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Applied Microbiology and Biotechnology (v.56, #1-2)

Fungal hydrophobins in medical and technical applications by K. Scholtmeijer; J. Wessels; H. Wösten (pp. 1-8).

Class I and class II hydrophobins are small secreted fungal proteins that self-assemble at hydrophilic–hydrophobic interfaces into amphipathic films. Apart from eight conserved cysteine residues, the amino acid sequences between and within both classes have diverged considerably, and this is reflected in the biophysical properties of these proteins. For instance, assemblages of class I hydrophobins are highly insoluble, while those of class II hydrophobins readily dissolve in a variety of solvents. The properties of hydrophobins make them interesting candidates for use in a wide range of medical and technical applications. Each application has its own requirements, which may be met by using specific natural variants of hydrophobins or by modifying hydrophobins chemically or genetically. Applications also require high production systems for hydrophobins. In this respect, filamentous fungi that naturally secrete hydrophobins into the medium seem to be the hosts of choice.

Pharmaceutically relevant metabolites from lichens by K. Müller (pp. 9-16).

Lichen metabolites exert a wide variety of biological actions including antibiotic, antimycobacterial, antiviral, antiinflammatory, analgesic, antipyretic, antiproliferative and cytotoxic effects. Even though these manifold activities of lichen metabolites have now been recognized, their therapeutic potential has not yet been fully explored and thus remains pharmaceutically unexploited. In this mini-review, particular attention is paid to the most common classes of small-molecule constituents of lichens, from both the chemical viewpoint and with regard to possible therapeutic implications. In particular, aliphatic acids, pulvinic acid derivatives, depsides and depsidones, dibenzofuans, anthraquinones, naphthoquinones as well as epidithiopiperazinediones are described. An improved access to these lichen substances in drug discovery high-throughput screening programs will provide impetus for identifying novel lead-compounds with therapeutic potential and poses new challenges for medicinal chemistry.

Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration by J. Zaldivar; J. Nielsen; L. Olsson (pp. 17-34).

With industrial development growing rapidly, there is a need for environmentally sustainable energy sources. Bioethanol (ethanol from biomass) is an attractive, sustainable energy source to fuel transportation. Based on the premise that fuel bioethanol can contribute to a cleaner environment and with the implementation of environmental protection laws in many countries, demand for this fuel is increasing. Efficient ethanol production processes and cheap substrates are needed. Current ethanol production processes using crops such as sugar cane and corn are well-established; however, utilization of a cheaper substrate such as lignocellulose could make bioethanol more competitive with fossil fuel. The processing and utilization of this substrate is complex, differing in many aspects from crop-based ethanol production. One important requirement is an efficient microorganism able to ferment a variety of sugars (pentoses, and hexoses) as well as to tolerate stress conditions. Through metabolic engineering, bacterial and yeast strains have been constructed which feature traits that are advantageous for ethanol production using lignocellulose sugars. After several rounds of modification/evaluation/modification, three main microbial platforms, Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli, have emerged and they have performed well in pilot studies. While there are ongoing efforts to further enhance their properties, improvement of the fermentation process is just one of several factorsthat needs to be fully optimized and integrated to generate a competitive lignocellulose ethanol plant.

The use of alternative technologies to develop malolactic fermentation in wine by S. Maicas (pp. 35-39).

The development of the malolactic fermentation, bioconversion of L-malic acid to L-lactic acid, is a difficult and time-consuming process that does not always proceed favorably under the natural conditions of wine. Traditional fermentations are used worldwide to produce high-quality wines, although delay or failure is not an unusual outcome. During recent years several technologies have been proposed to induce biological deacidification of wines by using malolactic bacteria, principally Oenococcus oeni and Lactobacillus sp. These alternative technologies usually involve the use of high densities of cells or enzymes, free or immobilized onto different matrices. Immobilization materials, several types of bioreactors, and the properties of many specific systems are discussed in this review.

Application of rRNA-targeted oligonucleotide probes in biotechnology by A. Lipski; U. Friedrich; K. Altendorf (pp. 40-57).

Ribosomal RNA-targeted oligonucleotide probes have become valuable tools for the detection of microorganisms involved in important biotechnological processes. Microorganisms which are of major importance for processes such as wastewater treatment, microbial leaching or methane production can be detected and quantified in situ within a complex microbial community. For certain processes, such as nitrification or biological phosphate removal, new microorganisms have become the focus of interest and have led to an improved understanding of these bioremediation techniques. Hybridization techniques have become fast and reliable alternatives to conventional cultivation techniques in the food industry as a control method for starter cultures for fermentation processes or product control. Recent analytical tools such as flow cytometry and digital image processing have improved the efficiency of these techniques. This review is intended to present a summary of methodological aspects of rRNA-based hybridization techniques and their application in biotechnology.

