Identification and biotyping of clinical isolates of Acinetobacter

A total of 343 Acinetobacter strains, most isolated from hospital patients, were identified using a 16-test system (acid production from glucose, gelatin hydrolysis and utilization of 14 carbon sources) associated with tests for growth at 37, 41 and 44°C. Of 299 nosocomial isolates, 253 were identified as A. baumannii, 20 as Acinetobacter genospcies 3, 8 as A. haemolyticus, 8 as A. lwoffii, 4 as A. johnsonii and 6 as other (presently) unnamed species. A biotyping system based on the utilization of levulinate, citraconate, L-phenylanine, phenylacetate, 4-hydroxybenzoate and L-tartrate allowed recognition of 17 biotypes among 247 A. baumannii isolates. This biotyping system should be useful in epidemiological studies of Acinetobacter strains.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.     

Biosynthesis of (R)-3-hydroxyalkanoic acids by metabolically engineered Escherichia coli

An efficient system for the production of (R)-hydroxyalkanoicacids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli. Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of RHAs. In recombinant E. coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA. When the recombinant E. coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose, (R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers. R3HB dimers could be efficiently converted to monomers by mild alkaline heat treatment. A stable recombinant E. coli strain in which the R. eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB. When the R. eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced.     

Influence of fadA G203R and ΔflbA mutations on morphology and physiology of submerged Aspergillus nidulans cultures

Morphologic and physiologic changes taking place in carbon-limited submerged cultures of Aspergillus nidulans ΔflbA and fadA ^G203R strains were studied. Loss-of-function mutation of the flbA gene resulted in an altered germination with unusually thick germination tubes, “fluffy” pellet morphology, as well as a reduced fragmentation rate of hyphae during autolysis. In the fadA ^G203R mutant strain, conidiophores formed in the stationary phase of growth, and the size of pellets shrank considerably. There were no significant differences in the generation of reactive oxygen species (ROS) and in the specific catalase and superoxide dismutase activities by the tested mutants and the appropriate parental strains. Therefore, the participation of ROS or antioxidative enzymes in FadA/FlbA signaling pathways seems to be unlikely in submerged cultures. On the other hand, earlier increases in the extracellular protease and ammonia production were recorded with the ΔflbA strain, whereas the protease and ammonia production of the fadA ^G203R mutant lagged behind those of the wild-type strains. Similar changes in the time courses of the induction of γ-glutamyltranspeptidase and the degradation of glutathione were observed. These results suggest that FadA/FlbA signaling may be involved in the mobilization of protein and peptide reserves as energy sources during carbon starvation.     

Production, purification, and characterization of a polygalacturonase from a new strain of kluyveromyces marxianus isolated from coffee wet-processing wastewater

A new high polygalacturonase (PG)-producing Kluyveromyces marxianus strain was isolated from coffee wet-processing wastewater. PG production in this strain is not repressed in the presence of 100g/L of glucose and, being growth-associated, reached its maximum accumulation in the culture medium at the beginning of the stationary phase. Oxygen and galacturonic acid negatively regulated enzyme synthesis, and glucose as the carbon source afforded better enzyme yields than lactose. The data reported here show that this strain exhibits the highest index of PG production among the wild-type strains reported so far (18.8U/mL). PG was readily purified by ion-exchange chromatography on SP-Sepharose FF. The activity corresponded to a single protein with an M _r of 41.7 kDa according to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was stable in the pH range of 3.0–5.0 and displayed an optimal temperature of 55°C; it showed a typical endo-splitting way of substrate hydrolysis and exhibited a fair degree of activity on pectin with a high degree of esterification.     

Reduction of furfural to furfuryl alcohol by ethanologenic strains of bacteria and its effect on ethanol production from xylose

The ethanologenic bacteria Escherichia coli strains KO11 and LYO1, and Klebsiella oxytoca strain P2, were investigated for their ability to metabolize furfural. Using high performance liquid chromatography and ¹³C-nuclear magnetic resonance spectroscopy, furfural was found to be completely biotransformed into furfuryl alcohol by each of the three strains with tryptone and yeast extract as sole carbon sources. This reduction appears to be constitutive with NAD(P)H acting as electron donor. Glucose was shown to be an effective source of reducing power. Succinate inhibited furfural reduction, indicating that flavins are unlikely participants in this process. Furfural at concentrations >10 mM decreased the rate of ethanol formation but did not affect the final yield. Insight into the biochemical nature of this furfural reduction process may help efforts to mitigate furfural toxicity during ethanol production by ethanologenic bacteria.     

