n-Amyl alcohol as a substrate for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacteria

Abstract n -Amyl alcohol was examined as a source for the synthesis of the 3-hydroxyvalerate (3HV) unit of the biopolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), by Alcaligenes sp., Pseudomonas sp. and several methylotrophic bacteria. A. eutrophus and Ps. lemoignei synthesized P(3HB-co-3HV) from glucose and n -amyl alcohol under nitrogen-deficient conditions. Many of methylotrophic bacteria grown on methanol synthesized the copolyester from methanol and n -amyl alcohol under nitrogen-deficient conditions. The content and composition of the polyester varied from strain to strain. Paracoccus denitrificans differed from all others in having a higher content of 3-hydroxyvalerate units in the copolyester synthesized.     

Trimethylamine metabolism in obligate and facultative methylotrophs

1. Twelve bacterial isolates that grow with trimethylamine as sole source of carbon and energy were obtained in pure culture. All the isolates grow on methylamine, dimethylamine and trimethylamine. One isolate, bacterium 4B6, grows only on these methylamines whereas another isolate, bacterium C2A1, also grows on methanol but neither grows on methane; these two organisms are obligate methylotrophs. The other ten isolates grow on a variety of C(i) and other organic compounds and are therefore facultative methylotrophs. 2. Washed suspensions of the obligate methylotrophs bacteria 4B6 and C2A1, and of the facultative methylotrophs bacterium 5B1 and Pseudomonas 3A2, all grown on trimethylamine, oxidize trimethylamine, dimethylamine, formaldehyde and formate; only bacterium 5B1 and Ps. 3A2 oxidize trimethylamine N-oxide; only bacterium 4B6 does not oxidize methylamine. 3. Cell-free extracts of trimethylamine-grown bacteria 4B6 and C2A1 contain a trimethylamine dehydrogenase that requires phenazine methosulphate as primary hydrogen acceptor, and evidence is presented that this enzyme is important for the growth of bacterium 4B6 on trimethylamine. 4. Cell-free extracts of eight facultative methylotrophs, including bacterium 5B1 and Ps. 3A2, do not contain trimethylamine dehydrogenase but contain instead a trimethylamine monooxygenase and trimethylamine N-oxide demethylase. It is concluded that two different pathways for the oxidation of trimethylamine occur amongst the isolates.     

Genetics of carbon metabolism in methylotrophic bacteria

Abstract The application of genetic techniques to the methylotrophic bacteria has greatly enhanced studies of these important organisms. Two methylotrophic systems have been studied in some detail, the serine cycle for formaldehyde assimilation and the methanol oxidation system. In both cases, genes have been cloned and mapped in Methylobacterium species (facultative serine cycle methanol-utilizers). In addition, methanol oxidation genes have been studied in an autotrophic methanol-utilizer ( Paracoccus denitrificans ) and three methanotrophs ( Methylosporovibrio methanica, Methylomonas albus and Methylomonas sp. A4). Although much remains to be learned in these systems, it is becoming clear that the order of C₁ genes has been conserved to some extent in methylotrophic bacteria, and that many C₁ genes are loosely clustered on the chromosome. Operons appear to be rare, but some examples have been observed. The extension of genetic approaches to both the obligate and facultative methylotrophs holds much promise for the future in understanding and manipulating the activities of these bacteria.     

Microbial oxidation of gaseous hydrocarbons: epoxidation of C2 to C4 n-alkenes by methylotrophic bacteria.

Over 20 new cultures of methane-utilizing microbes, including obligate (types I and III) and facultative methylotrophic bacteria were isolated. In addition to their ability to oxidize methane to methanol, resting cell-suspensions of three distinct types of methane-grown bacteria (Methylosinus trichosporium OB3b [type II, obligate]; Methylococcus capsulatus CRL M1 NRRL B-11219 [type I, obligate]; and Methylobacterium organophilum CRL-26 NRRL B-11222 [facultative]) oxidize C2 to C4 n-alkenes to their corresponding 1,2-epoxides. The product 1,2-epoxides are not further metabolized and accumulate extracellularly. Methanol-grown cells do not have either the epoxidation or the hydroxylation activities. Among the substrate gaseous alkenes, propylene is oxidized at the highest rate. Methane inhibits the epoxidation of propylene. The stoichiometry of the consumption of propylene and oxygen and the production of propylene oxide is 1:1:1. The optimal conditions for in vivo epoxidation are described. Results from inhibition studies indicate that the same monooxygenase system catalyzes both the hydroxylation and the epoxidation reactions. Both the hydroxylation and epoxidation activities are located in the cell-free particulate fraction precipitated between 10,000 and 40,000 x g centrifugation.     