The fungal biocontrol agent Coniothyrium minitans: production by solid-state fermentation, application and marketing by T. de Vrije; N. Antoine; R. Buitelaar; S. Bruckner; M. Dissevelt; A. Durand; M. Gerlagh; E. Jones; P. Lüth; J. Oostra; W. Ravensberg; R. Renaud; A. Rinzema; F. Weber; J. Whipps (pp. 58-68).

Biological control agents (BCAs) are potential alternatives for the chemical fungicides presently used in agriculture to fight plant diseases. Coniothyrium minitans is an example of a promising fungal BCA. It is a naturally occurring parasite of the fungus Sclerotinia sclerotiorum, a wide-spread pathogen which substantially reduces the yield of many crops. This review describes, exemplified by C. minitans, the studies that need to be carried out before a fungal BCA is successfully introduced into the market. The main aspects considered are the biology of C. minitans, the development of a product by mass production of spores using solid-state fermentation technology, its biocontrol activity and marketing of the final product.

Basic and applied aspects in the microbial degradation of azo dyes by A. Stolz (pp. 69-80).

Azo dyes are the most important group of synthetic colorants. They are generally considered as xenobiotic compounds that are very recalcitrant against biodegradative processes. Nevertheless, during the last few years it has been demonstrated that several microorganisms are able, under certain environmental conditions, to transform azo dyes to non-colored products or even to completely mineralize them. Thus, various lignolytic fungi were shown to decolorize azo dyes using ligninases, manganese peroxidases or laccases. For some model dyes, the degradative pathways have been investigated and a true mineralization to carbon dioxide has been shown. The bacterial metabolism of azo dyes is initiated in most cases by a reductive cleavage of the azo bond, which results in the formation of (usually colorless) amines. These reductive processes have been described for some aerobic bacteria, which can grow with (rather simple) azo compounds. These specifically adapted microorganisms synthesize true azoreductases, which reductively cleave the azo group in the presence of molecular oxygen. Much more common is the reductive cleavage of azo dyes under anaerobic conditions. These reactions usually occur with rather low specific activities but are extremely unspecific with regard to the organisms involved and the dyes converted. In these unspecific anaerobic processes, low-molecular weight redox mediators (e.g. flavins or quinones) which are enzymatically reduced by the cells (or chemically by bulk reductants in the environment) are very often involved. These reduced mediator compounds reduce the azo group in a purely chemical reaction. The (sulfonated) amines that are formed in the course of these reactions may be degraded aerobically. Therefore, several (laboratory-scale) continuous anaerobic/aerobic processes for the treatment of wastewaters containing azo dyes have recently been described.

Microbial decolourisation and degradation of textile dyes by G. McMullan; C. Meehan; A. Conneely; N. Kirby; T. Robinson; P. Nigam; I. Banat; R. Marchant; W. Smyth (pp. 81-87).

Dyes and dyestuffs find use in a wide range of industries but are of primary importance to textile manufacturing. Wastewater from the textile industry can contain a variety of polluting substances including dyes. Increasingly, environmental legislation is being imposed to control the release of dyes, in particular azo-based compounds, into the environment. The ability of microorganisms to decolourise and metabolise dyes has long been known, and the use of bioremediation based technologies for treating textile wastewater has attracted interest. Within this review, we investigate the mechanisms by which diverse categories of microorganisms, such as the white-rot fungi and anaerobic bacterial consortia, bring about the degradation of dyestuffs.

Changes in morphology of Paecilomyces japonica and their effect on broth rheology during production of exo-biopolymers by J. Sinha; J. Bae; J. Park; K. Kim; C. Song; J. Yun (pp. 88-92).

The influence of Paecilomyces japonica pellet morphology on fermentation broth rheology and exo-biopolymer production was investigated in a 5-l jar fermenter. Rapid formation of pellets was observed after the first day of fermentation; and these slowly increased in size and roughness. This, together with the increase in biomass concentration, altered the transport characteristics and broth rheology towards a pseudoplastic nature which, in turn, influenced cell growth and exo-biopolymer production. At mild agitation, high aeration and optimum substrate concentration, pellets were the most predominant morphological form, compared with free mycelia. The broth rheology showed pseudoplastic behavior; and the fungal morphology was closely related to the rheological properties.

Changes of in vivo fluxes through central metabolic pathways during the production of nystatin by Streptomyces noursei in batch culture by E. Jonsbu; B. Christensen; J. Nielsen (pp. 93-100).