Conformational studies of intermolecular hydorgen bonding through induced crystal G phase in nBA:7HB

This paper exploits the physical investigation on liquid crystal complexes obtained by self-organisation of p-n-alkyl benzoic acid (nBA) mesogens with non-mesogenic materials heptyl p-hydroxy benzoate (7HB). Intermolecular interactions of the molecules result the hydrogen bond between the proton donor (COOH) of nBA and proton acceptor (OH) of 7HB. The formation of hydrogen bond is attributed to the quenching of the nematic phase and inducement of crystal G phase in liquid crystal complex. A comparative study of phase abundance is presented with respect to the pure nBAs and other hydrogen bonded liquid crystal complexes of nBAs. Thermal and phase behaviour of the complexes are determined by polarising optical microscope (POM), differential scanning calorimetry (DSC) and image moments approach. Intermolecular interactions which result the hydrogen bond in complexes are investigated using Fourier transform infrared (FTIR) spectroscopy. Molecular structure of the liquid crystal complexes in the solid phase was elucidated using powder X-ray diffraction and proton nuclear magnetic resonance (¹HNMR).     

On-line Characterization of Physiological State in Poly(β-Hydroxybutyrate) Production by <Emphasis Type="Italic">Wautersia eutropha</Emphasis>

Culture fluorescence measurement technique has the potential for on-line characterization of metabolic status of fermentation processes. Many fluorophores present inside the living cells such as NADH + H⁺, tryptophan, pyridoxine, and riboflavin fluoresce at specific excitation and emission wavelength combinations. Since these key intracellular metabolites are involved in cell growth and metabolism, their concentration change at any time inside the cell could reflect the changes in cell metabolic activity. NADH + H⁺ spectrofluorometry was used for on-line characterization of physiological state during batch cultivation of poly-β-hydroxybutyric acid (PHB) production by Wautersia eutropha. The culture fluorescence increased with an increase in the biomass concentration with time. A linear correlation between cell mass concentration and net NADH + H⁺ fluorescence was established during active growth phase (13 to 38 h) of batch cultivation. The rate of change of culture fluorescence (dF/dt) exhibited a gradual increase during the predominantly growth phase of batch cultivation (till 20 h). Thereafter, a sudden drop in the dF/dt rate and its leveling was recorded indicating major changes in culture metabolism status which synchronized with the start-up of accumulation of PHB. After 48 h, yet another decrease in the rate of change of fluorescence (dF/dt) was observed primarily due to severe substrate limitation in the reactor. On-line NADH + H⁺ fluorescence signal and its rate (dF/dt) could therefore be used to distinguish the growth, product formation, and nutrient depletion stage (the metabolic state marker) during the batch cultivation of W. eutropha.     

Stress Response Kinetics of Two Nisin Producer Strains of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>

The purpose of this study is to determine the survival and nisin production behaviors of two strains of Lactococcus lactis under different stress conditions that represent the food ecosystem. In this respect, the survival ratios of two nisin producers were determined under different pH, temperature, NaCl, and bile salt concentrations. Then, nisin production levels of the strains were determined at each stress conditions. Both strains had similar growth or inactivation patterns under the same stress conditions. NaCl and bile salt stresses on the survival ratio of the strains could be successfully described by the exponential decay function, whereas Gaussian function produced good fits for temperature and pH stresses. The nisin activity of two nisin producers (in their mid-exponential and/or early stationary phase) decreased dramatically under all stress conditions, except osmotic (NaCl) and low temperature applications. The results of this study showed that two nisin producers had similar adaptive responses under severe stress conditions, which could be described by appropriate mathematical equations. Moreover, the effect of harsh environment on the nisin activity of L. lactis strains depends on the stress factors applied.     

Umsetzungen von Metall- und Metalloidverbindungen mit mehrfunktionellen Molekülen, 16. Mitt.

The reaction of trimethylsilylcarbonamides with halogeno-diorganyl-boranes resp. trihalogenoboranes or organodihalogenoboranes gives monomeric resp. dimeric amidoboranes (borylcarbonamides) and derivatives of 4,8-diaza-1,5-dibora-2,6,9-trioxabicyclo[3.3.1]nonadienes. By reaction of the free acylamides with halogenoboranes in most cases the imide halides could be isolated as the only products. By reaction of the hydrochloride of bis(dimethylamino)-hydroxyborane withn-butyl-lithium followed by addition of the imide halides, the corresponding imidoylamines were formed.¹H,¹¹B, and¹⁹F-nmr spectra, mass spectra and characteristic ir group frequencies are reported.

