Lanpepsy is a novel lanthanide-binding protein involved in the lanthanide response of the obligate methylotroph Methylobacillus flagellatus.

Lanthanides were recently discovered as metals required in the active site of certain methanol dehydrogenases. Since then, the characterization of the lanthanome, that is, proteins involved in sensing, uptake, and utilization of lanthanides, has become an active field of research. Initial exploration of the response to lanthanides in methylotrophs has revealed that the lanthanome is not conserved and that multiple mechanisms for lanthanide utilization must exist. Here, we investigated the lanthanome in the obligate model methylotroph Methylobacillus flagellatus. We used a proteomic approach to analyze differentially regulated proteins in the presence of lanthanum. While multiple known proteins showed induction upon growth in the presence of lanthanum (Xox proteins, TonB-dependent receptor), we also identified several novel proteins not previously associated with lanthanide utilization. Among these was Mfla_0908, a periplasmic 19 kDa protein without functional annotation. The protein comprises two characteristic PepSY domains, which is why we termed the protein lanpepsy (LanP). Based on bioinformatic analysis, we speculated that LanP could be involved in lanthanide binding. Using dye competition assays, quantification of protein-bound lanthanides by inductively coupled plasma mass spectrometry, as well as isothermal titration calorimetry, we demonstrated the presence of multiple lanthanide binding sites that showed selectivity over the chemically similar calcium ion. LanP thus represents the first member of the PepSY family that binds lanthanides. Although the physiological role of LanP is still unclear, its identification is of interest for applications toward the sustainable purification and separation of rare-earth elements.

Methylobacillus methanolivorans sp. nov., a novel non-pigmented obligately methylotrophic bacterium.

Three strains of obligately methylotrophic Betaproteobacteria (ZT, SP and M3) with the ribulose monophosphate pathway of C1 assimilation are described. The isolates were strictly aerobic, Gram-stain-negative, asporogenous, motile (strains ZT and M3) or non-motile (strain SP) rods that multiplied by binary fisson, and were mesophilic and neutrophilic. All three strains utilized methanol but only strains SP and M3 utilized methylamine as carbon and energy sources. The prevailing cellular fatty acids were straight-chain saturated C16 : 0 and unsaturated C16 : 1ω7c acids. The major ubiquinone was Q-8. The predominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Ammonia was assimilated by glutamate dehydrogenase. The DNA G+C contents of strains ZT, SP and M3 were 51.0, 52.0 and 52.0 mol% (Tm), respectively. Levels of 16S rRNA gene sequence similarity between the three strains were very high (99.9-100 %), and they shared high levels of DNA-DNA relatedness (88-98 %). Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (19-30 %) with the type strains of the genus Methylobacillus, the novel isolates ZT, SP and M3 are classified as representing a novel species of this genus, for which the name Methylobacillus methanolivorans sp. nov. is proposed. The type strain is ZT (=VKM B-3037T=JCM 31401T=CCUG 68999T).

Enhanced fed-batch production of pyrroloquinoline quinine in Methylobacillus sp. CCTCC M2016079 with a two-stage pH control strategy.

The effects of pH control strategy and fermentative operation modes on the biosynthesis of pyrroloquinoline quinine (PQQ) were investigated systematically with Methylobacillus sp. CCTCC M2016079 in the present work. Firstly, the shake-flask cultivations and benchtop fermentations at various pH values ranging from 5.3 to 7.8 were studied. Following a kinetic analysis of specific cell growth rate (µ x ) and specific PQQ formation rate (µ p ), the discrepancy in optimal pH values between cell growth and PQQ biosynthesis was observed, which stimulated us to develop a novel two-stage pH control strategy. During this pH-shifted process, the pH in the broth was controlled at 6.8 to promote the cell growth for the first 48 h and then shifted to 5.8 to enhance the PQQ synthesis until the end of fermentation. By applying this pH-shifted control strategy, the maximum PQQ production was improved to 158.61 mg/L in the benchtop fermenter, about 44.9% higher than that under the most suitable constant pH fermentation. Further fed-batch study showed that PQQ production could be improved from 183.38 to 272.21 mg/L by feeding of methanol at the rate of 11.5 mL/h in this two-stage pH process. Meanwhile, the productivity was also increased from 2.02 to 2.84 mg/L/h. In order to support cell growth during the shifted pH stage, the combined feeding of methanol and yeast extract was carried out, which brought about the highest concentration (353.28 mg/L) and productivity (3.27 mg/L/h) of PQQ. This work has revealed the potential of our developed simple and economical strategy for the large-scale production of PQQ.