Microbial oxidation of gaseous hydrocarbons: epoxidation of C2 to C4 n-alkenes by methylotrophic bacteria.

Over 20 new cultures of methane-utilizing microbes, including obligate (types I and III) and facultative methylotrophic bacteria were isolated. In addition to their ability to oxidize methane to methanol, resting cell-suspensions of three distinct types of methane-grown bacteria (Methylosinus trichosporium OB3b [type II, obligate]; Methylococcus capsulatus CRL M1 NRRL B-11219 [type I, obligate]; and Methylobacterium organophilum CRL-26 NRRL B-11222 [facultative]) oxidize C2 to C4 n-alkenes to their corresponding 1,2-epoxides. The product 1,2-epoxides are not further metabolized and accumulate extracellularly. Methanol-grown cells do not have either the epoxidation or the hydroxylation activities. Among the substrate gaseous alkenes, propylene is oxidized at the highest rate. Methane inhibits the epoxidation of propylene. The stoichiometry of the consumption of propylene and oxygen and the production of propylene oxide is 1:1:1. The optimal conditions for in vivo epoxidation are described. Results from inhibition studies indicate that the same monooxygenase system catalyzes both the hydroxylation and the epoxidation reactions. Both the hydroxylation and epoxidation activities are located in the cell-free particulate fraction precipitated between 10,000 and 40,000 x g centrifugation.     

Methanol metabolism in thermotolerant methylotrophic Bacillus species

Abstract For a number of years we have tried to isolate versatile methylotrophic bacteria employing the ribulose monophosphate (RuMP) cycle of formaldehyde fixation. Recently this has resulted in the development of techniques for the selective enrichment and isolation in pure culture of Bacillus strains able to grow in methanol mineral medium over a temperature range between 35 and 60°C. At the optimum growth temperatures (50–55°C), these isolates display doubling times between 40 and 80 min. The metabolism of the strains studied is strictly respiratory. Methanol assimilation is exclusively via the RuMP cycle variants with the fructose bisphosphate (FBP) aldolase cleavage and transketolase (TK)/transaldolase (TA) rearrangement. Whole cells were unable to oxidize formate, and no activities of NAD-(in)dependent formaldehyde and formate dehydrogenases were detected. Formaldehyde oxidation most likely proceeds via the so-called dissimilatory RuMP cycle. The initial oxidation of methanol is catalyzed by an NAD-dependent methanol dehydrogenase present as an abundant protein in all strains. The enzyme from Bacillus sp. C1 has been purified and characterized.     

Nature, nomenclature and taxonomy of obligate methanol utilizing strains

In a screening program, a number of different bacterial strains with the ability to utilize methanol as a sole carbon and energy source were isolated and described. They are well known methanol utilizing genera Pseudomonas, Klebsiella, Micrococcus, Methylomonas or, on the contrary, the new, unknown genera and species of methylotrophic bacteria. In the last category, Acinetobacter and Alcaligenes are the new reported genera of organisms able to use methanol as a sole carbon and energy source. The present paper reports the very complex physiological and biochemical modifications when very versatile bacteria such as Pseudomonas aeruginosa and Acinetobacter calcoaceticus are cultured on methanol and when the obligate methylotrophic state is compared with the facultative methylotrophic state of the same bacterial strain. Based on experiments and comparisons with literature data, it seems that Methylomonas methanica is the obligate methylotrophic state of Pseudomonas aeruginosa and that Acinetobacter calcoaceticus is the facultative methylotrophic state of Methylococcus capsulatus, an obligate methylotroph. The relationship of the obligate to the facultative and of the facultative to the obligate methylotrophy were established. These new methylotrophic genera and species, the profound physiological and biochemical modifications as well as the new data concerning nature, nomenclature and taxonomy of methanol utilizing bateria were reported for the first time in 1983.     

