Indigo production by Escherichia coli carrying the phenol hydroxylase gene from Acinetobacter sp strain ST-550 in a water-organic solvent two-phase system.

Acinetobacter sp. strain ST-550 produces indigo from indole in the presence of a large volume of diphenylmethane and a high level of indole. Particular proteins increased remarkably in strain ST-550 grown in the two-phase culture system for indigo production. One of the proteins showed a N-terminal amino acid sequence that was identical to that of the largest subunit of phenol hydroxylase (MopN) from A. calcoaceticus NCBI8250. The indigo-producing activity was strongly induced when ST-550 was grown with phenol as a sole carbon source. Genes coding for the multicomponent phenol hydroxylase were cloned, based on the homology with mopKLMOP from A. calcoaceticus NCBI8250. Escherichia coli carrying the genes produced indigo from indole. E. coli JA300 and its cyclohexane-resistant mutant, OST3410, carrying the hydroxylase genes and the NADH regeneration system were grown in the two-phase culture system for indigo production. The OST3410 recombinant produced 52 microg indigo ml(-1) of medium in the presence of diphenylmethane. This productivity was 4.3-fold higher than that of the JA300 recombinant.

Isolation of an Acinetobacter sp. ST-550 which produces a high level of indigo in a water-organic solvent two-phase system containing high levels of indole.

Acinetobacter sp. strain ST-550 was isolated from fumus soil as an efficient indigo producer in the presence of organic solvent. The minimum inhibitory concentration of indole was 0.4 mg/ml for ST-550. ST-550 produced only a small amount of indigo (less than 0.01 microg/ml) when grown in the presence of indole at concentrations of 0.05 to 0.3 mg/ml without any organic solvent. However. ST-550 produced indigo effectively when grown in the presence of a large volume of diphenylmethane and a high level of indole: optimized conditions were 3 ml of a medium containing 0.3 ml diphenylmethane and 2.7 mg indole. Under these conditions, ST-550 produced 0.88 mg indigo (292 microg/mI medium).

Cloning, sequence analysis and expression of a gene encoding an organic solvent- and detergent-tolerant cholesterol oxidase of Burkholderia cepacia strain ST-200.

Burkholderia cepacia strain ST-200 produces an extracellular cholesterol oxidase which is stable and highly active in the presence of organic solvents. This cholesterol oxidase produces 6beta-hydroperoxycholest-4-en-3-one from cholesterol, with the consumption of two moles of O2 and the formation of one mole of H2O2. The structural gene encoding the cholesterol oxidase was cloned and sequenced. The primary translation product was predicted to be 582 amino acid residues. The mature product is composed of 539 amino acid residues and is preceded by a signal sequence of 43 residues. The cloned gene was expressed as an active product in Escherichia coli and the product was localized in the periplasmic space. The cholesterol oxidase produced from E. coli was purified to homogeneity from the periplasmic fraction. The purified enzyme was highly stable in the presence of various organic solvents or detergents, as compared with the commercially available cholesterol oxidases tested.

Synthesis of oligosaccharide with controlled sequence.

A simple method for the synthesis of alternate heteropolysaccharide was investigated. Two types of modified glucose were combined into a cellobiose-type disaccharide monomer having only acetyl groups except a free hydroxyl group at C-3'. Polycondensation of this monomer, using stannic tetrachloride as catalyst, easily gave a novel copolysaccharide having regularly alternating 1,3-beta and 1,4-beta linkages.

Evaluation of the growth and inhibition strength of hydrocarbon solvents against Escherichia coli and Pseudomonas putida grown in a two-liquid phase culture system consisting of a medium and organic solvent.

The growth of microorganisms is often inhibited in a two-liquid phase culture system consisting of an aqueous medium and a large volume of hydrophobic solvent. Escherichia coli and Pseudomonas putida were cultured in a two-phase system containing a solvent with a log Pow value in a range of 2.1 to 6.0. The increase in the cell mass was monitored by increase in turbidity of the medium phase. We devised a semiquantitative method to evaluate the growth inhibition strength of solvents based on the relative amount of bacterial growth occurring in the two-phase system. Analyses of growth of the bacteria by this method showed that the growth inhibition strength of a given solvent was usually but not always correlated inversely with its polarity. It is clear that growth inhibition strength is not determined simply by polarity of the solvent.

