Dinitrophenol influences the rate and yield of Acetobacter methanolicus during the growth on glucose

Acetobacter methanolicuswas grown on glucose in the presence of dinitrophenol (DNP) under carbon/energy-limited conditions. DNP affected both the growth yield and the growth rate (D_sh) at which the energy generation was shifted from a complete to an incomplete substrate oxidation by using the PQQ-linked glucose dehydrogenase. The more the growth yield was decreased, the higher both the DNP concentration and the growth rate became. At about 0.53 mM DNP, growth was completely stopped. D_sh decreased from 0.21h^?1in the absence of DNP to 0.175 h^?1and 0.075 h^?1in the presence of 0.2 mM and 0.4 mM DNP, respectively. The experimental data are discussed in terms of the limitations in the generation of energy and some stress situations which are exerted by the presence of the uncoupler.     

Zur Gewinnung von Ubichinon-10 aus Acetobacter methanolicus IMET B 346

Mixtures of acetone/water (93 : 7 ··· 90 : 10, v/v) are favoured solvents for the extraction of ubiquinone-10 from Acetobacter methanolicus IMET B 346. Using these solvent mixtures ubiquinone-10 was extracted nearly completely. Other lipids were extracted partially. Extracts were obtained by optimal conditions with a ubiquinone content > 5%.     

Construction of broad-host-range plasmids for the expression of heterologous genes in Acetobacter methanolicus B58

The construction of different plasmids reported here on the basis of a broad-host-range RSF1010 replicon allows an efficient expression of heterologous genes in the acidophilic methanol-assimilating bacterium Acetobacter methanolicus B58. The promoter-probe vector pRS201 was used for the identification and isolation of the promoter containing sequences derived from the DNA of the Acetobacter phage Acm1. Further, this plasmid was coupled with the Escherichia coli promoters tac and p_r creating the expression vectors pRS201tac and pRS201p_r, respectively. After the insertion of the chloramphenicol acetyltransferase (cat) gene of the cloned promoters downstream, the chloramphenicol acetyltransferase (CAT) was determined in a cell-free extract of both E. coli and A. methanolicus. Using E. coli promoters as well as promoters of the Acetobacter phage Acm1 arranged in tandem with the promoters of the heterologous genes to be expressed, the pectat lyase gene (ptlB) of Erwinia carotovora and the threonine A gene (thrA) of E. coli were successfully expressed in A. methanolicus. The stability of recombinant plasmids under various conditions in A. methanolicus strains was tested using antibiotic-free media.     

Methanol and ethanol oxidase respiratory chains of the methylotrophic acetic acid bacterium, Acetobacter methanolicus.

Acetobacter methanolicus is a unique acetic acid bacterium which has a methanol oxidase respiratory chain, as seen in methylotrophs, in addition to its ethanol oxidase respiratory chain. In this study, the relationship between methanol and ethanol oxidase respiratory chains was investigated. The organism is able to grow by oxidizing several carbon sources, including methanol, glycerol, and glucose. Cells grown on methanol exhibited a high methanol-oxidizing activity and contained large amounts of methanol dehydrogenase and soluble cytochromes c. Cells grown on glycerol showed higher oxygen uptake rate and dehydrogenase activity with ethanol but little methanol-oxidizing activity. Furthermore, two different terminal oxidases, cytochrome c and ubiquinol oxidases, have been shown to be involved in the respiratory chain; cytochrome c oxidase predominates in cells grown on methanol while ubiquinol oxidase predominates in cells grown on glycerol. Both terminal oxidases could be solubilized from the membranes and separated from each other. The cytochrome c oxidase and the ubiquinol oxidase have been shown to be a cytochrome co and a cytochrome bo, respectively. Methanol-oxidizing activity was diminished by several treatments that disrupt the integrity of the cells. The activity of the intact cells was inhibited with NaCl and/or EDTA, which disturbed the interaction between methanol dehydrogenase and cytochrome c. Ethanol-oxidizing activity in the membranes was inhibited with 2-heptyl-4-hydroxyquinoline N-oxide, which inhibited ubiquinol oxidase but not cytochrome c oxidase. Alcohol dehydrogenase has been purified from the membranes of glycerol-grown cells and shown to reduce ubiquinone-10 as well as a short side-chain homologue in detergent solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth rate,not carrying capacity,determines extinction in simple stochastic model

Summary A simple stochastic model of logistic population growth is considered. The criterion for eventual extinction is a function of population growth rate, not of carrying capacity.     

