Methyl methanesulphonate (MMS) is clearly mutagenic in S. typhimurium strain TA1535; a comparison with strain TA100

No mutagenicity or an uncertain mutagenic response has been reported in the literature for methyl methanesulphonate (MMS) in S. typhimurium strain TA1535 when using the plate assay. In our studies we found a reproducible mutagenic activity of 62 revertants/mumole and plate for MMS in strain TA1535 when using the preincubation assay. A dose-dependent increase in revertants was, however, observed only at fairly high doses (exceeding 4 mumole). Two different slopes were observed in the dose-response curve when testing MMS with strain TA100. Slope A is dependent on the error-prone response, possible only in strain TA100 due to the pKm101 plasmid (R factor) but not possible in strain TA1535 due to its umuDC deficiency. Slope B observed at higher doses (as in strain TA1535) could be explained through a GC----AT transition initiated by the O6-methylation of guanine. Our findings demonstrate that MMS induces back mutation in S. typhimurium strains carrying the hisG46 missense mutation due to the formation of O6-methylguanine. In the case of strain TA100 the pKm101 plasmid-mediated error-prone mechanism is, however, the predominant process in MMS mutagenesis which leads to a higher mutagenic response at much lower doses than the GT----AT transition in strain TA1535.     

Plasmid-controlled mercury biotransformation by Clostridium cochlearium T-2.

A strain of Clostridium cochlearium having methylmercury-decomposing ability was isolated. The ability was cured by the treatment with acridine dye and recovered by the conjugation of the cured strain with the parent strain. The cured strain then showed the activity to methylate mercuric ion as previously reported (M. Yamada and K. Tonomura, J. Ferment. Technol. 50:159-166, 1971). These results and the agarose gel electrophoretic pattern of the deoxyribonucleic acids from the lysates indicate a possible role of plasmids in controlling the mercury biotransformation of the two opposite directions in a single bacterial strain: methylation in the absence of the plasmid and demethylation in the presence of it. A possible mechanism for mercury resistance involving hydrogen sulfide is discussed.     

Novel Allylic Oxidation of α-Cedrene to sec-Cedrenol by a Rhodococcus Strain

A bacterial strain, designated KSM-7358, that can use α-cedrene for growth was isolated. The strain was identified as a member of the genus Rhodococcus and catalyzed the novel allylic oxidation of α-cedrene regiospecifically to produce (R)-10-hydroxycedrene (sec-cedrenol) with a very high yield. α-Curcumene was also produced as a possible metabolite of sec-cedrenol. A possible pathway for the microbial conversion of α-cedrene to sec-cedrenol and α-curcumene is proposed.     

New woody and ambery notes from cedarwood and turpentine oil

The development of a new product in the chemical industry is still driven by needs like technical properties, price/performance ratio, biodegradability, or product safety. However, in terms of improving more and more on ecological criteria, summarized under such catchphrases as sustainable development or green chemistry, another important aspect is to use renewable resources as starting materials. This is not significantly new in fragrance chemistry, and there are a lot of raw materials in the perfume oils that are derived from molecules of renewable resources. Two commonly used materials are: longifolene (from turpentine oil) and cedrene (from cedarwood oil). These compounds are very suitable for the synthesis of woody and ambery notes, and even though it seemed that all possibilities were exhausted, it is actually still feasible to discover new molecules with excellent olfactory properties such as Ambrocenide (50a), which is available in three steps from alpha-cedrene. Some of these molecules will be treated in this review, both with respect to synthesis as well as structural and sensory aspects.     

Mutagenic and comutagenic effects of ethionine in Escherichia coli K12

A possible mutagenic and comutagenic activity of ethionine, an analog of the amino acid methionine, was investigated in several mutant strains of E. coli K12. Ethionine was found to act as a weak mutagen only in a mismatch repair deficient mutator strain (mutL) and as a comutagen with 2-aminopurine (2AP) in a wild type E. coli. The latter effect was nor observed in a restriction-deficient strain (r-) nor in a recombination or SOS-deficient recA strain. These effects are interpreted as a consequence of restriction-induced double-strand breaks in hypomethylated E. coli DNA resulting in induction of the SOS mutator effect which generates predominantly mismatch correctable untargeted mutations.     