15. Mitt.:W. Maringgele undA. Meller, Z. anorg. allg. Chem., im Druck.     

Construction of recombinant Escherichia coli strains for polyhydroxybutyrate production using soy waste as nutrient

Construction and comparison of recombinant Escherichia coli strains harboring the polyhydroxybutyrate (PHB) operon from Ralstonia entropha using vectors possessing different promotors, as well as the production of PHB from soy waste by the recombinant strain, are reported. The lac promotor was the most efficient on expression of the phb operon among the three promotors studied: i.e., lac promotor, T7 promotor and the normal σ⁷⁰ promotor. The pKS/PHB was the most efficient plasmid for phboperon expression among the three plasmids used: i.e., pKS⁻, pAED4, and pJM9131. It was observed that isopropyl-β-d-thiogalactopyranoside was not required for the induction of the expression of phb operon. The cell dry wt and polyhydroxyalkan cote content by E. coli XL-1 Blue (pKS/PHB) were 3.025 g/L and 27.83%, respectively.     

Optimization of culture parameters for production of podophyllotoxin in suspension culture of Podophyllum hexandrum

The root explants of the germinated seedlings of Podophyllum hexandrum were grown in MS medium supplemented with indole acetic acid (IAA) (2 mg/L) and activated charcoal (0.5%), and healthy callus culture was obtained after incubation for 3 wk at 20°C. The cultivation of plant cells in shake flask was associated with problems such as clumping of cells and browning of media, which were solved by the addition of pectinase and polyvinylpyrrolidone. The effect of major media components and carbon source was studied on the growth and podophyllotoxin production in suspension culture. It was found that glucose was a better carbon source than sucrose and that NH₄ ⁺:NO₃ ⁻ ratio (total nitrogen concentration of 60 mM) and PO₄ ³⁻ did not have much effect on the growth and product formation. The relative effect of culture parameters (inoculum level, pH, IAA, glucose, NH₄ ⁺:NO₃ ⁻ ratio, and PO₄ ³⁻) on the overall growth and product response of the plant cell suspension culture was further investigated by Plackett-Burman design. This indicated that inoculum level, glucose, IAA, and pH had significant effects on growth and production of podophyllotoxin. To identify the exact optimum concentrations of these parameters on culture growth and podophyllotoxin production, central composite design experiments were formulated. The overall response equations with respect to growth and podophyllotoxin production as a function of these culture parameters were developed and used to determine the optimum concentrations of these parameters, which were pH 6.0, 1.25 mg/L of IAA, 72 g/L of glucose, and inoculum level of 8 g/L.     

Relationship between GLC retention data and topological indices for a wide variety of solutes on five stationary phases of different polarity

Summary Numerical correlations between the specific retention volumes of several dozen solutes (hydrocarbons and derivatives containing oxygen, nitrogen and halogens) and between the retention indices, from the literature, of 26 chemical derivatives of benzene on several stationary phases have been studied. Although significant linear correlations were usually obtained between logV _g and the valence connectivity indices,¹ X ^v , for hydrocarbons and non-hydrocarbons with the same chemical function, the relationship between logV _g and the Wiener,W, and Balaban,J, indices for hydrocarbons was found to be non-linear. Correlations between retention index increments and valence connectivity index increments for the 26 chemical derivatives of benzene were linear for alkyl substituents only.     

Microbial Baeyer-Villiger Oxidation of Ketones by Cyclohexanone and Cyclopentanone Monooxygenase – A Computational Rational for Biocatalyst Performance

Summary. Recombinant Escherichia coli overexpressing Pseudomonas sp. NCIMB 9872 cyclopentanone monooxygenase (CPMO, EC 1.14.13.16) and Acinetobacter sp. NCIMB 9871 cyclohexanone monooxygenase (CHMO, EC 1.14.13.22) have been utilized in whole-cell Baeyer-Villiger biotransformations of prochiral bicycloketones. A significant difference in substrate acceptance and stereoselectivity was observed for bicyclo[3.3.0] and bicyclo[4.3.0] substrates. A plausible mechanism of these transformations was established by means of high level DFT/B3LYP calculations suggesting an essential difference in electronic requirements for a successful enzymatic conversion, which was similarly encountered in recombinant whole-cell mediated biooxidations. Some of the lactones produced in the biocatalytic Baeyer-Villiger oxidation represent key intermediates for the synthesis of indole alkaloids.     