Biochemical Properties and Phylogeny of Hydroxypyruvate Reductases from Methanotrophic Bacteria with Different C1-Assimilation Pathways.

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C1-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C1-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.

Screening and Degradation Mechanism of a Cold-Resistant Nitrobenzene-Degrading Microorganism.

A cold-resistant nitrobenzene-degrading strain was screened from river sediment. The strain was identified as Methylobacillus glycogens, which has never been reported to be capable of degrading nitrobenzene. The degradation rates of 900 µg/L nitrobenzene reached respectively 99.3% and 88.6% in 144 h under both aerobic and anaerobic environments (30 mL inoculation volume at 12 ± 0.5 °C and pH7.0 ± 0.1). When aerobically degraded, nitrobenzene was firstly oxidized into o-nitrophenol, which was further oxidized into 1,2-benzenediol, meanwhile releasing NO2-. Then the 1,2-benzenediol was metabolized through either the ortho-cleavage into succinic acid and acetyl-CoA, or meta-cleavage into pyruvic acid and acetaldehyde, as well as other small molecule substances of non-toxicity or low-toxicity, which were finally decomposed into CO2 and H2O. When anaerobically degraded, nitrobenzene was firstly degraded into aniline (C6H5NH2), which was further degraded into 4-amino benzoic acid. The benzoic acid was degraded into benzoyl, which was finally metabolized and decomposed.

Medium optimization for pyrroloquinoline quinone (PQQ) production by Methylobacillus sp. zju323 using response surface methodology and artificial neural network-genetic algorithm.

Methylobacillus sp. zju323 was adopted to improve the biosynthesis of pyrroloquinoline quinone (PQQ) by systematic optimization of the fermentation medium. The Plackett-Burman design was implemented to screen for the key medium components for the PQQ production. CoCl2 · 6H2O, ρ-amino benzoic acid, and MgSO4 · 7H2O were found capable of enhancing the PQQ production most significantly. A five-level three-factor central composite design was used to investigate the direct and interactive effects of these variables. Both response surface methodology (RSM) and artificial neural network-genetic algorithm (ANN-GA) were used to predict the PQQ production and to optimize the medium composition. The results showed that the medium optimized by ANN-GA was better than that by RSM in maximizing PQQ production and the experimental PQQ concentration in the ANN-GA-optimized medium was improved by 44.3% compared with that in the unoptimized medium. Further study showed that this ANN-GA-optimized medium was also effective in improving PQQ production by fed-batch mode, reaching the highest PQQ accumulation of 232.0 mg/L, which was about 47.6% increase relative to that in the original medium. The present work provided an optimized medium and developed a fed-batch strategy which might be potentially applicable in industrial PQQ production.

Novel and efficient screening of PQQ high-yielding strains and subsequent cultivation optimization.

A novel and efficient screening method for pyrroloquinoline quinone (PQQ) high-yielding methylotrophic strains was developed by using glucose dehydrogenase apoenzyme (GDHA) which depended on PQQ as the cofactor. Using this high-throughput method, PQQ high-yielding strains were rapidly screened out from thousands of methylotrophic colonies at a time. The comprehensive phylogenetic analysis revealed that the highest PQQ-producing strain zju323 (CCTCC M 2016079) could be assigned to a novel species in the genus Methylobacillus of the Betaproteobacteria. After systematic optimization of different medium components and cultivation conditions, about 33.4 mg/L of PQQ was obtained after 48 h of cultivation with Methylobacillus sp. zju323 at the shake flask scale. Further cultivations of Methylobacillus sp. zju323 were carried out to investigate the biosynthesis of PQQ in 10-L bench-top fermenters. In the batch operation, the PQQ accumulation reached 78 mg/L in the broth after 53 h of cultivation. By adopting methanol feeding strategy, the highest PQQ concentration was improved up to 162.2 mg/L after 75 h of cultivation. This work developed a high-throughput strategy of screening PQQ-producing strains from soil samples and also demonstrated one potential bioprocess for large-scale PQQ production with the isolated PQQ strain.

Methylobacillus rhizosphaerae sp. nov., a novel plant-associated methylotrophic bacterium isolated from rhizosphere of red pepper.

