l-threonine transport in the obligate methylotroph Methylobacillus flagellatum KT

Abstract The accumulation of l -threonine by the methylotrophic bacterium Methylobacillus flagellatum KT occurs via a specific system that is capable of transporting l -threonine against a 100-fold concentration gradient. This transport system demonstrates the following kinetic parameters: K _m= 0.2 mM and V _max= 2.5 nmol/min/mg of cells (dry weight). The activity of the system is inhibited by oxidative phosphorylation uncouplers and valinomycin. Cytoplasmic l -threonine does not leak from the cell, but bacteria are capable of exchanging exogenous l -threonine for its intracellular counterpart.     

Properties of glucose 6-phosphate and 6-phosphogluconate dehydrogenases of the obligate methylotroph Methylobacillus flagellatum KT

Abstract Extracts from the obligate methylotroph Methylobacillus flagellatum KT and its temperature-sensitive (ts) glucose 6-phosphate dehydrogenase (GPD) mutants were analysed by electrophoresis, isoelectrofocusing and chromatography methods. GPD is present in two forms differing in the isoelectric point (IEP) values, but identical in other properties. Both forms are specific to NAD and NADP, have similar affinity to substrates, exhibit equal levels of inhibition by NAD(P)H and ATP and have the same dependence of activity on temperature. The synthesis of both forms is controlled by one gene. 6-phosphogluconate dehydrogenase (GND) is represented by two proteins with different IEP values. One is specific both to NAD and NADP, is stable and inhibited by NADH and NADPH to a similar extent. The second is specific to NAD only, unstable and inhibited by NADH to a greater extent than by NADPH.     

Mapping of pgi and gpd genes involved in C-1 assimilation in the obligate methylotroph Methylobacillus flagellatum

Homologous matings with plasmids R68.45 and pULB113, and also with Hfr type donor were employed for mapping pgi and gpd genes involved in C-1 metabolism in the obligate methylotroph Methylobacillus flagellatum. A preliminary map of the late chromosomal region was constructed on the basis of these experimental results. The C-1 markers were linked to methionine and leucine auxotrophy and nalidixic acid resistance markers. The phenomenon of retrotransfer, or shuttle transfer of chromosomal markers by Inc P1 plasmids, revealed earlier, was demonstrated for M. flagellatum.     

Effect of formaldehyde on growth of obligate methylotroph Methylobacillus flagellatum in a substrate non-limited continuous culture

The ribulose monophosphate cycle methylotroph Methylobacillus flagellatum was grown under oxyturbidostat conditions on mixtures of methanol and formaldehyde. Formaldehyde when added at low concentration (50 mg/l) increased the methanol consumption and the yield of biomass. The presence of 150–300 mg/l of formaldehyde resulted in an increase of the growth rate from 0.74 to about 0.79–0.82 h^-1. The presence of 500 mg/l of formaldehyde in the inflow decreased culture growth characteristics. Activities of methanol dehydrogenase and enzymes participating in formaldehyde oxidation and assimilation were measured. The enzymological profiles obtained are discussed.Abbreviations MDH methanol dehydrogenase - NAD-linked FDDH NAD-linked formaldehyde dehydrogenase - DLFDDH dye-linked formaldehyde dehydrogenase - DLFDH dye-linked formate dehydrogenase - GPDH glucose-6-phosphate dehydrogenase - PGDH 6-phosphogluconate dehydrogenase - RuMP cycle ribulose monophosphate cycle     

Refined genetic map of the obligate methylotroph Methylobacillus flagellatum

We present a refined genetic map of the obligate methylotroph Methylobacillus flagellatum. New, Hfr (high-frequency-of-transfer) donors, and pulsed-field gel electrophoresis, were used to determine that M.␣flagellatum contains one ∼3.1-Mb circular chromosome, and no plasmids. A correlation between time-of-entry units and DNA length was established. Using in vivo and in vitro cloning, and sequencing, a number of new genetic markers were identified and mapped; in addition, the nature of some of the previously mapped markers was elucidated. Received: 11 August 1997 / Accepted: 11 December 1997     

Regulation of ammonia assimilation in an obligate methylotroph Methylobacillus flagellatum under steady-state and transient growth conditions

The obligate methylotroph Methylobacillus flagellatum was grown in the presence of different ammonium concentrations and the regulation of the enzymes associated with ammonium assimilation was investigated in steady-state and transient growth regimes. As the medium changed from C-limitation to dual C/N- and finally to N-limitation, the culture passed through three definite growth phases. The NADP⁺-dependent glutamate dehydrogenase (GDH) was present under ammonium limitation of the culture growth (at 2 mmol l^-1 of ammonium in the growth medium) and increased in response to an increase in nitrogen availability. Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were negligible during C- and C/N-limitation. In N-limited cells the GOGAT activity increased as the dilution rate increased up to 0.35 h^-1, and then sharply dropped. In the N-sufficient cultures both NAD⁺- and NADP⁺-dependent isocitrate dehydrogenase (NAD-ICDH and NADP-ICDH) activities were up-regulated as dilution rate increased, but in the N-limited culture the NAD-ICDH activity was up-regulated whereas NADP-ICDH one was down-regulated. Pulse additions of ammonium and methanol demonstrated the coordinate regulation of the GDH and ICDHs activities. When pulses were added to the C/N-limited cultures, there was an immediate utilization of the nutrients, resulting in an increase in biomass; at the same time the GDH and ICDH activities increased and the GS and GOGAT activities decreased. When the same ammonium/methanol pulse was added into the N-limited culture, there was a 3-hours delay in the culture response, after which the substrates were utilized at rates close to the ones shown by the C/N-limited culture after the analogous pulse.     