Coping with formaldehyde during C1 metabolism of Paracoccus denitrificans

Methylotrophic bacteria are capable of growth using reduced one-carbon (C1) compounds like methanol or methylamine as free energy sources. Paracoccus denitrificans, which is a facultative methylotrophic organism, switches to this type of autotrophic metabolism only when it experiences a shortage of available heterotrophic free energy sources. Since the oxidation of C1 substrates is energetically less favourable than that of the heterotrophic ones, a global regulatory circuit ensures that the enzymes involved in methylotrophic growth are repressed during heterotrophic growth. Once the decision is made to switch to methylotrophic growth, additional regulatory proteins ensure the fine-tuned expression of the participating enzymes such that the steady-state concentration of formaldehyde, the oxidation product of C1 substrates, is kept below cytotoxic levels.     

Methylotrophic autotrophy in Beijerinckia mobilis

Representatives of the genus Beijerinckia are known as heterotrophic, dinitrogen-fixing bacteria which utilize a wide range of multicarbon compounds. Here we show that at least one of the currently known species of this genus, i.e., Beijerinckia mobilis, is also capable of methylotrophic metabolism coupled with the ribulose bisphosphate (RuBP) pathway of C1 assimilation. A complete suite of dehydrogenases commonly involved in the sequential oxidation of methanol via formaldehyde and formate to CO2 was detected in cell extracts of B. mobilis grown on CH3OH. Carbon dioxide produced by oxidation of methanol was further assimilated via the RuBP pathway as evidenced by reasonably high activities of phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Detection and partial sequence analysis of genes encoding the large subunits of methanol dehydrogenase (mxaF) and form I RubisCO (cbbL) provided genotypic evidence for methylotrophic autotrophy in B. mobilis.     

Identification of putative methanol dehydrogenase (moxF) structural genes in methylotrophs and cloning of moxF genes from Methylococcus capsulatus bath and Methylomonas albus BG8.

An open-reading-frame fragment of a Methylobacterium sp. strain AM1 gene (moxF) encoding a portion of the methanol dehydrogenase structural protein has been used as a hybridization probe to detect similar sequences in a variety of methylotrophic bacteria. This hybridization was used to isolate clones containing putative moxF genes from two obligate methanotrophic bacteria, Methylococcus capsulatus Bath and Methylomonas albus BG8. The identity of these genes was confirmed in two ways. A T7 expression vector was used to produce methanol dehydrogenase protein in Escherichia coli from the cloned genes, and in each case the protein was identified by immunoblotting with antiserum against the Methylomonas albus methanol dehydrogenase. In addition, a moxF mutant of Methylobacterium strain AM1 was complemented to a methanol-positive phenotype that partially restored methanol dehydrogenase activity, using broad-host-range plasmids containing the moxF genes from each methanotroph. The partial complementation of a moxF mutant in a facultative serine pathway methanol utilizer by moxF genes from type I and type X obligate methane utilizers suggests broad functional conservation of the methanol oxidation system among gram-negative methylotrophs.     

Respiratory resistance of methylotrophic bacteria to formate and cyanide

Whole cells and cell-free preparations of the methylotrophic bacteria, Pseudomonas sp. AM 1 and Achromobacter parvulus, can oxidize formate at tis concentration in the reaction medium up to 1 M. The respiration of whole cells is registered at a concentration of formate greater than 10(-2) M, while that of cell-free extracts at a formate concentration greater than 5 X 10(-5) M. This seems to be due to the presence of a permeability barrier in cells for formate. The oxidation of reduced TMPD and exogenous cytochrome c by the membrane preparations of the two bacteria is inhibited by formate and cyanide; Ki50% = 2.5 X 10(-2) and 10(-6) M, respectively. The oxidation of NADH by the membrane preparations of the bacteria is not inhibited by 1 M formate and 5 X 10(-4) M cyanide but is inhibited by formaldehyde with Ki50% = 3 X 10(-2) M. Formaldehyde has no effect on the oxidation of reduced TMPD and cytochrome c at concentrations greater than 2 X 10(-1) M. These data indicate that respiration of the studied methylotrophic bacteria in the presence of high formate concentrations should be attributed in the presence of a branched electron transport chain in them; one branch of the chain is resistant to formate and cyanide, but is sensitive to formaldehyde.     