The central carbon metabolism of the nystatin-producing strain Streptomyces noursei ATCC 11455 was evaluated by ¹³C-labelling experiments. A batch fermentation was examined during the idiophase by GC-MS measurements of the labelling patterns of amino acids in the biomass. The labelling patterns of the amino acids and calculated fluxes of the central metabolism showed that changes in the primary and secondary metabolisms occurred simultaneously. Changes in the profiles for the integrated fluxes showed a decreased flux through the pentose phosphate pathway and an increased flux in the tricarboxylic acid cycle relative to the glucose uptake rate when the culture entered a phase with reduced specific growth rate and enhanced nystatin yield. The flux through the pentose phosphate pathway seemed to be adjusted according to the NADPH requirement during the different phases of the batch fermentation.

Bioconversion of limonene to increased concentrations of perillic acid by Pseudomonas putida GS1 in a fed-batch reactor by A. Mars; J. Gorissen; I. van den Beld; G. Eggink (pp. 101-107).

Pseudomonas putida GS1 is able to convert limonene to perillic acid (up to 64 mM,(11 g/l) when the bacteria is cultivated in fed-batch culture with non-limiting amounts of glycerol, ammonium, and limonene. P. putida GS1 can use p-cymene as a single source of carbon and energy, and the enzymes that are responsible for the conversion of limonene to perillic acid belong to the degradation pathway of p-cymene. The p-cymene pathway of P. putida GS1 is very similar, if not identical, to the cym pathway of P. putida F1. The latter strain, and a recombinant Escherichia coli strain that carried the genes of the cym pathway of P. putida F1, also converted limonene to perillic acid. However, the final concentrations that were obtained in batch cultures with these two strains were lower than those obtained with P. putida GS1.

Effect of directional switching frequency on toluene degradation in a vapor-phase bioreactor by J. Song; K. Kinney (pp. 108-113).

A potential method to improve biomass distribution and the stability of vapor-phase bioreactors is to operate them in a directionally switching mode such that the contaminant air stream direction is periodically reversed through the reactor. In this study, the effect of switching frequency (SF) on bioreactor performance and biodegradation activity was investigated at 1-, 3- and 7-day SFs using toluene as a model compound. Rapid losses of biodegradation capacity and serious bioreactor instability were observed in the bioreactor operated at a 1-day SF. It is hypothesized that the frequent dynamic loading conditions at the 1-day SF hindered biofilm development and ultimately bioreactor stability. In contrast, bioreactors operated at the 3- and 7-day SFs achieved overall removal efficiencies of greater than 99% for 72 and 59 days of operation, respectively. Following each air-stream reversal, the bioreactor operated at the 7-day SF required 48 h to fully restore biodegradation capacity in the inlet bioreactor section. The 1-day SF bioreactor required no such reacclimation period. The toluene-degrading activity in the inlet section of the 7-day SF bioreactor dropped by 71% during the 7-day cycle, whereas it decreased by only 11% in the inlet of the 3-day SF bioreactor. These declines suggest that continuous or near-continuous exposure to toluene can inhibit microbial activity. Of the three SFs examined, the 3-day SF yielded the most efficient bioreactor performance by balancing reacclimation requirements with biodegradation activity losses.

Production and characterization of anti-nisin Z monoclonal antibodies: suitability for distinguishing active from inactive forms through a competitive enzyme immunoassay by L. Daoudi; C. Turcotte; C. Lacroix; I. Fliss (pp. 114-119).

As a pre-requisite to monoclonal antibody development, an efficient purification strategy was devised that yielded 72 mg of nisin Z from 14.5 l of Lactococcus lactis subsp. lactis biovar. diacetylactis UL 719 (L. diacetylactis UL719) culture in supplemented whey permeate. Specific monoclonal antibodies (mAbs) were produced in mice against the purified nisin Z using keyhole limpet hemocyanin as a carrier protein. These antibodies did not recognize nisin A, suggesting that the asparagine residue at position 27 is involved in antibody recognition to nisin Z. However, the high reactivity of mAbs against biologically inactive nisin Z degradation products, produced during storage of freeze-dried pure nisin Z at –70 °C, indicated that the dehydroalanine residue at position 5 (Dha₅), required for biological activity, is not necessary in nisin Z recognition by the mAb. A competitive enzyme immunoassay (cEIA) using the specific anti-nisin Z mAb was developed and used for rapid and sensitive detection and quantification of nisin Z in fresh culture supernatant, milk and whey. Detection limits of 78 ng/ml in phosphate-buffered saline, 87 ng/ml in culture supernatant, 106 ng/ml in milk and 90.5 ng/ml in whey were obtained for this assay. The cEIA using specific mAbs can be used to quantify nisin Z in food products.

Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol by N. Nichols; B. Dien; R. Bothast (pp. 120-125).

Use of agricultural biomass, other than cornstarch, to produce fuel ethanol requires a microorganism that can ferment the mixture of sugars derived from hemicellulose. Escherichia coli metabolizes a wide range of substrates and has been engineered to produce ethanol in high yield from sugar mixtures. E. coli metabolizes glucose in preference to other sugars and, as a result, utilization of the pentoses in hemicellulose-derived sugar mixtures is delayed and may be incomplete. Residual sugar lowers the ethanol yield and is problematic for downstream processing of fermentation products. Therefore, a catabolite repression mutant that simultaneously utilizes glucose and pentoses would be useful for fermentation of complex substrate mixtures. We constructed ethanologenic E. coli strains with a glucose phosphotransferase (ptsG) mutation and used the mutants to ferment glucose, arabinose, and xylose, singly and in mixtures, to ethanol. Yields were 87–94% of theoretical for both the wild type and mutants, but the mutants had an altered pattern of mixed sugar utilization. Phosphotransferase mutants metabolized the pentoses simultaneously with glucose, rather than sequentially. Based upon fermentations of sugar mixtures, a catabolite-repression mutant of ethanologenic E. coli is expected to provide more efficient fermentation of hemicellulose hydrolysates by allowing direct utilization of pentoses.

Optimisation of media and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441 by M. Hujanen; S. Linko; Y.-Y. Linko; M. Leisola (pp. 126-130).

Process variables and concentration of carbon in media were optimised for lactic acid production by Lactobacillus casei NRRL B-441. Lactic acid yield was inversely proportional to initial glucose concentration within the experimental area (80–160 g l^–1). The highest lactic acid concentration in batch fermentation, 118.6 g l^–1, was obtained with 160 g l^–1 glucose. The maximum volumetric productivity, 4.4 g l^–1 h^–1 at 15 h, was achieved at an initial glucose concentration of 100 g l^–1. Similar lactic acid concentrations were reached with a fed-batch approach using growing cells, in which case the fermentation time was much shorter. Statistical experimental design and response surface methodology were used for optimising the process variables. The temperature and pH optima for lactic acid production were 35 °C, pH 6.3. Malt sprout extract supplemented with yeast extract (4 g l^–1) appeared to be an economical alternative to yeast extract alone (22 g l^–1) although the fermentation time was a little longer. The results demonstrated both the separation of the growth and lactic acid production phases and lactic acid production by non-growing cells without any nutrient supplements. Resting L. casei cells converted 120 g l^–1 glucose to lactic acid with 100% yield and a maximum volumetric productivity of 3.5 g l^–1 h^–1.

Comparative study of the relationship between monomer structure and reactivity for two polyhydroxyalkanoate synthases by S. Zhang; M. Kamachi; Y. Takagi; R. Lenz; S. Goodwin (pp. 131-136).

Using organically synthesized hydroxyalkanoate coenzyme A thioesters, the activities of two short-chain polyhydroxalkanoate (PHA) synthases were investigated – Ralstonia eutropha PHA synthase (a type I PHA synthase) and Ectothiorhodospira shaposhnikovii PHA synthase (a type III synthase). The results indicate that the two synthases have similar activities towards most of the monomers tested. 3-Hydroxybutyryl CoA was found to be the most efficient substrate for both synthases. Changes in the side-chain length of the monomers affect monomer reactivity, with shortening of the side-chain length having the more severe effect. Hydrophobicity in the side chain appears to play an important role in the catalytic reaction. The configuration and the position of the hydroxyl group also affect the reactivity of a monomer. Monomers with the [S] configuration can not be recognized by either synthase. Moving the hydroxyl group from the β carbon to the α carbon has a much more severe effect on the reactivity of the monomer than moving the hydroxyl group to the γ carbon. The results demonstrate that the in vitro system can be used to prepare entirely novel polymers that may not be obtainable from living cells because of metabolic restrictions.

Characterization of a chitinase and an endo-β-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii by M. El-Katatny; M. Gudelj; K.-H. Robra; M. Elnaghy; G. Gübitz (pp. 137-143).

Of 24 Trichoderma isolates, T. harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and β-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa β-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (β-1,3-glucanase). K _m and K _cat were 3.8 g l^–1 and 0.71 s^–1 for the chitinase (chitin) and 1.1 g l^–1 and 52 s^–1 for the β-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the β-1,3-glucanase was specific for β-1,3-linkages in polysaccharides. Both enzymes were stable at 30°C, while at 60°C the chitinase and the β-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED₅₀(50% effective dose) value of 2.7 µg/ml.

Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium freudenreichii by P. Kiatpapan; Y. Murooka (pp. 144-149).