A novel plant-associated obligate methylotrophic bacterium, designated strain Ca-68(T), was isolated from the rhizosphere soil of field-grown red pepper from India. The isolates are strictly aerobic, Gram negative, motile rods multiplying by binary fission and formaldehyde is assimilated via the ribulose monophosphate pathway. A comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Methylobacillus flagellatus, Methylobacillus glycogens and Methylobacillus pratensis, with which it showed pairwise similarity of 97.8, 97.4 and 96.2 %, respectively. The major fatty acids are C(16:0), C(10:0) 3OH and C(16:1) ω7c. The G+C content of the genomic DNA is 59.7 mol%. The major ubiquinone is Q-8. Dominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (14-19 %) with type strains of the genus Methylobacillus, the novel isolate was classified as a new species of this genus and named Methylobacillus rhizosphaerae Ca-68(T) (=KCTC 22383(T) = NCIMB 14472(T)).

Membrane topology and functional analysis of Methylobacillus sp. 12S genes epsF and epsG, encoding polysaccharide chain-length determining proteins.

The EpsF and EpsG of the methanol-assimilating bacterium Methylobacillus sp. 12S are involved in the synthesis of a high molecular weight exopolysaccharide, methanolan. These proteins share homology with chain-length determiners in other polysaccharide-producing bacteria. The N- and C-termini of EpsF were found to locate to the cytoplasm, and EpsF was predicted to have two transmembrane regions. EpsG showed both ATPase and autophosphorylation activities.

Purification and characterization of azurin from the methylamine-utilizing obligate methylotroph Methylobacillus flagellatus KT.

Methylamine dehydrogenase (MADH) and azurin were purified from the periplasmic fraction of the methylamine-grown obligate methylotroph Methylobacillus flagellatus KT. The molecular mass of the purified azurin was 16.3 kDa, as measured by SDS-PAGE, or 13 920 Da as determined by MALDI-TOF mass spectrometry. Azurin of M. flagellatus KT contained 1 copper atom per molecule and had an absorption maximum at 620 nm in the oxidized state. The redox potential of azurin measured at pH 7.0 by square-wave voltammetry was +275 mV versus normal hydrogen electrode. MADH reduced azurin in the presence of methylamine, indicating that this cupredoxin is likely to be the physiological electron acceptor for MADH in the electron transport chain of the methylotroph. A scheme of electron transport functioning in M. flagellatus KТ during methylamine oxidation is proposed.

Expressed genome of Methylobacillus flagellatus as defined through comprehensive proteomics and new insights into methylotrophy.

In recent years, techniques have been developed and perfected for high-throughput identification of proteins and their accurate partial sequencing by shotgun nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS), making it feasible to assess global protein expression profiles in organisms with sequenced genomes. We implemented comprehensive proteomics to assess the expressed portion of the genome of Methylobacillus flagellatus during methylotrophic growth. We detected a total of 1,671 proteins (64% of the inferred proteome), including all the predicted essential proteins. Nonrandom patterns observed with the nondetectable proteins appeared to correspond to silent genomic islands, as inferred through functional profiling and genome localization. The protein contents in methylamine- and methanol-grown cells showed a significant overlap, confirming the commonality of methylotrophic metabolism downstream of the primary oxidation reactions. The new insights into methylotrophy include detection of proteins for the N-methylglutamate methylamine oxidation pathway that appears to be auxiliary and detection of two alternative enzymes for both the 6-phosphogluconate dehydrogenase reaction (GndA and GndB) and the formate dehydrogenase reaction (FDH1 and FDH4). Mutant analysis revealed that GndA and FDH4 are crucial for the organism's fitness, while GndB and FDH1 are auxiliary.

Crystal structure of an aldehyde oxidase from Methylobacillus sp. KY4400.