Simultaneous Determination of Phytohormones in Plant Extracts using SPME and HPLC

Solid-phase microextraction followed by HPLC was used for the determination of indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) in plant samples. Parameters influencing performance, including pH, salinity, extraction time, fiber coating and temperature, were optimized. A Carbowax-coated fiber was chosen for determination due to much higher extraction efficiency compared to polyacrylate fibers. The dynamic ranges spanned over three orders of magnitude. The LOD/(LOQ) values of the target compounds in pure water were 0.149(0.497), 0.442(1.472), 0.121(0.403), 0.058(0.193) μg L⁻¹ for IAA, ABA, IBA and NAA respectively. The method was successfully applied to the analysis of xylem fluid from Musa basjoo stem obtaining recoveries of 98.85% (IAA), 94% (IBA) and 94.30% (NAA). The method was also successfully applied to the analysis of these four target compounds in the hyperaccumulating plant, Viola baoshanensis. The results matched quite well with ones obtained by solid phase extraction followed by HPLC. The method developed was superior when applied to liquid samples because matrix effects could be eliminated.     

Study of Bulk Membrane Transport of Some Heavy Metal Cations Using Three Macrocyclic Ligands Containing Oxygen and Nitrogen Heteroatoms

The transport experiments of Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Ag⁺ and Pb²⁺ metal cations were carried out by dibenzo-18-crown-6 (DB18C6), dibenzyl-diaza-18-crown-6 (Dibenzyl-diaza-18C6) and di-tert-butyl-dibenzo-18-crown-6 (Di-tert-butyl-DB18C6) using chloroform (CHCl₃), 1,2-dichloroethane (1,2-DCE) and nitrobenzene (NB) organic solvents as liquid membranes. The source phase contained equimolar concentration of these metal cations and the source and receiving phases being buffered at pH=5 and pH=3, respectively. The obtained results show that the selectivity and the efficiency of transport for these heavy metal cations change with the nature of the ligand and also the organic solvents, which were used as liquid membranes in these experiments. A good selectivity was observed for silver (I) ion by dibenzyl-diaza-18C6 in all membrane systems. Dibenzo-18C6 and di-tert-butyl-DB18C6 showed the highest transport efficiency for cobalt (II) ion. The effect of stearic acid on transport efficiency was also investigated and the results show that the efficiency of transport of the heavy metal cations increases in the presence of this organic acid.     

Umsetzung von Metall- und Metalloidverbindungen mit mehrfunktionellen Molekülen, 22. Mitt.: Die Reaktion von Trimethylsilylcarbonsäureamiden mit Halogen(diorganyl)boranen

Trimethylsilylacetamide and trimethylsilyltrifluoroacetamide react with halogenodiorganylboranes to monomeric or dimeric amidoboranes depending on the organyl substituents. The compounds were characterized analytically and spectroscopically (NMR:¹H,¹⁹F,¹¹B; MS; IR).

21. Mitt.:A. Meller, W. Maringgele, K. Hennemuth undU. Sicker, J. Z. anorg. allg. Chem., im Druck.     

Effect of ecological factors on conjugal transfer of chromium-resistant plasmid in Escherichia coli isolated from tannery effluent

The influence of total organic carbon (TOC), pH, and mating temperature on transfer of chromium-resistant plasmid between Escherichia coli strains in terms of variation in the number of transconjugants formed and variation in transfer frequency was investigated. In vitro transfer was studied in five chromate-tolerant E. coli strains isolated from tannery effluent using E. coli K12 J62 (Nal^r Lac⁻) as a recipient. Conjugal transfer of different selection markers was observed in three strains. The study was carried out in sterile wastewater. A gradual decrease was observed both in the number of transconjugants and in transfer frequencies as the concentration of TOC in the mating medium descended from 10,095 to 1.2 mg of C/L, obtaining the maximum values with a TOC concentration of 10,095 mg of C/L. The number of transconjugants and the transfer frequency were maximum at 30°C. However, neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. The strains were also found resistant to different heavy metals and antibiotics. Curing of these strains resulted in loss of one or more resistance markers indicating the plasmid-borne resistance. It is inferred that plasmid transfer by conjugation occurs in wastewater bodies within a wide range of conditions.     

A study of early colour change due to simulated accelerated sunlight exposure in Scots pine (Pinus sylvestris)

A detailed study of early colour change in Scots pine (Pinus sylvestris) due to accelerated simulated sunlight exposure was undertaken focusing on the first 24 h of change. Colour changes were monitored with a Datacolor check spectrophotometer and compared with a set of controls. Measurements on both samples and controls were performed hourly for the first 24 h and there after daily until 168 h’ exposure with extra measurements at 200, 350 and 500 h. A subset of samples was extracted prior to exposure to check the effects of any colour change due to the presence of extractives. Data was analysed using the reflectance spectra (400–700 nm) as well as the CIE-L^*a^*b^* system and ΔE. The majority of colour changes were found to occur within the first 24 h. This was unaffected by the removal of extractives from the wood and was independent of temperature. Mechanical properties and weight changes were also monitored to allow a comparison of sensitivity between the differing methods.