Repression of lipopolysaccharide biosynthesis in Escherichia coli by an antisense RNA of Acetobacter methanolicus phage Acm1

Lysogenic Acetobacter methanolicus strains carrying the prophage Acm1 were found to be unable to synthesize both the capsutar polysaccharide (CPS) and the O-specific side-chain of lipopolysaccharide (LPS) and to represent rough variants of the host bacterium. A 262 bp DNA fragment of phage Acm1, obviously required for interference with LPS biosynthesis, was cloned and expressed in Escherichia coli Independently of the O-type, transformation of various E. coli strains with the recombinant DNA resulted in a suppression of biosynthesis of the O-specific chains. The DNA fragment of phage Acm1 contained three very short open reading frames of 21, 24, and 36 bp. However, attempts to express phage-encoded peptides were not successful. Instead, the Acm1-derived DNA fragment was shown to code for the synthesis of a trans-acting RNA molecule of 97 nucleotides, designated lbi (L PS b iosynthesis-i nterfering) RNA. This RNA contains sequence complementarity to E. coli target RNA sequences and appears to have the ability to form intracellularly RNA hybrid duplexes with mRNA. The data presented in this study support the hypothesis that the phenotypic effect of conversion to rough-type LPS is accompanied by the expression of an antisense RNA of phage Acm1.     

Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors

Summary Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids. Offprint requests to: R. Schröder     

Entropy capacity determines protein folding

Search and study of the general principles that govern kinetics and thermodynamics of protein folding generate a new insight into the factors controlling this process. Here, based on the known experimental data and using theoretical modeling of protein folding, we demonstrate that there exists an optimal relationship between the average conformational entropy and the average energy of contacts per residue-that is, an entropy capacity-for fast protein folding. Statistical analysis of conformational entropy and number of contacts per residue for 5829 protein structures from four general structural classes (all-alpha, all-beta, alpha/beta, alpha+beta) demonstrates that each class of proteins has its own class-specific average number of contacts (class alpha/beta has the largest number of contacts) and average conformational entropy per residue (class all-alpha has the largest number of rotatable angles phi, psi, and chi per residue). These class-specific features determine the folding rates: alpha proteins are the fastest folding proteins, then follow beta and alpha+beta proteins, and finally alpha/beta proteins are the slowest ones. Our result is in agreement with the experimental folding rates for 60 proteins. This suggests that structural and sequence properties are important determinants of protein folding rates.     

The linear oxidation of formaldehyde to CO2 as the proper energy generating sequence for the assimilation of methanol in Acetobacter methanolicus MB58

Acetobacter methanolicus MB58 can grow on methanol. Since this substrate exhibits to be energy deficient there must be a chance to oxidize methanol to CO₂ merely for purpose of energy generation. For the assimilation of methanol the FBP variant of the RuMP pathway is used. Hence methanol can be oxidized cyclically via 6-phosphogluconate. Since Acetobacter methanolicus MB58 possesses all enzymes for a linear oxidation via formate the question arises which of both sequences is responsible for generation of the energy required. In order to clarify this the linear sequence was blocked by inhibiting the formate dehydrogenase with hypophosphite and by mutagenesis inducing mutants defective in formaldehyde or formate dehydrogenase. It has been shown that the linear dissimilatory sequence is indispensable for methylotrophic growth. Although the cyclic oxidation of formaldehyde to CO₂ has not been influenced by hypophosphite and with mutants both the wild type and the formaldehyde dehydrogenase defect mutants cannot grown on methanol. The cyclic oxidation of formaldehyde does not seem to be coupled to a sufficient energy generation, probably it operates only detoxifying and provides reducing equivalents for syntheses. The regulation between assimilation and dissimilation of formaldehyde in Acetobacter methanolicus MB58 is discussed.Abbreviations ATP Adenosine-5-triphosphate - DCPIP 2,6-dichlorphenolindophenol - DW dry weight - ETP electron transport phosphorylation - FBP fructose-1,6-bisphosphate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PMS phenazine methosulfate - RuMP ribulose monophosphate - Ru5P ribulose-5-phosphate - SDS sodiumdodecylsulphate - TCA tricarboxylic acid - TYB toluylene blue Dedicated to Prof. Dr. Dr. S. M. Rapoport on occasion of his 75th birthday     

Regulation of alcohol-oxidizing capacity in chemostat cultures of Acetobacter pasteurianus