Hetero-trimeric aldehyde oxidases of bacterial origin, which use O2 to catalyse the oxidation of various aldehydes but not those of aromatic N-heterocycles, belong to the xanthine oxidase family. In the present study, the crystal structure of a recombinant aldehyde oxidase from Methylobacillus sp. KY4400 (Mb-AOX) was determined at 2.5 Å resolution. The structures of its subunits resemble those of the corresponding subunits or domains of other structurally characterised enzymes belonging to the family, and include a [4Fe-4 S] cluster in the medium subunit like that found in Escherichia coli periplasmic aldehyde oxidoreductase (EP-AOR). A funnel leading to the si-face of the isoalloxazine ring of FAD, which is narrower than those in mouse liver AOX3 and human AOX1, is also present and it is even narrower than that in EP-AOR. The environment surrounding the ring in Mb-AOX and EP-AOR is subtly different, which might account for their different abilities to use O2. A remarkable characteristic of the Mo catalytic centre in Mb-AOX is a tryptophan situated near the centre instead of the alanine present in other xanthine oxidase family members. The tryptophan residue together with other residue differences might play an important role in binding to aldehydes such as n-heptylaldehyde in Mb-AOX.

Characterization of an iron- and manganese-containing superoxide dismutase from Methylobacillus sp. strain SK1 DSM 8269.

A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.

Expression and purification of HtpX-like small heat shock integral membrane protease of an unknown organism related to Methylobacillus flagellatus.

The M48 conserved family of peptidases contains a single catalytic zinc ion tetrahedrally co-ordinated by two histidines within an HEXXH motif. The proteases of this class are generally toxic to the cell and thus difficult to express and purify. Here, we report the expression and purification of the small HtpX-like heat shock metalloprotease from an unknown organism related to the obligate methylotrophic anaerobic bacterium, Methylobacillus flagellatus. The protease was expressed in the Escherichia coli vector - pT7. Optimization of expression was done to increase the yield and solubility of the expressed protein. Improved refolding procedures from inclusion bodies of pT7 E. coli system were devised to get the protease in an active and stable form. The protease was purified to near homogeneity in its active form from the refolded proteins of the inclusion bodies by a two-step (cation exchange followed by gel filtration) high performance liquid chromatography (HPLC). The purified protease was active on zymography and casein hydrolysis assays. The activity of the protease was found to be optimum at pH 7.4 and at a temperature of 37 degrees C but significant activity was also retained at higher temperatures of 45-50 degrees C. Centrifugal fractionation showed that it is a membrane localized endopeptidase. The methods described here can serve as guidelines to express and purify other homologues of M48 family of proteases for functional and structural studies.

[Establishment of the screening method and isolation of PQQ producing strains].

Pyrroloquinoline quinone (PQQ) is a cofactor of some oxido-reductases with many important physiological effects and potential pharmaceutical applications. The glucose dehydrogenase of Escherichia coli, being a candidate for enzymic detection of PQQ, is known to be a quinoprotein which is obligately dependant on PQQ as cofactor. The gdh gene of E. coli was amplified and cloned into plasmid pET28a. The recombinant GDH was overexpressed in soluble form in E. coli BL21 (DE3). A bioassay method was established for determination of PQQ by the purified GDH. A screening model was set up for the enrichment of methylotrophic bacteria. Together with the above bioassay method, over 2000 soil samples were screened for the isolation of high-yielding PQQ producing strains. A methylotrophic strain, named MP606, was thus isolated. The PQQ production of MP606 is determined to be 113mg/L without conditional optimization and genetic breeding. The PQQ crystal was obtained from the culture supernatant which has been identified by HPLC, absorption spectra assay, and enzymatic analysis. The 16S rDNA of MP606 was amplified and sequenced. According to the comparison of 16S rDNA sequences, overall similarity value between strain MP606 and 12 typical methylotrophic bacteria is above 95% . The highest value is with two strains of Methylovorus, which reached at 99%.

Biodiversity of aerobic methylobacteria associated with the phyllosphere of the southern Moscow Oblast.

During the summer period (15­25°C), 34 strains of methylotrophic bacteria associated with different species of herbs, shrub, and trees in Pushchino (Moscow oblast, Russia) were isolated on the medium with methanol. Predominance of pink-colored Methylobacterium strains in the phyllosphere of many plants was confirmed by microscopy, enumeration of the colonies from grass leaves, and sequencing of the 16S rRNA genes. Colorless and yellow-pigmented methylotrophs belonged to the genera Methylophilus, Methylobacillus, Hansschlegelia, Methylopila, Xanthobacter, and Paracoccus. All isolates were able to synthesize plant hormones auxins from L-tryptophan (5−50 µg/mL) and are probably plant symbionts.

Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus.