Biosorption of cadmium by a Cd2+-hyperresistant Bacillus cereus strain HQ-1 newly isolated from a lead and zinc mine

A Cd²⁺-hyperresistant bacterial strain HQ-1 was isolated from a lead–zinc mine. The strain was characterized and identified as Bacillus cereus based on morphology, physiological tests and 16S rRNA gene analysis. The minimal inhibitory concentration of Cd²⁺ for the bacterium was 0.012 mol/l. Isotherms for cadmium (Cd) biosorption by cells of B. cereus strain HQ-1 were investigated. The equilibrium data could be fitted by a Langmuir isotherm equation. The possible functional sites that might be influenced by the sorption were determined. The results indicate that this B. cereus strain has excellent potential for biosorption of Cd. Physiological characterization of the isolate also indicates possible application of this strain for bioremediation of sites with Cd contamination.     

Alternate pathway for isoleucine biosynthesis in Escherichia coli

A threonine dehydrataseless mutant of Escherichia coli, Crookes strain, was observed to grow on an acetate minimal medium without the usual requirement for isoleucine supplementation. Both the wild-type Crookes strain and a threonine auxotroph metabolized l-glutamate-1-(14)C to l-isoleucine-1-(14)C with no appreciable randomization, suggesting that a pathway for isoleucine formation from glutamate via beta-methylaspartate, beta-methyloxaloacetate, and alpha-ketobutyrate was possible in addition to the pathway from threonine and alpha-ketobutyrate. Crude cell-free extracts formed (14)C-beta-methylaspartate from (14)C-glutamate, and the conversion of beta-methylaspartate to alpha-ketobutyrate was also demonstrated, thus supporting the conclusion that glutamate can serve as a precursor of alpha-ketobutyrate (and isoleucine) without the necessary involvement of threonine as an intermediate.     

Lysis and fractionation of Mycobacterium paratuberculosis and Escherichia coli by matrix solid-phase dispersion.

A novel method for the lysis and subsequent fractionation of bacterial constituents from Mycobacterium paratuberculosis strain 19698 (M. paratuberculosis) and Escherichia coli strain DH5 alpha utilizing the technique of matrix solid-phase dispersion (MSPD) is described. Bacteria were blended with octadecylsilyl (C18) derivatized silica to obtain cellular lysis. The blended material was used to prepare a column which was sequentially eluted with solvents of increasing polarity. Fractionation of cellular components was confirmed by analysis of the solvent extracts. The possible applicability of the MSPD technique as a general method for the lysis and fractionation of bacterial components is proposed.     

Isolation and characterization of 3-N-trimethylamino-1-propanol-degrading Rhodococcus sp. strain A2

The aerobic degradation of 3- N -trimethylamino-1-propanol (homocholine) as a sole source of carbon and nitrogen has been found for a Rhodococcus sp. bacterium isolated from soil. The isolate was identified as Rhodococcus sp. strain A2 based on its phenotypic features, physiological and biochemical characteristics, and results of phylogenetic analysis. The washed cells of strain A2 completely degraded homocholine within 6 h, with concomitant formation of several metabolites. Analysis of the metabolites using capillary electrophoresis, fast atom bombardment–MS, and GC–MS showed that trimethylamine was the major metabolite, in addition to β-alanine betaine (β-AB) and trimethylaminopropionaldehyde. Therefore, the possible degradation pathway of homocholine in the isolated strain is through consequent oxidation of the alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH). Thereafter, the cleavage of β-AB C–N bonds yielded trimethylamine and alkyl chain.     