Several promoters from Propionibacterium freudenreichii subsp. shermanii were isolated using a promoter probe vector, pCVE1, containing the Streptomyces cholesterol oxidase gene (choA) as a reporter gene. Three of four promoters isolated exhibiting a strong activity in Escherichia coli also expressed a strong activity in P. freudenreichii subsp. shermanii IFO12426. Using two promoters with a strong activity and a previously constructed shuttle vector, pPK705, shuttling between E. coli and Propionibacterium, we constructed expression vectors for propionibacteria. To overproduce 5-aminolevulinic acid (ALA), which is the first intermediate in the synthesis of porphyrins, the ALA synthase gene (hemA) from Rhodobacter sphaeroides was recombined with the expression vectors. The activity of ALA synthase in the recombinant P. freudenreichii subsp. shermanii increased about 70-fold that in the strain without a vector. The recombinant Propionibacterium produced ALA at a maximum concentration of 8.6 mM in the absence of levulinic acid, an inhibitor of ALA dehydratase, with 1% glucose as a carbon source. The recombinant P. freudenreichii accumulated 18.8 mmol/g cells ALA in the presence of 1 mM levulinic acid and 30 mM glycine. The construction of an efficient expression vector will facilitate genetic studies of a vitamin B₁₂ producer, Propionibacterium.

Efficiency of delivery of DNA to cells by bovine papillomavirus type-1 L1/L2 pseudovirions by Y. Liu; I. Frazer; W. Liu; X. Liu; N. McMillan; K.-N. Zhao (pp. 150-156).

To investigate the efficiency of encapsidation of plasmid by papillomavirus virus-like particles (PV VLPs), and the infectivity of the resultant PV pseudovirions, Cos-1 cells were transfected with an 8-kb plasmid incorporating a green fluorescent protein (GFP) reporter gene (pGSV), and infected with bovine PV (BPV-1) L1/L2 recombinant vaccinia virus to produce BPV1 pseudovirions. Approximately 1 in 1.5×10⁴ of dense (1.35 g/ml) PV pseudovirions and 0.3 in 10⁴ of less-dense (1.29 g/ml) pseudovirions packaged an intact pGSV plasmid. The majority (>75%) of packaged plasmids contained deletions, and the deletions affected all tested genes. After exposure of Cos-1 cells to BPV-1 pseudovirions at an MOI of 40,000:1, 6% of cells expressed GFP, giving a calculated efficiency of delivery of the pGSV plasmid, by pseudovirions which had packaged an intact plasmid, of approximately 5%. Plasmid delivery was not effected by purified pGSV plasmid, was blocked by antiserum against BPV-1, and was not blocked by DNase treatment of pseudovirions, confirming that delivery was mediated by DNA within the pseudovirion. We conclude that a major limitation to the use of PV pseudovirions as a gene delivery system is that intact plasmid DNA is not efficiently selected for packaging by VLPs in cell-based pseudovirions production systems.

Expression of mouse anticreatine kinase (MAK33) monoclonal antibody in the yeast Hansenula polymorpha by H. Abdel-Salam; T. El-Khamissy; G. Enan; C. Hollenberg (pp. 157-164).

The methylotrophic yeast Hansenula polymorpha HM1-39 (ura 3 and leu 2) was used as a host strain for the expression of the F_ab fragment of the MAK33 monoclonal antibody. The MAK33 antibody reacts specifically with creatine kinase-M. The cDNA of kappa and gamma chains were inserted between the FMD or MOX promoter and the MOX terminator within the expression plasmids. In addition, the secretion signal sequence of the mating factor-alpha (prepro segment) and a fragment from glucoamylase with its secretion signal peptide, were also inserted in the expression plasmids for efficient secretion and production of the MAK33 monoclonal antibody. The co-expression of kappa and gamma chains was achieved by double transformation with kappa and then with gamma chain-expressing plasmids. The cells of H. polymorpha HM1-39 showed high mitotic stability and both uracil⁺ and leucine⁺ phenotypic stability after double transformation. Northern analysis showed a high rate of transcription of either kappa or gamma chain mRNA but not both, when the cells were grown in an induction medium. Protein analysis of double-transformed cells showed the monomers of the MAK33 antibody (kappa and gamma chains) were not assembled into a heterodimeric functional form. The expressed proteins of light and heavy chains represent about 11–12% of total cell protein and are found more inside than outside the cell. The expressed monomers show antigen-binding affinity in the Ouchterlony diffusion test; and the binding activity exhibited by cell-free extract was more than that of the cell culture supernatant.

Expression, processing and high level secretion of a virus toxin in fission yeast by T. Heintel; T. Zagorc; M. Schmitt (pp. 165-172).