Cloning and expression of mosquitocidal endotoxin gene cryIVBfrom Bacillus thuringiensis var israelensis in the obligate methylotroph Methylobacillus flagellatum

The cryIVB gene from a new isolate of Bacillus thuringiensis var israelensis was cloned and sequenced. Two nucleotide replacements resulted in changing Asp³⁸⁵-Thr³⁸⁶ to Glu³⁸⁵-Ser³⁸⁶ were found in comparison with the previously sequenced cryIVB gene. Two genetic constructions were designed for expression of cryIVB in the obligate methylotroph Methylobacillus flagellatum. In the first construction, cryIVB was cloned under the strong inducible lac promoter and contained original ribosome binding site and 150 bp of 5′ transcribed but untranslated region. In the second construct, the first five codons of the lacZ gene were fused to the second codon of the cryIVB gene. Both E. coli and M. flagellatum harboring both constructs were toxic to insect larvae of Anopheles stephensi and Aedes aegypti. However, the toxicity of the methylotroph was about 450 times less. This study is the first attempt to use methylotrophs as an insecticidal endotoxin producer. Journal of Industrial Microbiology & Biotechnology (2000) 24, 14–18. Received 02 April 1999/ Accepted in revised form 17 August 1999     

Growth of the obligate methylotroph Methylobacillus flagellatum under stationary and nonstationary conditions during continuous cultivation

The growth characteristics of a chemostat culture of the obligate methylotrophic bacterium Methylobacillus flagellatum have been determined. Steady-state cultures growing at a rate of 0.73-0.74 h(-1), equal to the maximal growth rate, were obtained under oxyturbidostat cultivation conditions. The response of a chemostat culture to a pulse increase of methanol concentration was studied. It was shown that slow and rapidly growing cultures of M. flagellatum responded differently to pulse methanol addition. The growth characteristics of slow-growing cultures decreased after methanol addition compared to those of stationary chemostat cultures. The growth characteristics of rapidly growing cultures were practically unchanged with and without pulse methanol addition.     

Expression of the human interferon alpha F gene in the obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida

The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter. A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E. coli trp-operon intercystronic region. Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l. This plasmid was cloned in broad-host-range vector plasmid pAYC31. The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6. Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E. coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host.     

Complementation of Methylobacterium organophilum mutants affected in pyrroloquinoline quinone biosynthesis genes pqqE and pqqF by cloned Escherichia coli chromosomal DNA

The hybrid plasmid pBGT3, a derivative of pLA2917 containing a 7.8-kb fragment of Escherichia coli DNA, was found to complement pqqE and pqqF mutants of Methylobacterium organophilum, both impaired in PQQ biosynthesis. The cloned fragment of E. coli DNA did not hybridize with DNA fragments containing pqqE or pqqF previously cloned from M. organophilum. Yet, in M. organophilum mutants, expression of pqqE and pqqF genes from E. coli resulted in a PQQ production estimated at 9-16% of the production observed in M. organophilum wild-type. The growth rate in methanol medium of the complemented M. organophilum mutants was about 60% of that of the wild-type.     

Regulation of the activity and synthesis of 3-deoxy-D-arabinoheptuloso-7-phosphate synthase in the obligate methylotroph Methylobacillus flagellatum KT]

The activity of the first enzyme of aromatic path 3-deoxy-D-arabino-heptuloso-7-phosphate-synthase (DAHP-synthase) is regulated by retro-inhibition and is a subject of repression. Analysis of partially purified preparations of the enzyme has revealed three isoenzymes: DAHP-synthase-Tyr, DAHP-synthase-Trp and DAHP-synthase-Phe, each of them being regulated by a corresponding amino acid. DAHP-synthase-Phe is a dominant isoenzyme presenting 70% of the enzyme activity, 30% inhibition of which is possible by 7.0 mkM of phenylalanine. DAHP-synthase-Tyr and DAHP-synthase-Trp are minor isoenzymes (sharing 15% of enzyme activity each) and are controlled by tyrosine and tryptophane correspondingly. 50% of inhibition of activity is possible by adding 0.7 and 0.8 mkM of corresponding amino acid. Regulation of the enzyme synthesis was studied in the Trp-, Phe- and Tyr- mutants. The enzyme activity was registered under the conditions of limiting and surplus of each aromatic amino acid. The synthesis of DAHP-synthase in M. flagellatum KT is repressed by tryptophane and tyrosine decreasing the synthesis 18.8 and 15.6 fold.     

