Formaldehyde incorporation by a new methylotroph (L3).

A number of bacterial strains have been isolated and investigated in our search for a promising organism in the production of single-cell protein from methanol. Strain L3 among these isolates was identified as an obligate methylotroph which grew only on methanol and formaldehyde as the sole sources of carbon and energy. The organism also grew well in batch and chemostat mixed-substrate cultures containing methanol, formaldehyde, and formate. Although formate was not utilized as a sole carbon and energy source, it was readily taken up and oxidized by either formaldehyde- or methanol-grown cells. The organism incorporated carbon by means of the ribulose monophosphate pathway when growing on either methanol, formaldehyde, or various mixtures of C1 compounds. Its C1-oxidation enzymes included phenazine methosulfate-linked methanol and formaldehyde dehydrogenase and a nicotinamide adenine dinucleotide-linked formate dehydrogenase. Identical inhibition by formaldehyde of the first two dehydrogenases suggested that they are actually the same enzyme. The organism had a rapid growth rate, a high cell yield in the chemostat, a high protein content, and a favorable amino acid distribution for use as a source of single-cell protein. Of special interest was the ability of the organism to utilize formaldehyde via the ribulose monophosphate cycle.     

Characterization of a new marine methylotroph

Abstract A methanol-oxidizing bacterium from a marine environment has been isolated and characterized. The bacterium was a Gram-negative rod, capable of growth on methanol and methylamine, but not on multicarbon compounds. It showed a temperature optimum of 30°C, a salt optimum of 0.4% (w/v) and the mol % G + C of its DNA was 46%. Carbon was assimilated via the ribulose monophosphate pathway for formaldehyde fixation during growth on methanol. This bacterium superficially resembled other obligate methylotrophs requiring NaCl reported previously which were designated Methylomonas thalassica . It also appeared similar to many strains of obligate freshwater methylotrophs, except for its NaCl requirement and its lower mol % G + C.     

Growth dynamics of a methylotroph (Methylomonas L3) in continuous cultures. I. Fast transients induced by methanol pulses and methanol accumulation

The dynamic behavior of the Ribulose Monophosphate-type Methylomonas L3 in continuous cultures was studied, using methanol pulses to induce fast transients in steady-state cultures of single (methanol) and mixed (methanol plus formaldehyde) substrates. In several experiments, the methanol-uptake rate (MUR) profiles displayed negative MUR values for a time period following the methanol pulse, and significant amounts of methanol disappeared immediately following the pulse. These phenomena suggested the accumulation of methanol in the cells upon pulsing, apparently due to an active transport system. Accordingly, and in order to estimate the potential of the transport system for methanol accumulation, accumulation profiles were calculated for several pulse experiments. The calculations are based on a methanol balance and experimentally determined values of the cell volume and the true transient biomass yields. It is calculated that methanol accumulates up to 200-fold to very high intracellular concentrations. The accumulation is calculated to be much higher in single- (methanol) substrate cultures of low dilution rate than in cultures of high dilution rate or of mixed substrates. The specific growth rate immediately following the methanol pulse decreased in single-substrate cultures and increased in mixed-substrate ones. The biomass yield decreased after the methanol addition in all experiments; however, the drop was less severe in the mixed-substrate experiments. It is also suggested that formaldehyde as a methanol cosubstrate may be an effective means of providing more stable biomass yields and growth rates in reactors with imperfect mixing, and of protecting the reactor against accidentally induced methanol accumulation.     

Transformation of a new Gram-positive methylotroph,Brevibacterium methylicum,by plasmid DNA

Summary Brevibacterium methylicum is a newly isolated Gram-positive facultatively methylotrophic bacterium that uses the NAD⁺-dependent methanol dehydrogenase for methanol oxidation and assimilates its carbon via the ribulose monophosphate cycle. Protoplasts prepared by lysozyme treatment of B. methylicum cells grown in the presence of glycine were transformed by plasmid shuttle vectors pCEM500 (16.5 kb; Sm^r/Sp^r, Km^r/Gm^r) and pEC71 (7.1 kb; Km^r/Nm^r) constructed on the basis of B. lactofermentum plasmid pAM330 and replicating in Escherichia coli and in amino-acid-producing coryneform bacteria. The resistance markers were found to be expressed in B. methylicum and autonomous plasmid DNAs of various size were isolated from the transformants. The presence of the pAM330 replicon in these plasmids was demonstrated by DNA-DNA hybridization experiments. Offprint requests to: J. Nevera     

