisawaense were identified to colonize the phyllosphere of these crop plants. Among these, M. populi found to be the dominating species followed by M. aminovorans. The diversity indices like Shannon index of diversity, Pielou index of evenness, and Margalef index of richness calculated based on combined data of ARDRA, 16S rRNA gene sequence, and differential carbon-substrate utilization revealed that sunflower leaves showed richest methylobacterial diversity, followed by soybean, while cotton and mentha leaves recorded the lowest diversity. The outcome of this study indicates that diversity of Methylobacterium in phyllosphere depends upon the host plant species colonized.