Metabolism of hexadecyltrimethylammonium chloride in Pseudomonas strain B1.

A bacterium (strain B1) utilizing hexadecyltrimethylammonium chloride as a carbon and energy source was isolated from activated sludge and tentatively identified as a Pseudomonas sp. This bacterium only grew on alkyltrimethylammonium salts (C12 to C22) and possible intermediates of hexadecyltrimethylammonium chloride breakdown such as hexadecanoate and acetate. Pseudomonas strain B1 did not grow on amines. Simultaneous adaptation studies suggested that the bacterium oxidized only the alkyl chain of hexadecyltrimethylammonium chloride. This was confirmed by the stoichiometric formation of trimethylamine from hexadecyltrimethylammonium chloride. The initial hexadecyltrimethylammonium chloride oxygenase activity, measured by its ability to form trimethylamine, was NAD(P)H and O2 dependent. Finally, assays of aldehyde dehydrogenase, hexadecanoyl-coenzyme A dehydrogenase, and isocitrate lyase in cell extracts revealed the potential of Pseudomonas strain B1 to metabolize the alkyl chain via beta-oxidation.     

Metabolism of hexadecyltrimethylammonium chloride in Pseudomonas strain B1.

A bacterium (strain B1) utilizing hexadecyltrimethylammonium chloride as a carbon and energy source was isolated from activated sludge and tentatively identified as a Pseudomonas sp. This bacterium only grew on alkyltrimethylammonium salts (C12 to C22) and possible intermediates of hexadecyltrimethylammonium chloride breakdown such as hexadecanoate and acetate. Pseudomonas strain B1 did not grow on amines. Simultaneous adaptation studies suggested that the bacterium oxidized only the alkyl chain of hexadecyltrimethylammonium chloride. This was confirmed by the stoichiometric formation of trimethylamine from hexadecyltrimethylammonium chloride. The initial hexadecyltrimethylammonium chloride oxygenase activity, measured by its ability to form trimethylamine, was NAD(P)H and O2 dependent. Finally, assays of aldehyde dehydrogenase, hexadecanoyl-coenzyme A dehydrogenase, and isocitrate lyase in cell extracts revealed the potential of Pseudomonas strain B1 to metabolize the alkyl chain via beta-oxidation.     

Characteristics of Anabaena variabilis influencing plaque formation by cyanophage N-1.

Phage N-1 grown in Anabaena strain 7120 [N-1 . 7120] forms plaques on A. variabilis about 10(-7) to 10(-6) as efficiently as on Anabaena 7120. By manipulating different characteristics of the interaction between phage and host, it was possible to increase the relative efficiency of plaque formation to 0.38. Growth of A. variabilis at 40 degrees C for at least three generations resulted in an increase in the rate of phage adsorption and a 10-fold increase in the efficiency of plaque formation. The efficiency of plaque formation was further increased about 42-fold, with little or no further increase in rate of adsorption, in a variant strain. A. variabilis strain FD, isolated from a culture of A. variabilis which had grown for more than 30 generations at 40 degrees C. The low relative efficiency of plaque formation by N-1 . 7120 on A. variabilis could be partially accounted for if A. variabilis contains a deoxyribonucleic acid restriction endonuclease which is absent from Anabaena 7120. Indirect evidence for such an endonuclease included the following: (i) phage N-1 grown in A. variabilis (N-1 . Av) had approximately a 7 X 10(3)-fold higher relative efficiency of plaque formation on A. variabilis than had N-1 . 7120; and (ii) the efficiency of plaque formation by N-1 . 7120 on A. variabilis strain FD was increased by up to 146-fold after heating the latter organism at 51 degrees C.     