The virally encoded K28 toxin of Saccharomyces cerevisiae kills sensitive yeast cells in a multi-step receptor-mediated fashion by cell cycle arrest and inhibition of DNA synthesis. In vivo, the toxin is translated as a 38 kDa preprotoxin (pptox) which is enzymatically processed to the biologically active α/β heterodimer during passage through the yeast secretory pathway. Here, we demonstrate that Schizosaccharomyces pombe, a yeast from which no natural toxin-secreting killer strains are known, is perfectly capable of expressing a killer phenotype. Episomal as well as integrating K28 pptox gene cassettes were constructed that allowed a tightly thiamine-regulated killer phenotype expression under transcriptional control of the Sch. pombe nmt1 promotor. Northern analysis of the toxin-coding transcript as well as Western analysis of the secreted toxin indicated that fission yeast is capable of expressing a correctly processed and fully functional virus toxin. Moreover, toxin secretion in recombinant Sch. pombe was at least ten-fold higher than in any natural and/or recombinant Sac. cerevisiae killer strain, indicating that pptox-derived vectors might be attractive in the fast growing field of heterologous protein expression and secretion in yeast.

Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis by H.-G. Yoon; H.-Y. Kim; Y.-H. Lim; H.-K. Kim; D.-H. Shin; B.-S. Hong; H.-Y. Cho (pp. 173-180).

The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues (Glu-50, Glu-62, and Asp-66) was changed to Asp and Gln or Asn and Glu by site-directed mutagenesis, respectively. The Asp-66→Asn and Asp-66→Glu mutation remarkably decreased kinetic parameters such as V _max and k _cat to approximately 1/1,000 those of the wild-type enzyme, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three Cys residues at positions 49, 72, and 211. The Cys-49→Ser/Tyr and Cys-72→Ser/Tyr mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However, the half-life of the Cys-211→Ser/Tyr mutant enzyme was less than 10 min at 80 °C, while that of the wild-type enzyme was about 90 min. Moreover, the residual activity of Cys-211→Ser/Tyr enzyme was substantially decreased by 8 M urea; and it lost all catalytic activity in 40% ethanol. These results show that the substitution of Cys with any amino acid residues at position 211 seems to affect the conformational stability of the chitosanase.

Polyketide synthase genes in insect- and nematode-associated fungi by T. Lee; S.-H. Yun; K. Hodge; R. Humber; S. Krasnoff; G. Turgeon; O. Yoder; D. Gibson (pp. 181-187).

Production of polyketides is accomplished through complex enzymes known as polyketide synthases (PKS); these enzymes have highly conserved domains that might be useful in screens for PKSs in diverse groups of organisms. A degenerate PCR-based approach was used to amplify PKS fragments of the ketosynthase domain from genomic DNA of a group of insect- and nematode-associated fungi. Of 157 isolates (representing 73 genera and 144 species) screened, 92 isolates generated PCR products of predicted size (~ 300 bp). The ability to detect PKS domains was a function of the number of different primer pairs employed in the screen. Cloning and sequencing revealed that 66 isolates had at least one unique PKS sequence; ten members of this set contained multiple PKS fragments, for a total of 76 unique PKS fragments. Since PKS genes appear to be widespread among fungi, a PCR-based screening system appears to be an efficient, directed means to identify organisms having the potential to produce polyketides.

The physiological effects and metabolic alterations caused by the expression of Rhizobium etli pyruvate carboxylase in Escherichia coli by R. Gokarn; J. Evans; J. Walker; S. Martin; M. Eiteman; E. Altman (pp. 188-195).

Oxaloacetate (OAA) plays an important role in the tricarboxylic acid cycle and for the biosynthesis of a variety of cellular compounds. Some microorganisms, such as Rhizobium etli and Corynebacterium glutamicum, are able to synthesize OAA during growth on glucose via either of the enzymes pyruvate carboxylase (PYC) or phosphoenolpyruvate carboxylase (PPC). Other microorganisms, including Escherichia coli, synthesize OAA during growth on glucose only via PPC because they lack PYC. In this study we have examined the effect that the R. etli PYC has on the physiology of E. coli. The expressed R. etli PYC was biotinylated by the native biotin holoenzyme synthase of E. coli and displayed kinetic properties similar to those reported for α₄ PYC enzymes from other sources. R. etli PYC was able to restore the growth of an E. coli ppc null mutant in minimal glucose medium, and PYC expression caused increased carbon flow towards OAA in wild-type E. coli cells without affecting the glucose uptake rate or the growth rate. During aerobic glucose metabolism, expression of PYC resulted in a 56% increase in biomass yield and a 43% decrease in acetate yield. During anaerobic glucose metabolism, expression of PYC caused a 2.7-fold increase in succinate concentration, making it the major product by mass. The increase in succinate came mainly at the expense of lactate formation. However, in a mutant lacking lactate dehydrogenase activity, expression of PYC resulted in only a 1.7-fold increase in succinate concentration. The decreased enhancement of succinate formation in the ldh mutant was hypothesized to be due to accumulation of pyruvate and NADH, metabolites that affect the interconversion of the active and inactive form of the enzyme pyruvate formate-lyase.