An investigation of biooxidation ability of Acidithiobacillus ferrooxidans using NMR relaxation measurement

NMR relaxation measurements can provide a simple means for understanding biological activity of cells in solution with known composition. It has the advantage that it is an in situ, non-intrusive technique, and the acquisition is fast. The iron oxidation ability of Acidithiobacillus ferrooxidans was investigated using NMR relaxation measurements. The transversal relaxation is characterized by a time constant, T?, which is sensitive to the chemical environment. Fe3? ion has more significant T? shortening than Fe2? ion. In the presence of A. ferrooxidans in solutions containing Fe2? ion, T? shortening was found with increasing time as the bacteria oxidize Fe2? to Fe3? ions. In the optimal growth medium, the bacteria concentration increased 80 times and high iron oxidation rate was found. In 10 mM K?SO? medium, however, bacteria concentration remained almost unchanged and the iron oxidation rate was significantly lower.     

New unified nomenclature for genes involved in the oxidation of methanol in Gram-negative bacteria

Abstract The system involving the oxidation of methanol to formaldehyde in Gram-negative methylotrophic bacteria is complex. A total of 32 genes have been reported, termed mox , for methanol oxidation, and it is possible that more will be identified. Some mox genes carrying out completely different functions have been given the same designations by different laboratories and others have been given separate designations that were later discovered to be the same. It is now important to change the mox nomenclature to remedy this confusing situation. This communication proposes a new nomenclature for genes involved in methanol oxidation based on currently known linkage groups.     

Distribution of dissimilatory enzymes in methane and methanol oxidizing bacteria

Methanol oxidation was studied in several RuMP and serine type methylotrophic bacteria. On the basis of the distribution of the dissimilatory enzymes and the electrophoretic mobility of the methanol dehydrogenases, the methanol and methane oxidizers of the RuMP type belong to two different taxonomic groups. The pink pigmented facultative serine type methylotrophs represent another taxon.     

Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol

Formaldehyde is toxic for all organisms from bacteria to humans due to its reactivity with biological macromolecules. Organisms that grow aerobically on single-carbon compounds such as methanol and methane face a special challenge in this regard because formaldehyde is a central metabolic intermediate during methylotrophic growth. In the alpha-proteobacterium Methylobacterium extorquens AM1, we found a previously unknown enzyme that efficiently catalyzes the removal of formaldehyde: it catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin, a reaction which also proceeds spontaneously, but at a lower rate than that of the enzyme-catalyzed reaction. Formaldehyde-activating enzyme (Fae) was purified from M. extorquens AM1 and found to be one of the major proteins in the cytoplasm. The encoding gene is located within a cluster of genes for enzymes involved in the further oxidation of methylene tetrahydromethanopterin to CO(2). Mutants of M. extorquens AM1 defective in Fae were able to grow on succinate but not on methanol and were much more sensitive toward methanol and formaldehyde. Uncharacterized orthologs to this enzyme are predicted to be encoded by uncharacterized genes from archaea, indicating that this type of enzyme occurs outside the methylotrophic bacteria.     

XoxF is required for expression of methanol dehydrogenase in Methylobacterium extorquens AM1

In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins, which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ～50% identical to MxaF and ～90% identical to each other. It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM, which are required for the expression of the methanol dehydrogenase genes.     

Consumption of tropospheric levels of methyl bromide by C(1) compound-utilizing bacteria and comparison to saturation kinetics.

Pure cultures of methylotrophs and methanotrophs are known to oxidize methyl bromide (MeBr); however, their ability to oxidize tropospheric concentrations (parts per trillion by volume [pptv]) has not been tested. Methylotrophs and methanotrophs were able to consume MeBr provided at levels that mimicked the tropospheric mixing ratio of MeBr (12 pptv) at equilibrium with surface waters ( approximately 2 pM). Kinetic investigations using picomolar concentrations of MeBr in a continuously stirred tank reactor (CSTR) were performed using strain IMB-1 and Leisingeria methylohalidivorans strain MB2(T) - terrestrial and marine methylotrophs capable of halorespiration. First-order uptake of MeBr with no indication of threshold was observed for both strains. Strain MB2(T) displayed saturation kinetics in batch experiments using micromolar MeBr concentrations, with an apparent K(s) of 2.4 microM MeBr and a V(max) of 1.6 nmol h(-1) (10(6) cells)(-1). Apparent first-order degradation rate constants measured with the CSTR were consistent with kinetic parameters determined in batch experiments, which used 35- to 1 x 10(7)-fold-higher MeBr concentrations. Ruegeria algicola (a phylogenetic relative of strain MB2(T)), the common heterotrophs Escherichia coli and Bacillus pumilus, and a toluene oxidizer, Pseudomonas mendocina KR1, were also tested. These bacteria showed no significant consumption of 12 pptv MeBr; thus, the ability to consume ambient mixing ratios of MeBr was limited to C(1) compound-oxidizing bacteria in this study. Aerobic C(1) bacteria may provide model organisms for the biological oxidation of tropospheric MeBr in soils and waters.     