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.     

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.     

Genetic mapping of the obligate methylotroph Methylobacillus flagellatum: characteristics of prime plasmids and mapping of the chromosome in time-of-entry units.

A pULB113 (RP4::mini-Mu cts) plasmid was used to generate a library of prime plasmids carrying fragments of the Methylobacillus flagellatum genome. The genes carried by these prime plasmids were identified by complementation after transfer to suitably marked Escherichia coli and Pseudomonas aeruginosa strains. The hybrid plasmids were used for complementation mapping with a range of E. coli, M. flagellatum, and P. aeruginosa mutants. A preliminary map of the M. flagellatum genome section with seven groups of linked markers was obtained. Three of seven groups contain an overlapping sequence of cloned genes and can be considered as one large group of linked genes. A high-frequency-of-recombination donor of M. flagellatum (strain MFK64) mobilized the chromosome in a polarized manner from a single transfer origin. The donor was used to construct a time-of-entry map of the M. flagellatum chromosome. This was achieved by determining the time of entry of six randomly dispersed markers, four of which are included in known groups of linked markers. The linear map of M. flagellatum reported here consists of 44 markers.     

Construction and characterization of a linked-dimeric pyrroloquinoline quinone glucose dehydrogenase

Using the structural gene of a homo dimeric enzyme, the water-soluble pyrroloquinoline quinone glucose dehydrogenase (PQQGDH-B), a gene consisting of two identical subunits linked together by a DNA segment coding linker peptide region was constructed. Using the constructed gene, a linked-dimeric PQQGDH-B was produced in Escherichia coli as the active soluble enzyme. Linked-dimeric PQQGDH-B showed a larger increase in thermal stability than the native dimeric enzyme. During incubation over 45 °C, the residual activity of linked-dimeric PQQGDH-B was more than twice that of the native dimeric enzyme. The potential application of linked-dimeric PQQGDH-B for glucose enzyme sensor is also discussed.     

吡咯喹啉醌合成及生产工艺研究进展

吡咯喹啉醌(pyrroloquinoline quinone, PQQ)是继烟酰胺和核黄素之后发现的第三类氧化还原酶辅因子,普遍存在于生物体中参与呼吸链电子传递,具有促进线粒体产生、清除自由基、增强细胞代谢和预防心肌损伤等生理功能,在医药、食品和农业领域具有广泛的应用前景。微生物发酵法是PQQ生产的主要方式,解析PQQ生物合成途径及其调控机制,通过代谢工程选育短周期、高产量的生产菌是PQQ工业化的研究方向之一。本文综述了PQQ的合成途径、高产菌株选育以及微生物发酵生产与分离纯化的研发工作,为深入阐释PQQ的生物合成机制和工业化生产菌株的选育提供参考。     

Oxidative and assimilative enzyme activities in continuous cultures of the obligate methylotrophMethylobacillus flagellatum

In methanol-limited continuous cultures of the obligate methylotrophic bacteriumMethylobacillus flagellatum grown at rates from 0.05 to 0.63 h^-1, and also in an oxyturbidostat culture ofM. flagellatum growing at the rate of 0.73 h^-1, levels of methanol dehydrogenase, enzymes of formaldehyde oxidation (both linear and cyclic) and assimilation (RuMP cycle), a number of intermediary metabolism and TCA cycle enzymes and also dye-linked formaldehyde dehydrogenase were determined. It was shown that the activities of dissimilatory enzymes, with the exception of dye-linked formaldehyde dehydrogenase, decreased with increasing growth rate. Activities of assimilative enzymes and activities of the TCA cycle enzymes detected as well as the dye-linked formaldehyde dehydrogenase activity, increased with increasing growth rate. A periplasmic location was shown for the latter enzyme and a role in formaldehyde detoxification was proposed.     

Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.     

Hepatoprotective effect of pyrroloquinoline quinone against alcoholic liver injury through activating Nrf2-mediated antioxidant and inhibiting TLR4-mediated inflammation responses

The present study was aimed at investigating the hepatoprotective effect of pyrroloquinoline quinone (PQQ) against acute alcoholic liver injury in mice. Acute alcoholic liver injury model was established in mice, and they were administrated with PQQ to investigate its hepatoprotective effect. Our results shows that PQQ can significantly ameliorate acute alcoholic liver injury by decreasing the hepatic marker enzymes, including serum alanine transaminase (ALT) and aspartate transaminase (AST), and increasing the levels of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in the liver. And PQQ can also significantly reduce the content of hepatic triglyceride (TG) and malondialdehyde (MDA). Moreover, PQQ attenuated alcohol-induced oxidative damage by activating NF-E2-related factor 2 (Nrf2)-mediated signaling pathway, and inhibiting Toll-like receptor 4 (TLR4)-mediated nuclear factor-kappa B (NF-κB) signaling pathway. Our findings have elucidated the liver protection mechanism of PQQ, which would encourage the further exploitation of PQQ as a hepatoprotective functional food.