Microbial desulfurization of alkylated dibenzothiophene and alkylated benzothiophene by recombinant Rhodococcus sp. strain T09 by T. Matsui; K. Hirasawa; J. Konishi; Y. Tanaka; K. Maruhashi; R. Kurane (pp. 196-200).

The dibenzothiophene (DBT) desulfurizing operon, dsz, was introduced into various benzothiophene (BT)-desulfurizing bacteria using a Rhodococcus-E. coli shuttle vector. Of the tested recombinant bacteria, only those from Rhodococcus sp. strain T09 grew with both DBT and BT as the sole sulfur source. These recombinant cells desulfurized not only alkylated BTs, but also various alkylated DBTs, producing alkylated hydroxybiphenyls as the desulfurized products. Recombinant strain T09 also desulfurized alkylated DBT in an oil-water, two-phase resting-cell reaction. The dsz operon had the same desulfurizing activity when inserted into the vector in either orientation, indicating that the promoter region of the operon was functional in strain T09.

Genetic transformation of Trametes versicolor to phleomycin resistance with the dominant selectable marker shble by K. Bartholomew; G. Dos Santos; T. Dumonceaux; T. Charles; F. Archibald (pp. 201-204).

We have developed a stable, DNA-mediated transformation system for the white-rot basidiomycete Trametes versicolor based on the dominant selectable marker shble (phleomycin resistance). We employed a vector containing the selectable marker under control of expression sequences from the basidiomycete Schizophyllum commune and a polyethylene glycol/CaCl₂ protoplast-fusion technique to introduce the transforming DNA. This transformation system generated stable phleomycin-resistant transformants at a frequency of four to seven transformants/µg of transforming DNA.

Cloning, expression, and carbon catabolite repression of the bamM gene encoding β-amylase of Bacillus megaterium DSM319 by J.-S. Lee; K.-D. Wittchen; C. Stahl; J. Strey; F. Meinhardt (pp. 205-211).

The bamM gene from Bacillus megaterium DSM319 encoding an extracellular β-amylase was isolated and completely sequenced. Chromosomal inactivation by deletion mutagenesis resulted in total loss of amylolytic activity, indicative of a single starch-degrading enzyme. Functional characterization of the expressed protein revealed a maltogenic enzyme exhibiting optimal activities at pH 7.5 and 50 °C. Amylase expression is subject to catabolite repression by glucose. A putative cis-acting catabolite-responsive element (CRE) was identified; it is located within the bamM coding region, matching the position of the predicted signal peptide processing site. Base substitutions introduced by site-directed mutagenesis within the bamM-CRE – retaining unchanged the amino acid sequence – provoked a remarkable relief from carbon catabolite repression (CCR), thereby proving functionality of the CRE for CCR.

Functional patterns and temperature response of cellulose-fermenting microbial cultures containing different methanogenic communities by X.-L. Wu; K.-J. Chin; S. Stubner; R. Conrad (pp. 212-219).

The effect of microbial composition on the methanogenic degradation of cellulose was studied using two lines of anaerobic cellulose-fermenting methanogenic microbial cultures at two different temperatures: that at 15 °C being dominated by Methanosaeta and that at 30 °C by Methanosarcina . In both cultures, CH₄ production and acetate consumption were completely inhibited by either 2-bromoethanesulfonate or chloroform, whereas H₂ consumption was only inhibited by chloroform, suggesting that homoacetogens utilized H₂ concomitantly with methanogens. Hydrogen was the intermediate that was consumed first, while acetate continued to accumulate. At 15 °C, acetoclastic methanogenesis smoothly followed H₂-dependent CH₄ production. Fluorescence in situ hybridization showed that populations of Methanosaeta steadily increased with time from 5 to 25% of total cell counts. At 30 °C, two phases of CH₄ production were obtained, with acetate consumed after the abrupt increase of Methanosarcina from 0 to 45% of total cell counts. Whereas populations of Methanosaeta were able to adapt after transfer from 15 to 30 °C, those of Methanosarcina were not, irrespective of during which phase the cultures were transferred from 30 °C to 15 °C. Our results thus show that the community structure of methanogens indeed affects the function of a cellulose-fermenting community with respect to temperature response.