Suppression of hypersensitivity of Escherichia coli acrB mutant to organic solvents by integrational activation of the acrEF operon with the IS1 or IS2 element.

The AcrAB-TolC efflux pump plays an intrinsic role in resistance to hydrophobic solvents in Escherichia coli. E. coli OST5500 is hypersensitive to solvents due to inactivation of the acrB gene by insertion of IS30. Suppressor mutants showing high solvent resistance were isolated from OST5500. These mutants produced high levels of AcrE and AcrF proteins, which were not produced in OST5500, and in each mutant an insertion sequence (IS1 or IS2) was found integrated upstream of the acrEF operon, coding for the two proteins. The suppressor mutants lost solvent resistance on inactivation of the acrEF operon. The solvent hypersensitivity of OST5500 was suppressed by introduction of the acrEF operon with IS1 or IS2 integrated upstream but not by introduction of the operon lacking the integrated IS. It was concluded that IS integration activated acrEF, resulting in functional complementation of the acrB mutation. The acrB mutation was also complemented by a plasmid containing acrF or acrEF under the control of Plac. The wild-type tolC gene was found to be essential for complementation of the acrB mutation by acrEF. Thus, it is concluded that in these cells a combination of the proteins AcrA, AcrF, and TolC or the proteins AcrE, AcrF, and TolC is functional in solvent efflux instead of the AcrAB-TolC efflux pump.

Entry into and release of solvents by Escherichia coli in an organic-aqueous two-liquid-phase system and substrate specificity of the AcrAB-TolC solvent-extruding pump.

Growth of Escherichia coli is inhibited upon exposure to a large volume of a harmful solvent, and there is an inverse correlation between the degree of inhibition and the log P(OW) of the solvent, where P(OW) is the partition coefficient measured for the partition equilibrium established between the n-octanol and water phases. The AcrAB-TolC efflux pump system is involved in maintaining intrinsic solvent resistance. We inspected the solvent resistance of delta acrAB and/or delta tolC mutants in the presence of a large volume of solvent. Both mutants were hypersensitive to weakly harmful solvents, such as nonane (log P(OW) = 5.5). The delta tolC mutant was more sensitive to nonane than the delta acrAB mutant. The solvent entered the E. coli cells rapidly. Entry of solvents with a log P(OW) higher than 4.4 was retarded in the parent cells, and the intracellular levels of these solvents were maintained at low levels. The delta tolC mutant accumulated n-nonane or decane (log P(OW) = 6. 0) more abundantly than the parent or the delta acrAB mutant. The AcrAB-TolC complex likely extrudes solvents with a log P(OW) in the range of 3.4 to 6.0 through a first-order reaction. The most favorable substrates for the efflux system were considered to be octane, heptane, and n-hexane.

Contribution of the cell wall component teichuronopeptide to pH homeostasis and alkaliphily in the alkaliphile Bacillus lentus C-125.

A teichuronopeptide (TUP) is one of major structural components of the cell wall of the facultative alkaliphilic strain Bacillus lentus C-125. A mutant defective in TUP synthesis grows slowly at alkaline pH. An upper limit of pH for growth of the mutant was 10.4, while that of the parental strain C-125 was 10.8. Gene tupA, directing synthesis of TUP, was cloned from C-125 chromosomal DNA. The primary translation product of this gene is likely a cytoplasmic protein (57. 3 kDa) consisting of 489 amino acid residues. Introduction of the tupA gene into the TUP-defective mutant complemented the mutation responsible for the pleiotropic phenotypes of the mutant, leading to simultaneous disappearance of the defect in TUP synthesis, the diminished ability for cytoplasmic pH homeostasis, and the low tolerance for alkaline conditions. These results demonstrate that the acidic polymer TUP in the cell wall plays a role in pH homeostasis in this alkaliphile.

Two moles of O2 consumption and one mole of H2O2 formation during cholesterol peroxidation with cholesterol oxidase from Pseudomonas sp. strain ST-200.