The isocitrate dehydrogenase (MfIDH) with unique double coenzyme specificity from Methylobacillus flagellatus was purified and characterized, and its gene was cloned and overexpressed in E. coli as a fused protein. This enzyme is homodimeric,-with a subunit molecular mass of 45 kDa and a specific activity of 182 U mg -1 with NAD+ and 63 U mg -1 with NADP+. The MfIDH activity was dependent on divalent cations and Mn2+ enhanced the activity the most effectively. MfIDH exhibited a cofactor-dependent pH-activity profile. The optimum pH values were 8.5 (NAD+) and 6.0 (NADP+).The Km values for NAD+ and NADP+ were 113 µM and 184 µM respectively, while the Km values for DL-isocitrate were 9.0 µM (NAD+), 8.0 µM (NADP+). The MfIDH specificity (kcat/Km) was only 5-times higher for NAD+ than for NADP+. The purified MfIDH displayed maximal activity at 60°C. Heat-inactivation studies showed that the MfIDH was remarkably thermostable, retaining full activity at 50°C and losting ca. 50% of its activity after one hour of incubation at 75°C. The enzyme was insensitive to the presence of intermediate metabolites, with the exception of 2 mM ATP, which caused 50% inhibition of NADP+-linked activity. The indispensability of the N6 amino group of NAD(P)+ in its binding to MfIDH was demonstrated. MfIDH showed high sequence similarity with bacterial NAD(P)+-dependent type I isocitrate dehydrogenases (IDHs) rather than with eukaryotic NAD+-dependent IDHs. The unique double coenzyme specificity of MfIDH potentially resulted from the Lys340, Ile341 and Ala347 residues in the coenzyme-binding site of the enzyme. The discovery of a type I IDH with double coenzyme specificity elucidates the evolution of this subfamily IDHs and may provide fundamental information for engineering enzymes with desired properties.

A distinct holoenzyme organization for two-subunit pyruvate carboxylase.

Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the ß subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4ß4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2ß4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis.

Methylobacillus flagellatus KT contains a novel cbo-type cytochrome oxidase.

The o-type oxidase from the methanol-grown obligate methylotroph Methylobacillus flagellatus KT has been purified to homogeneity. The complex is composed of four subunits (57, 40, 35 and 30 kDa). It contains six haems (4C:1B:1O) and one copper atom per molecule. It is proposed that the haem O-Cu(B) binuclear centre and a low-spin haem B are located in subunit I (57 kDa), two haems C reside in the cytochrome c homodimer (35 kDa), two haems C belong to the dihaem cytochrome c (30 kDa). The presented data provide evidence that cytochrome cbo is a novel representative of the haem-copper oxidase superfamily.

Purification and some properties of a blue copper protein from Methylobacillus sp. strain SK1 DSM 8269.

A blue protein was purified from the Methylobacillus sp. strain SK1 that is grown on methanol in the presence of copper ion. This protein was found to be a monomer with a molecular weight of 13,500. The Isoelectric point of the protein was estimated to be 8.8. The spectrum of the protein that was treated with ferricyanide showed a broad peak around 620 nm, but that of the dithionite-treated protein revealed no peaks. It contained 0.83 mol of EDTA-stable copper per mol protein. Under air, the protein accelerated the inactivation of methanol dehydrogenase (MDH). The protein was reducible by phenazine methosulfate or by active MDH that was prepared from cells that were grown in the absence of added copper, but not by methanol, dichlorophenol indophenol, or inactive MDH that was prepared from cells that were grown in the presence of added copper. It was also reducible by active MDH in the presence of methanol. The absorption peak at 340 nm of the active MDH disappeared after the enzyme was treated with ferricyanide, hydrogen peroxide, or the purified blue protein. The inactive MDH also showed no peak at 340 nm. The 340-nm peak was not recovered after incubation of the inactive MDH and blue protein-treated active MDH with dithionite or methanol. The inactive MDH and blue protein-treated active MDH co-migrated with the active MDH preparation on nondenaturing polyacrylamide gel, and contained two non-identical subunits with molecular weights that were identical to those of the active MDH. The N-terminal amino acid sequence of the protein was Ala-Gly-Cys-Ser-Val-Asp-Val-Glu-Ala-Asn-Asp-Ala-Met-Gln-Phe. An analysis of the amino acid composition revealed that the protein contained no tryptophan. It contained three cysteines per mol protein. The blue protein in Methylobacillus sp. strain SK1 was produced only in the cells that were grown in the copper-supplemented medium.