Growth dynamics of a methylotroph (Methylomonas L3) in continuous cultures. II. Growth inhibition and comparison against an unstructured model

High methanol concentrations have a negative effect on the growth rate and the biomass yield of growth transients induced by methanol pulses in continuous cultures of Methylomonas L3. The physiological basis of this effect is investigated by measuring the effect of the methanol pulse on the cell energy charge (EC) and ATP, ADP, and AMP concentrations, and by comparing the results of the pulse transients against an unstructured model. The methanol pulse is shown to lead to increased values of the cell EC and ATP concentration, and thus, inhibition and reduced availability of biosynthetic energy are excluded as causes of inhibition. When the biomass and methanol profiles of the transient experiments are compared in phase-plane diagrams against computer simulations based on the model, satisfactory agreement between experimental data and model predictions is found in single-substrate, high-dilution-rate experiments. Conversely, poor agreement between experimental data and simulation results indicates a more severe growth inhibition than the model predicts at low dilution rates and a less severe one in mixed-substrate experiments. Based on these findings and other relevant physiological information, we propose that the large variations in the negative effect of methanol on growth result from the fact that cells accumulate methanol to widely different concentrations depending on their physiological state. In their effort to detoxify from the high intracellular methanol and formaldehyde concentrations, cells oxidize considerably more methanol than they can incorporate into biomass. This leads to a useless ATP surplus, which the cells must hydrolyze without doing any useful biosynthetic work, and this results in lower biomass yields.     

(14C)acetate assimilation by a type I obligate methylotroph, Methylococcus capsulatus.

Methanol and formate oxidation supported the assimilation of [14C]acetate by cell suspensions of Methylococcus capsulatus; oxidation of other primary alcohols, except ethanol, did not. The extent of [1-14C]acetate assimilation supported by methanol oxidation was decreased in the presence of primary alcohols, except ethanol. Potassium cyanide (0.33 mM) completely inhibited the oxidation of formate and its stimulation of [1-14C]acetate assimilation. The amount of [1-14C]acetate assimilation supported by methanol oxidation was significantly inhibited by cyanide.     

(14C)acetate assimilation by a type I obligate methylotroph, Methylococcus capsulatus.

Methanol and formate oxidation supported the assimilation of [14C]acetate by cell suspensions of Methylococcus capsulatus; oxidation of other primary alcohols, except ethanol, did not. The extent of [1-14C]acetate assimilation supported by methanol oxidation was decreased in the presence of primary alcohols, except ethanol. Potassium cyanide (0.33 mM) completely inhibited the oxidation of formate and its stimulation of [1-14C]acetate assimilation. The amount of [1-14C]acetate assimilation supported by methanol oxidation was significantly inhibited by cyanide.     

l-Serine production by a methylotroph and its related enzymes

The production process of l-serine from methanol and glycine has been developed using a methylotroph with the serine pathway. Consecutive reactions of two enzymes, methanol dehydrogenase (MDH) and serine hydroxymethyltransferase (SHMT) are involved in the production. We screened a high producer, Hyphomicrobium methylovorum, which is an obligate methylotroph. With resting cells of the bacterium, 24 mg/ml of l-serine was produced from 100 mg/ml of glycine and 48 mg/ml of methanol in 3 days under optimal conditions. Next, a glycine-resistant mutant GM2 showed improved serine production (32–34 mg/ml). The mutant GM2 was found to have elevated activities of MDH and SHMT. Since there has so far been little report on the systematic characterization of enzymes of the serine pathway in methylotrophs, not only the above two enzymes but also the other three enzymes in H. methylovorum were purified and characterized: MDH, SHMT and hydroxypyruvate reductase (HPR) were crystallized; serine-glyoxylate aminotransferase (SGAT) and glycerate kinase (GK) were purified to homogeneity. As a result, all these enzymes were found to be stable against preservation and to exist abundantly in the bacterium. The gene of SHMT was cloned and its deduced amino acid sequence had homology to those of Escherichia coli (55%) and rabbit liver (44%), whereas the enzyme of the bacterium was immunochemically distinguishable from those of microorganisms other than Hyphomicrobium strains and mammalian livers. Correspondence to: Y. Izumi     