Construction and characterization of an oxalic acid nonproducing strain of Aspergillus niger

Aspergillus niger produces oxalic acid as a by-product which causes problems with downstream processing of industrial enzymes. To overcome this problem the oah gene encoding oxaloacetate hydrolase (EC 3.7.1.1) was disrupted in a glucoamylase-producing strain of A. niger and the resulting strain was incapable of producing oxalic acid. The strain with the disrupted gene was compared with the wild-type strain producing oxalic acid in batch cultivations. The specific growth rate of both strains was 0.20 h(-1). The citric acid yields were identical, but the glucoamylase yield was only 50% in the disruptant compared with the wild-type strain. Batch experiments with 13C-labeled glucose as substrate were carried out to determine the metabolic fluxes through the central metabolism. The two strains had almost identical metabolic fluxes, which suggested that it was possible to disrupt the oah gene without pleiotropic consequences. The flux through the pentose phosphate pathway was around 60% of the glucose uptake for both strains, which suggested that a sufficient supply of NADPH was available for biosynthesis.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. V. Biochemical characterization of a strain (ebony) that is UV- and X-ray sensitive and deficient in photorepair

We investigated larval sensitivity to UV and repair of UV- and X-ray-induced lesions in the DNA of the ebony strain compared to a wild-type strain (Canton S). The ebony strain was previously characterized as being more sensitive to UV-induced killing of embryos than Canton S. Also the ebony strain is more sensitive to X-rays for induction of larval killing, dominant lethals and recessive lethals. In this paper it is demonstrated that (1) ebony larvae are more sensitive to killing by UV and less proficient in photoreactivation (PR) ability than Canton S larvae; (2) the ebony strain has a defect in PR repair of endonuclease-sensitive sites induced in the DNA of primary cell cultures by UV irradiation; (3) the ebony strain has a defect in the repair of single-strand breaks induced in the DNA by X-rays (again in primary cell cultures), at least early on in the repair incubation. A rough localization of the UV sensitivity and the PR ability is presented and the possible relevance of the biochemical to the genetic results is discussed.     

Acetogenesis from Dichloromethane by a Two-Component Mixed Culture Comprising a Novel Bacterium

A strictly anaerobic two-component culture able to grow exponentially with a doubling time of 20 h on a medium containing dichloromethane as the carbon and energy source was characterized. On a medium without sulfate, we observed (per mol of dichloromethane) a mass balance of 2 mol of chloride, 0.26 mol of acetate, 0.05 mol of formate, and 0.25 mol of carbon in biomass. One component of the culture, strain DMB, was identified by a 16S ribosomal DNA analysis as a Desulfovibrio sp. The other component, the gram-positive organism strain DMC, could not be isolated. It was possible, however, to associate strain DMC on a medium containing dichloromethane in a coculture with Acetobacterium woodii or Methanospirillum hungatei. Coculture of strain DMC with the Archaeon M. hungatei allowed us to specifically amplify by PCR the 16S rRNA gene of strain DMC. A phylogenetic analysis of the 16S ribosomal DNA sequence revealed that this organism groups within the radiation of the Clostridium-Bacillus subphylum and exhibits the highest levels of sequence similarity (89%) with Desulfotomaculum orientis and Desulfitobacterium dehalogenans. Since the novel organism strain DMC was able to grow acetogenically with dichloromethane when it was associated with one of three metabolically different partners and since, in contrast to strain DMB, strain DMC contained carbon monoxide dehydrogenase activity, this bacterium is responsible for both the dehalogenation of dichloromethane and the acetogenesis observed in the original two-component culture. The obligatory dependence of strain DMC on a partner during growth with dichloromethane is thought to stem from the need for a growth factor produced by the associated organism.     

Microplate bioassay for nisin in foods,based on nisin-induced green fluorescent protein fluorescence

A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 microg of nisin in cheese, and 1 microg of nisin per ml in salad dressings.     