Cholesterol oxidase from Pseudomonas sp. strain ST-200 oxidized cholesterol and cholestanol to 6beta-hydroperoxycholest-4-en-3-one and 5alpha-cholestan-3-one respectively. The former was converted spontaneously to several oxysteroids such as 6-hydroxycholest-4-en-3-one and cholest-4-ene-3,6-dione, with the consumption of 2 mol of O(2) and the formation of 1 mol of H(2)O(2) for each mole of cholesterol oxidized. An oxidized form of the cholesterol oxidase dehydrogenates cholesterol, probably to the 5-en-3-one derivative. A reduced form of the enzyme, yielded from the cholesterol dehydrogenation reaction, dioxygenated cholest-5-en-3-one to 6beta-hydroperoxycholest-4-en-3-one.

Organic solvent tolerance of Escherichia coli is independent of OmpF levels in the membrane.

The organic solvent tolerance of Escherichia coli was measured under conditions in which OmpF levels were controlled by various means as follows: alteration of NaCl concentration in the medium, transformation with a stress-responsive gene (marA, robA, or soxS), or disruption of the ompF gene. It was shown that solvent tolerance of E. coli did not depend upon OmpF levels in the membrane.

Deterioration of Tolerance to Hydrophobic Organic Solvents in a Toluene-Tolerant Strain of Pseudomonas putida under the Conditions Lowering Aerobic Respiration.

The growth curve (increase in the number of viable cells) of a toluene-tolerant strain Pseudomonas putida Px51T was not reproducible in the presence of harmful organic solvents, such as p-xylene and toluene. The survival often fluctuated the during late exponential phase of growth. The repetitive growth was obtained by maintaining pO2 20-40% (v/v) in the culture flask. However, even under these aerobic conditions, the cells starved for a carbon source were killed by exposure to harmful solvents. The tolerance to organic solvents was lowered greatly by treatment with a proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP), or an electron transport chain inhibitor, sodium azide. Px51T treated with CCCP lost tolerance to a wide variety of organic solvents with log P ow of 2.6-4.2, which the organism usually tolerates. These results indicate that the solvent tolerance of Px51T depends upon on energy produced by aerobic respiration.

Improvement of organic solvent tolerance level of Escherichia coli by overexpression of stress-responsive genes.

Water-immiscible organic solvents can be toxic to microorganisms. The tolerance levels differ among strains of Escherichia coli, suggesting that the organic solvent tolerance level is strain specific and determined genetically. We constructed several mutants from E. coli, of which the organic solvent tolerance levels were improved. The mutants were defective in the marR gene encoding a repressor protein for the mar operon that is responsible for environmental stress factors. High expression of stress-responsive genes, soxS, marA, and robA, elevated organic solvent tolerance levels of several strains of E. coli. These genes code for DNA-binding proteins that are transcriptional activators belonging to the AraC subfamily with the helix-turn-helix motif. It was shown that expression of the AcrAB-TolC system, a major efflux pump in E. coli, was positively regulated by the proteins. This system was highly expressed in the organic solvent-tolerant mutants. Strains defective in one of the genes, acrA, acrB, or tolC, were remarkably sensitive to organic solvents.

C-terminal domain of beta-1,3-glucanase H in Bacillus circulans IAM1165 has a role in binding to insoluble beta-1,3-glucan.

The deduced amino acid sequences of 72-kDa beta-1,3-glucanase from Bacillus circulans WL-12 (GIcA) and 91-kDa enzyme from B. circulans IAM1165 (BglH) are highly homologous, except that the latter has an additional long C-terminal region composed of 192 amino acid residues. Two mutant enzymes (BgIH deprived of the C-terminal region and GIcA with the C-terminal region added) were constructed. The enzymes possessing the C-terminal region bound more abundantly to pachyman (insoluble beta-1,3-glucan) and A.spergillus oryzae cell wall than those not possessing the region. This indicates that the C-terminal region participated in binding of the enzymes to insoluble beta-1,3-glucan.

Effect of magnetite on the hematocrit value in exsanguinated rats.