Quantifying 3H-thymidine incorporation rates by a phylogenetically defined group of marine planktonic bacteria (Bacteriodetes phylum)

The rate of [(3)H-methyl] thymidine ((3)H-TdR) incorporation into DNA has been applied extensively to measure cell production by bacterial communities in aquatic environments. Here we describe a method to quantify (3)H-TdR incorporation by specific, phylogenetically defined members of the bacterial community. The method involves selectively capturing DNA from targeted groups of bacteria and then quantifying its (3)H radioactivity. The method was applied to measure (3)H-TdR incorporation by the members of the phylum Bacteriodetes whose members, which include the Cytophaga-Flavobacter cluster, are ubiquitous in coastal waters. (3)H-labelled DNA from Bacteriodetes was selectively biotinylated in PCR-like reactions that contained a Bacteriodetes-specific 16S rRNA gene primer, thermostable DNA polymerase and biotinylated dUTP. The biotinylated DNA was then captured on streptavidin-coated beads and its (3)H radioactivity determined by scintillation counting. We have termed this method 'selective nucleic acid polymerase-biotinylation and capture' or 'SNAP-BAC'. Internal (33)P-labelled DNA standards were used to quantify the recovery of (3)H-labelled DNA from the SNAP-BAC reactions. The method was verified by successfully targeting Bacteriodetes in simple laboratory mixtures of (3)H-labelled DNA extracted from pure cultures of Bacteriodetes and gamma-proteobacteria. Field application of this method in Puget Sound and off the Washington coast determined that Bacteriodetes were responsible for 56 +/- 17% and 32 +/- 5% of community (3)H-TdR incorporation (1.3 +/- 0.3 and 9.9 +/- 1.7 pmol l(-1) h(-1)) at these two locations.     

Effect of growth conditions on enzyme activities,intracellular kinetics and biomass yields of a new RuMP-type methylotroph (T15)

Summary A new methylotrophic strain (T15), which employs the ribulose monophosphate (RuMP) cycle of formaldehyde assimilation, was isolated on the basis of high in vitro activities of formaldehyde and formate dehydrogenases (19 and 678 mU per mg protein, respectively). Serial subculturing of the strain in batch cultures, on 4 g/l CH₃OH for 6 months, led to loss of substantial percentages of the NAD-linked formaldehyde (25%) and formate (98%) dehydrogenases. The activities of these two enzymes were partially recovered when cells were grown continuously at very low dilution rate (0.03 h^–1). We found large variations (40 to 1000%) in the activities of other key enzymes of carbon-substrate oxidation (both linear and cyclic) and assimilation, in batch cultures with pure and mixed substrates, and in continuous cultures of different dilution rates. Key intracellular reaction rates, including those of the cyclic and linear substrate oxidation, were measured in vivo using a ¹⁴C-tracer technique in both continuous and batch cultures. The results indicate significant variations in these reaction rates, particularly those of linear and cyclic carbon oxidation. Overall, the cyclic oxidation appears to be employed to a larger (although not predominant) extent in strain T15 compared with another RuMP strain (L3) we have previously examined. T15 exhibits high biomass yields (up to 0.63 g cells per g CH₃OH) and growth rates (up to 0.46 h^–1) on CH₃OH in batch cultures. CH₃NH₂ can also be utilized as a substrate. In continuous culture, T15 could be grown at dilution rates up to 0.36 h^–1 with a corresponding biomass yield of 0.4. Examination of a large number of data on the biomass yields of strains T15 and L3 reveals that the large variations in yields derive from the variable branching of carbon flow between linear and cyclic oxidation and assimilation, rather than changes in the biosynthetic efficiency of carbon incorporation into biomass.     

