Characteristics of Methylobacillus flagellatum KT mutants, deficient for phosphoglucoisomerase, an enzyme of the ribulose monophosphate cycle of obligate methylotrophic bacteria

The activities of the key enzymes of ribulose monophosphate cycle for formaldehyde oxidation and assimilation were tested in crude extracts from temperature sensitive mutants of obligatemethylotroph M. flagellatum KT. Two mutants deficient in phosphoglucoisomerase activity were identified during this screening. Phosphoglucoisomerase of T525 pgi-1 mutant was active both at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. Complete inactivation of the enzyme at 42 degrees C occurred in 2 h in vitro, while in vivo incubation at nonpermissive temperature for more than 10 h was required for the enzyme inactivation. Phosphoglucoisomerase activity of T566 pgi-2 was 5-fold lower as compared with the one from the parent strain incubated at 30 degrees C. The enzyme was inactivated in 2 min. in crude extract at nonpermissive temperature.     

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.     

Citrate synthase from the obligate methylotroph Methylobacillus flagellatum

Abstract Formation of the lesion in the Escherichia coli inner membrane caused by λ lysis protein S was examined by electron microscopy. We also show that macromolecules exceeding the size of the λ R transglycosylase can pass through the S-dependent hole and that assembly of the S-dependent hole is independent of the proportion of acidic phospholipids in the inner membrane and of components of the cellular transport machinery.     

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.     

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.     

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.     

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     

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.     

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.     

Development of gene transfer systems in Methylobacillus flagellatum KT: Isolation of auxotrophic mutants

A collection of polyauxotrophic mutants of the obligate methylotroph Methylobacillus flagellatum KT was obtained. On the first step two stable auxotrophic mutants with a high requirement for amino acids supplements were isolated by treatment with nitrosoguanidine and selection on complete medium. Spontaneous variants of these mutants with a low requirement for nutrient supplements were the base for obtaining polyauxotrophic strains. It was shown, that the growth of mutants of M. flagellatum KT is inhibited by complete medium. Some amino acids and nucleotides are the inhibitor components of complete media. An approach for selection of auxotrophic mutants of individual genes was worked out on minimal medium. The optimal conditions for nitrosoguanidine mutagenesis of M. flagellatum KT were developed. The possible mechanisms of action of some of the nutrient supplements on the growth of M. flagellatum KT are discussed.     

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.     

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, sequencing, and mutation of a gene for azurin in Methylobacillus flagellatum KT.

The gene cluster for methylamine utilization (mau genes) has been cloned from the obligate methylotrophic bacterium Methylobacillus flagellatum KT. Partial sequence data showed that the organization of these genes was similar to that found in Methylophilus methylotrophus W3A1-NS, including the lack of a gene for amicyanin, which had been thought to be the electron acceptor for methylamine dehydrogenase in M. flagellatum KT. However, a gene encoding azurin was discovered at the 3' end of the mau gene cluster, transcribed in the opposite orientation. A mutant with a defect in this gene showed impaired growth on methylamine, suggesting that azurin is involved in methylamine oxidation in M. flagellatum KT.     

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.     

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     

Identification and characterization of the pqqDGC gene cluster involved in pyrroloquinoline quinone production in an obligate methylotroph Methylobacillus flagellatum

Abstract Pyrroloquinoline quinone is a prosthetic group of bacterial methanol dehydrogenases as well as some alcohol and glucose dehydrogenases. Genes involved in pyrroloquinoline quinone production have previously been cloned from the representatives of the α and γ subdivisions of the Proteobacteria. We report identification and the sequence of the pqqDGC gene cluster in the obligate methylotroph, Methylobacillus flagellatum , which belongs to the β subdivision. The deduced products of the pqq genes from M. flagellatum appear to be more similar to their counterparts from non-methylotrophic species of the γ subdivision than to a facultative methylotroph of the a subdivision. A non-polar mutation in pqqG was constructed and resulted in a strain impaired in growth on methanol. This mutant accumulated a detectable amount of intracellular pyrroloquinoline quinone, but in contrast to the wild type, did not excrete pyrroloquinoline quinone into the culture medium. The possible role of PqqG is discussed.     

The archaeon Pyrococcus horikoshii possesses a bifunctional enzyme for formaldehyde fixation via the ribulose monophosphate pathway

Pyrococcus horikoshii OT3, a hyperthermophilic and anaerobic archaeon, was found to have an open reading frame (PH1938) whose deduced amino acid sequence of the N-terminal and C-terminal halves showed significant similarity to two key enzymes of the ribulose monophosphate pathway for formaldehyde fixation in methylotrophic bacteria, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), respectively. The organism constitutively produced the encoded protein and exhibited activity of the sequential HPS- and PHI-mediated reactions in a particulate fraction. The full-length gene encoding the hybrid enzyme, the sequence corresponding to the HPS region, and the sequence corresponding to the PHI region were expressed in Escherichia coli and were found to produce active enzymes, rHps-Phi, rHps, or rPhi, respectively. Purified rHps-Phi and rHps were found to be active at the growth temperatures of the parent strain, but purified rPhi exhibited significant susceptibility to heat, suggesting that thermostability of the PHI moiety of the bifunctional enzyme (rHps-Phi) resulted from fusion with HPS. The bifunctional enzyme catalyzed the sequential reaction much more efficiently than a mixture of rHps and rPhi. These and other biochemical characterizations of the PH1938 gene product suggest that the ribulose monophosphate pathway plays a significant role in the archaeon under extreme environmental conditions.