Magnetite prepared by an enzyme-dependent reaction gradually released iron ion into the acidic-to-neutral buffer solution. A preparatory experiment was performed to examine the efficiency of magnetite as an iron supplement. Feeding exsanguinated rats with being magnetite resulted in the hematocrit value being recovered without any serious adverse effect on the digestive organs.

Purification of Extracellular Cholesterol Oxidase with High Activity in the Presence of Organic Solvents from Pseudomonas sp. Strain ST-200

Extracellular cholesterol oxidase of Pseudomonas sp. strain ST-200 was purified from the culture supernatant. This oxidase contained bound flavin and was categorized as a 3beta-hydroxysteroid oxidase, converting 3beta-hydroxyl groups to keto groups. The molecular mass was 60 kDa. The enzyme was stable at pH 4 to 11 and active at pH 5.0 to 8.5, showing optimal activity at pH 7 at 60 degreesC. The Michaelis constant of the ST-200 cholesterol oxidase was lower than those of commercially available oxidases. The cholesterol oxidation rate was enhanced 3- to 3.5-fold in the presence of organic solvents, with log Pow values (partition coefficients of the organic solvent between n-octanol and water), in the range of 2.1 to 4.2, compared with that in the absence of organic solvents.

Involvement of outer membrane protein TolC, a possible member of the mar-sox regulon, in maintenance and improvement of organic solvent tolerance of Escherichia coli K-12.

Escherichia coli mutants with improved organic solvent tolerance levels showed high levels of outer membrane protein TolC and inner membrane protein AcrA. The TolC level was regulated positively by MarA, Rob, or SoxS. A possible mar-rob-sox box sequence was found upstream of the tolC gene. These findings suggest that tolC is a member of the mar-sox regulon responsive to stress conditions. When a defective tolC gene was transferred to n-hexane- or cyclohexane-tolerant strains by P1 transduction, the organic solvent tolerance level was lowered dramatically to the decane-tolerant and nonane-sensitive level. The tolerance level was restored by transformation of the transductants with a wild-type tolC gene. Therefore, it is evident that TolC is essential for E. coli to maintain organic solvent tolerance.

Lithocholic acid side-chain cleavage to produce 17-keto or 22-aldehyde steroids by Pseudomonas putida strain ST-491 grown in the presence of an organic solvent, diphenyl ether.

We devised a method to screen for microorganisms capable of growing on bile acids in the presence of organic solvents and producing organic solvent-soluble derivatives. Pseudomonas putida biovar A strain ST-491 isolated in this study produced decarboxylated derivatives from the bile acids. Strain ST-491 grown on 0.5% lithocholic acid catabolized approximately 30% of the substrate as a carbon source, and transiently accumulated in the medium androsta-1,4-diene-3,17-dione in an amount of corresponding to 5% of the substrate added. When 20% (v/v) diphenyl ether was added to the medium, 60% of the substrate was converted to 17-keto steroids (androst-4-ene-3,17-dione-like steroid, androsta-1,4-diene-3,17-dione) or a 22-aldehyde steroid (pregna-1,4-dien-3-on-20-al). Amounts of the products were responsible for 45, 10, and 5% of the substrate, respectively. In the presence of the surfactant Triton X-100 instead of diphenyl ether, 40% of the substrate was converted exclusively to androsta-1,4-diene-3,17-dione.

Effects of Organic Solvents on Indigo Formation by Pseudomonas sp. strain ST-200 Grown with High Levels of Indole.

The indole tolerance level of Pseudomonas sp. strain ST-200 was 0.25 mg/ml. The level was raised to 4 mg/ml when diphenylmethane was added to the medium to 20% by volume. ST-200 grown in this two-phase culture system containing indole (1 mg/ml) and diphenylmethane (0.2 ml/ml) produced a water-soluble yellow pigment, isatic acid, and two water-insoluble and diphenylmethane-soluble pigments, blue indigo and purple indirubin. The amounts of the water-insoluble pigments corresponded to 0.5% (indigo) and 0.2% (indirubin) of the indole added to the medium. Of the conditions tried, indigo and indirubin were formed only when ST-200 was grown in the two-phase system overlaid with organic solvents with appropriate polarity.