一株新的产吡咯喹啉醌甲基营养菌全基因组测序和比较基因组学分析

【背景】由于甲基营养菌被发现的时间较短,而且可以生产吡咯喹啉醌(pyrroloquinoline quinone,PQQ)的甲基杆菌属细菌只有少数菌株的全基因组序列被公布,增加了该类细菌基因组学和生物代谢途径研究的难度。【目的】将本实验室筛选的PQQ生产菌经多种诱变方式处理,用于提高PQQ的发酵产量。对高产突变菌株进行全基因组解析,以探究甲基杆菌PQQ合成的分子机制,为后续分子育种提供序列背景信息。【方法】将野生型PQQ生产菌株进行紫外诱变、亚硝基胍诱变、甲基磺酸乙酯诱变、硫酸二乙酯诱变和紫外-氯化锂复合诱变。将突变菌株利用PromethION三代测序平台和MGISEQ-2000二代测序平台测序,然后进行组装和功能注释。组装得到的全基因组序列与模式菌株扭脱甲基杆菌AM1 (Methylobacterium extorquens AM1)进行比较基因组学分析。【结果】经11轮诱变获得一株突变菌株NI91,其PQQ产量为19.49mg/L,相较原始菌株提高44.91%。突变菌株NI91的基因组由一个5 409 262 bp的染色体组成,共编码4 957个蛋白,与模式菌株M. extorqu...     

Detoxification of Formaldehyde by the Spider Plant (Chlorophytum comosum L.) and by Soybean (Glycine max L.) Cell-Suspension Cultures

The phytotoxicity of formaldehyde for spider plants (Chlorophytum comosum L.), tobacco plants (Nicotiana tabacum L. cv Bel B and Bel W3), and soybean (Glycine max L.) cell-suspension cultures was found to be low enough to allow metabolic studies. Spider plant shoots were exposed to 7.1 [mu]L L-1 (8.5 mg m-3) gaseous [14C]-formaldehyde over 24 h. Approximately 88% of the recovered radioactivity was plant associated and was found to be incorporated into organic acids, amino acids, free sugars, and lipids as well as cell-wall components. Similar results were obtained upon feeding [14C]formaldehyde from aqueous solution to aseptic soybean cell-suspension cultures. Serine and phosphatidylcholine were identified as major metabolic products. Spider plant enzyme extracts contained two NAS+-dependent formaldehyde dehydrogenase activities with molecular mass values of about 129 and 79 kD. Only the latter enzyme activity required glutathione as an obligatory second cofactor. It had an apparent Km value of 30 [mu]M for formaldehyde and an isoelectric point at pH 5.4. Total cell-free dehydrogenase activity corresponded to 13 [mu]g formaldehyde oxidized h-1 g-1 leaf fresh weight. Glutathione-dependent formaldehyde dehydrogenases were also isolated from shoots and leaves of Equisetum telmateia and from cell-suspension cultures of wheat (Triticum aestivum L.) and maize (Zea mays L.). The results obtained are consistent with the concept of indoor air decontamination with common room plants such as the spider plant. Formaldehyde appears to be efficiently detoxified by oxidation and subsequent C1 metabolism.     

Mammalian selenoprotein in which selenocysteine (Sec) incorporation is supported by a new form of Sec insertion sequence element

Selenocysteine (Sec), the 21st amino acid in protein, is encoded by UGA. The Sec insertion sequence (SECIS) element, which is the stem-loop structure present in 3' untranslated regions (UTRs) of eukaryotic selenoprotein-encoding genes, is essential for recognition of UGA as a codon for Sec rather than as a stop signal. We now report the identification of a new eukaryotic selenoprotein, designated selenoprotein M (SelM). The 3-kb human SelM-encoding gene has five exons and is located on chromosome 22 but has not been correctly identified by either Celera or the public Human Genome Project. We characterized human and mouse SelM cDNA sequences and expressed the selenoprotein in various mammalian cell lines. The 3" UTR of the human, mouse, and rat SelM-encoding genes lacks a canonical SECIS element. Instead, Sec is incorporated in response to a conserved mRNA structure, in which cytidines are present in place of the adenosines previously considered invariant. Substitution of adenosines for cytidines did not alter Sec incorporation; however, other mutant structures did not support selenoprotein synthesis, demonstrating that this new form of SECIS element is functional. SelM is expressed in a variety of tissues, with increased levels in the brain. It is localized to the perinuclear structures, and its N-terminal signal peptide is necessary for protein translocation.     