Acetobacter pasteurianus LMG 1635 was studied for its potential application in the enantioselective oxidation of alcohols. Batch cultivation led to accumulation of acetic acid and loss of viability. These problems did not occur in carbon-limited chemostat cultures (dilution rate = 0.05 h^–1) grown on mineral medium supplemented with ethanol, L-lactate or acetate. Nevertheless, biomass yields were extremely low in comparison to values reported for other bacteria. Cells exhibited high oxidation rates with ethanol and racemic glycidol (2,3-epoxy-1-propanol). Ethanol- and glycidol-dependent oxygen-uptake capacities of ethanol-limited cultures were higher than those of cultures grown on lactate or acetate. On all three carbon sources, A. pasteurianus expressed NAD-dependent and dye-linked ethanol dehydrogenase activity. Glycidol oxidation was strictly dye-linked. In contrast to the NAD-dependent ethanol dehydrogenase, the activity of dye-linked alcohol dehydrogenase depended on the carbon source and was highest in ethanol-grown cells. Cell suspensions from chemostat cultures could be stored at 4°C for over 30 days without significant loss of ethanol- and glycidol-oxidizing activity. It is concluded that ethanol-limited cultivation provides an attractive system for production of A. pasteurianus biomass with a high and stable alcohol-oxidizing activity.     

Oxidative capacity in failing hearts

Although high-energy phosphate metabolism is abnormal in failing hearts [congestive heart failure (CHF)], it is unclear whether oxidative capacity is impaired. This study used the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP) to determine whether reserve oxidative capacity exists during the high workload produced by catecholamine infusion in hypertrophied and failing hearts. Left ventricular hypertrophy (LVH) was produced by ascending aortic banding in 21 swine; 9 animals developed CHF. Basal myocardial phosphocreatine (PCr)/ATP measured with 31P NMR spectroscopy was decreased in both LVH and CHF hearts (corresponding to an increase in free [ADP]), whereas ATP was decreased in hearts with CHF. Infusion of dobutamine and dopamine (each 20 microg. kg-1. min-1 iv) caused an approximate doubling of myocardial oxygen consumption (MVO2) in all groups and decreased PCr/ATP in the normal and LVH groups. During continuing catecholamine infusion, DNP (2-8 mg/kg iv) caused further increases of MVO2 in normal and LVH hearts with no change in PCr/ATP. In contrast, DNP caused no increase in MVO2 in the failing hearts; the associated decrease of PCr/ATP suggests that DNP decreased the mitochondrial proton gradient, thereby causing ADP to increase to maintain adequate ATP synthesis.     

Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection

Knowledge about the sensitivity of the test organism is essential for the evaluation of any disinfection method. In this work we show that sensitivity of Escherichia coli MG1655 to three physical stresses (mild heat, UVA light, and sunlight) that are relevant in the disinfection of drinking water with solar radiation is determined by the specific growth rate of the culture. Batch- and chemostat-cultivated cells from cultures with similar specific growth rates showed similar stress sensitivities. Generally, fast-growing cells were more sensitive to the stresses than slow-growing cells. For example, slow-growing chemostat-cultivated cells (D = 0.08 h(-1)) and stationary-phase bacteria from batch culture that were exposed to mild heat had very similar T(90) (time until 90% of the population is inactivated) values (T(90, chemostat) = 2.66 h; T(90, batch) = 2.62 h), whereas T(90) for cells growing at a mu of 0.9 h(-1) was 0.2 h. We present evidence that the stress sensitivity of E. coli is correlated with the intracellular level of the alternative sigma factor RpoS. This is also supported by the fact that E. coli rpoS mutant cells were more stress sensitive than the parent strain by factors of 4.9 (mild heat), 5.3 (UVA light), and 4.1 (sunlight). Furthermore, modeling of inactivation curves with GInaFiT revealed that the shape of inactivation curves changed depending on the specific growth rate. Inactivation curves of cells from fast-growing cultures (mu = 1.0 h(-1)) that were irradiated with UVA light showed a tailing effect, while for slow-growing cultures (mu = 0.3 h(-1)), inactivation curves with shoulders were obtained. Our findings emphasize the need for accurate reporting of specific growth rates and detailed culture conditions in disinfection studies to allow comparison of data from different studies and laboratories and sound interpretation of the data obtained.     

Detrital nutrient content determines growth rate and elemental composition of bromeliad‐dwelling insects

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