Reactions of direct formaldehyde oxidation to CO2 are non-essential for energy supply of yeast methylotrophic growth

Mutants of the methylotrophic yeast Hansenula polymorpha deficient in NAD-dependent formaldehyde or formate dehydrogenases have been isolated. They were more sensitive for exogenous methanol but retained the ability for methylotrophic growth. In the medium with methanol the growth yields of the mutant 356–83 deficient in formaldehyde dehydrogenase and of the wild-type strain were identical (0.34 g cells/g methanol) under chemostat cultivation. These results indicate that enzymes of direct formaldehyde oxidation are not indispensable for methylotrophic growth. At the same time inhibition of tricarboxylic acid cycle has resulted in suppression of growth in the media with multicarbon nonfermentable substrates such as glycerol, succinate, ethanol and dihydroxyacetone as well as with methanol, but not with glucose. In the experiments with the wild-type strain H. polymorpha it has been shown that citrate and dihydroxyacetone inhibit the radioactivity incorporation from ¹⁴C-methanol into CO₂. All obtained data indicate that for the dissimilation of methanol and the supplying of energy for methylotrophic growth, the functioning of tricarboxylic acid cycle reactions as oppossed to those of direct formaldehyde oxidation is essential.     

Efficient bioconversion of ethanol to acetaldehyde using a novel mutant strain of the methylotrophic yeast Hansenula polymorpha

We report the isolation of mutant strains of the methylotrophic yeast Hansenula polymorpha that are able to efficiently oxidize ethanol to acetaldehyde in an intact cell system. The oxidation reaction is catalyzed by alcohol oxidase (AOX), a key enzyme in the methanol metabolic pathway that is typically present only in H. polymorpha cells growing on methanol. At least three mutations were introduced in the strains. Two of the mutations resulted in high levels of AOX in glucose-grown cells of the yeast. The third mutation introduced a defect in the cell's normal ability to degrade AOX in response to ethanol, and thus stabilizing the enzyme in the presence of this substrate. Using these strains, conditions for bioconversion of ethanol to acetaldehyde were examined. In addition to pH and buffer concentration, we found that the yield of acetaldehyde was improved by the addition of the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) and by permeabilization of the cells with digitonin. Under optimal shake-flask conditions using one of the H. polymorpha mutant strains, conversion of ethanol to acetaldehyde was nearly quantitative.     

Molecular detection and isolation from antarctica of methylotrophic bacteria able to grow with methylated sulfur compounds

This study is the first demonstration that a diverse facultatively methylotrophic microbiota exists in some Antarctic locations. PCR amplification of genes diagnostic for methylotrophs was carried out with bacterial DNA isolated from 14 soil and sediment samples from ten locations on Signy Island, South Orkney Islands, Antarctica. Genes encoding the mxaF of methanol dehydrogenase, the fdxA for Afipia ferredoxin, the msmA of methanesulfonate monooxygenase, and the 16S rRNA gene of Methylobacterium were detected in all samples tested. The mxaF gene sequences corresponded to those of Hyphomicrobium, Methylobacterium, and Methylomonas. Over 30 pure cultures of methylotrophs were isolated on methanesulfonate, dimethylsulfone, or dimethylsulfide from ten Signy Island lakes. Some were identified from 16S rRNA gene sequences (and morphology) as Hyphomicrobium species, strains of Afipia felis, and a methylotrophic Flavobacterium strain. Antarctic environments thus contain diverse methylotrophic bacteria, growing on various C1-substrates, including C1-sulfur compounds.