Hexose phosphate synthase in trimethylamine-grown bacterium 2B2, a facultative methylotroph (Short Communication)

Hexose phosphate synthase and hexulose phosphate isomerase activities were found in trimethylamine-grown bacterium 2B2, a facultative methylotroph. The activities were separated by column chromatography of cell extracts on DEAE-cellulose. Hexulose phosphate isomerase activity was measured spectrophotometrically by using the product of the hexose phosphate synthase reaction as substrate.     

Carbon oxidation and incorporation patterns in batch cultures ofMethylomonas L3

Summary A recently developed methodology of directly measuring the oxidation and incorporation patterns of carbon substrate in continuous cultures of RuMP-type methylotrophs is extended to batch cultures of the obligate methylotrophMethylomonas L3. The ratio of cyclic to total substrate oxidation varies with the initial methanol concentration from 0 to 68%. Formaldehyde, as a methanol cosubstrate, enhances the net substrate oxidation. The substrate oxidation and incorporation pattern is also affected by the state of the culture inoculum.     

Microbial degradation of poly(d-3-hydroxybutyrate) by a new thermophilic Streptomyces isolate

A new thermophilic microorganism capable of degrading poly(D-3-hydroxybutyrate) (PHB) was isolated from soil. A phylogenetic analysis based on 16S rDNA sequences indicated that the new isolate belongs to genus Streptomyces. PHB film and powder were completely degraded after 6 and 3 d cultivation, respectively at 50 degrees C. Scanning micrographs showed adherence of the microbial cells to the entire film surface, indicating that biodegradation occurs by colonization of the PHB surface. The film was degraded both by microbial attack and by the action of an extracellular enzyme secreted by the microorganism. The strain can also degrade poly(ethylene succinate), poly(ester carbonate), polycaprolactone and poly(butylene succinate), but to a lesser extent.     

Evidence suggesting energy-dependent formaldehyde transport in an RuMP-type methylotroph (T15)

Formaldehyde accumulation ratios ([¹⁴CH₂O]_i/[¹⁴CH₂O]_o) as high as 12-fold were measured in anaerobic, CH₃OH-energized, whole cell suspensions of the ribulose monophosphate (RuMP)-type methylotrophic strain T15. Uptake kinetics were extremely rapid, enabling the attainment of equilibrium in only 10–30 s. Transport appears to be energy-dependent and associated with the protonmotive force (pmf). Anaerobic incubation with 5 M carbonyl p-(trifluoromethoxy)-phenylhydrazone (FCCP) led to 70%–90% reduction of the accumulation ratio. Though not as pronounced, diminished uptake was also observed in the presence of 140 M nigericin, 161 M valinomycin and 90 mM KSCN, commensurate with their effects on pmf. Accumulation of CH₂O as a function of external pH followed a trend more similar to that of pmf than either pH or . Preventing energization by incubation with 100 M N,N-dicyclohexylcarbodiimide (DCCD) led to nearly 80% inhibition of CH₂O transport. Over short time periods it was possible to chase accumulated ¹⁴CH₂O from previously loaded cells by collapsing pmf; however, this technique also indicated that significant ¹⁴CH₂O incorporation began to occur within 3 min.Abbreviations FCCP Carbonyl cyanide p-(trifluoromethyoxy)-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - RuMP ribulose monophosphate - TPP⁺ tetra[U-¹⁴C]phenylphosphonium - pmf protonmotive force     

Deamidation of Bcl-X(L): a new twist in a genotoxic murder mystery

Bcl-2 proteins are critical regulators of apoptosis following DNA damage. Recent studies have shown that genotoxic stress induces the deamidation of Bcl-X(L), inhibiting its antiapoptotic activities. Remarkably, Rb and p53 are important regulators of this novel modification of Bcl-X(L).