Heritability of flight distance for Cydia pomonella

Cydia pomonella L. (Lepidoptera: Tortricidae) is considered to be rather sedentary, but some individuals undertake flights of several kilometres in the field. This paper investigates the genetic influence on this variability. The flight capacity was measured in the laboratory by a flight mill and its heritability was estimated for two different strains. The laboratory strain was kept for more than 45 generations and the field strain from Embrach (northern Switzerland) was recently collected in the field.The multiple-trait-restricted-maximum-likelihood method was used for the estimation of genetic variances and covariances. A mixed full-sib/half-sib design was applied for the field strain and a full-sib design for the laboratory strain. The heritability of total distance was 0.57 for the field strain and 0.37 for the laboratory strain (both sexes). In addition, a heritability of 0.38 for total distance was estimated by parent-offspring regression for the laboratory strain. All three values were significantly different from zero P<0.05 and show that there is a significant additive genetic influence on flight capacity.The genetic correlations between total distance and other flight traits (total duration, flight velocity, longest flight) were between 0.84 and 1.00 for both strains and suggest that these traits actually belong to a single one. High genetic correlations were also found between total distance and the morphological traits body weight and wing length for the field strain, whereas a negative correlation was found between total flight distance and body weight for the laboratory strain. This difference between the two strains was interpreted as a possible trade-off between flight capacity and fecundity.     

D-apiose reductase from Aerobacter aerogenes

A strain of Aerobacter aerogenes PRL-R3 has been isolated which utilizes d-apiose as its sole source of carbon. A new enzyme, d-apiose reductase, was discovered in this strain. The enzyme was not present when the strain was grown on d-glucose. d-Apiose reductase catalyzes the nicotinamide adenine dinucleotide-dependent interconversion of d-apiose and d-apiitol. The enzyme is specific for d-apiose and d-apiitol, with a few possible exceptions. The K(m) for d-apiose is 0.02 m. The K(m) for d-apiitol is 0.01 m. The enzyme is almost completely specific for the reduced and oxidized forms of nicotinamide adenine dinucleotide. When cell-free extracts were centrifuged at 100,000 x g for 1 hr, the enzyme remained in solution. Optimal activity for the reduction of d-apiose was obtained at pH 7.5 in glycylglycine buffer, whereas for the oxidation of d-apiitol it was obtained at pH 10.5 in glycine buffer. Enzymatic reduction of d-apiose was not appreciably affected by the presence of 0.02 m ethylenediaminetetraacetate. Paper chromatography and specific spray reagents were used to identify d-apiitol and d-apiose as the products of this reversible reaction. d-Apiose and d-apiitol did not serve as substrates for ribitol dehydrogenase and d-arabitol dehydrogenase from A. aerogenes PRL-R3.     

The model legume Medicago truncatula A17 is poorly matched for N2 fixation with the sequenced microsymbiont Sinorhizobium meliloti 1021

Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021-M. truncatula symbiosis at fixing N(2) was evaluated. N(2) fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed. Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells. The Sm1021-M. truncatula symbiosis is poorly matched for N(2) fixation and the strain could possess broader N(2) fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N(2) fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.     

Oxidation of 1,5-anhydro-D-glucitol to 1,5-anhydro-D-fructose catalyzed by an enzyme from bacterial membranes

Bacteria which grow on 1,5-anhydro-D-glucitol (AG) were isolated from soil. One such strain showing the highest AG-assimilating activity was further characterized and identified as a new strain of the Pseudomonas family (named Pseudomonas sp. NK-85001). A subcellular membranous fraction obtained from this strain catalyzed the oxidation of AG to 1,5-anhydro-D-fructose. This oxidation reaction consumed molecular oxygen as the terminal electron acceptor. The AG-oxidizing activity was further purified after solubilization. The AG oxidation catalyzed by this solubilized enzyme utilized molecular oxygen only in the presence of an electron mediator such as 2,6-dichlorophenolindophenol or phenazine methosulfate. Thus, the enzyme was suggested to be a dehydrogenase rather than an oxidase. The solubilized enzyme preparation also showed a strict substrate specificity. The observed specificity indicated that application of the enzyme for AG assay in clinical samples might be possible.