The influence of supplemental components in nutrient medium on chitosan formation by the fungus Absidia orchidis by M. Jaworska; E. Konieczna (pp. 220-224).

Chitosan, a derivative of chitin, is a natural component of some fungus cell walls. It is formed by the complex action of chitin synthase and chitin deacetylase. The in vitro activity of these two enzymes is known to be influenced by several factors. We investigated the influence of ferrous ions, manganese ions, cobalt ions, trypsin, and chitin, as individual supplements to the nutrient medium, on the in vivo activity of chitin synthase and chitin deacetylase to form chitosan in the fungus Absidia orchidis. Manganese and ferrous ions gave the most significant results. These ions increase chitosan yields through an increase in biomass production rather than an increase of chitosan content in cell walls. Manganese and ferrous ions lowered the activity of chitin deacetylase; however, their influence on the activity of chitin synthase was more complex. The effects of trypsin and chitin on biomass and cell wall chitosan content were negligible, while cobalt ions completely inhibited the growth of fungi.

Enhanced formation of laccase activity by the white-rot fungus Trametes pubescens in the presence of copper by C. Galhaup; D. Haltrich (pp. 225-232).

The white-rot fungus Trametes pubescens MB 89 has been identified as an outstanding, although not-yet-described, producer of the industrially important enzyme laccase. Extracellular laccase formation could be greatly stimulated by the addition of Cu(II) to a simple, glucose-based culture medium. Using optimum Cu concentrations (1.5–2.0 mM), maximum values for laccase activity of approximately 65 U/ml were obtained. The synthesis of the laccase protein depended on the presence of Cu in the medium as shown by Western blot analysis. Copper had to be supplemented during the exponential phase of growth for its maximal effect; addition during the stationary phase, during which laccase activity is predominantly formed, resulted in markedly reduced laccase productivity. As was shown by X-ray microanalysis of T. pubescens mycelia obtained from a laboratory fermentation, Cu was rapidly taken up by the fungal biomass. A possible explanation for this stimulatory effect of Cu on laccase biosynthesis could be a role for this enzyme activity in melanin synthesis. The stimulatory effect of Cu on laccase synthesis was also effective for several other basidiomycetes and hence could be used as a simple method to boost the production of this enzyme.

Mycelial pellet intrastructure and visualization of mycelia and intracellular lipid in a culture of Mortierella alpina by T. Hamanaka; K. Higashiyama; S. Fujikawa; E. Park (pp. 233-238).

The intrastructure of mycelial pellets of Mortierella alpina, which accumulate fatty acids in mycelia, was visualized following labeling with fluorescein isothiocyanate (FITC) and Nile red using fluorescence microscopy. The pellet was an ellipse shape, but its intrastructure was shaped as a doughnut with a cave inside. Using three-dimensional image analysis, it was shown that the lipid was produced on the edge of the pellet, which corresponded to the area where the mycelial density was high. The cavity ratio of the pellet section was determined on the basis of the FITC fluorescence intensity, and in the early culture stage remained at 0.2 in a 10-kl fermentor culture, but finally increased to 0.35. Mycelial pellet volume paralleled the cavity ratio. Application of the technique used here allows analysis of the intrastructure of fungal pellets and new types of fungal biological study.

Accumulation of 2,5-dimethoxy-1,4-benzoquinone in suspension cultures of Panax ginseng by a fungal elicitor preparation and a yeast elicitor preparation by C. Kim; H. Im; H. Kim; H. Huh (pp. 239-242).

Suspension cultures of Panax ginseng C.A. Meyer (Araliaceae) were treated with either an elicitor preparation from the culture broth of the phytopathogenic hyphomycete Botrytis cinerea or a yeast elicitor preparation, and the accumulation of a new compound, which was not detected in non-elicited cultures, was observed. The accumulated compound was isolated and shown to be 2,5-dimethoxy-1,4-benzoquinone by ¹H-NMR, ¹³C-NMR and electron ionization (EI) mass spectra. While it is well known that this compound shows antibacterial activity against Staphylococcus aureus, its presence in ginseng root has not been reported to date. Levels of the compound in the media increased rapidly, reaching a maximum level of 65.10±4.96 µg/g fresh weight at approximately 12 h after treatment with the yeast elicitor preparation. The maximal level of the compound in medium from the culture treated with an elicitor preparation from the culture broth of B. cinerea was 46.13±10.42 µg/g fresh weight after 24 h of incubation.

Antimicrobial activity of argon fluoride (ArF) excimer laser on gram-negative bacteria by E. Charvalos; A. Karoutis (pp. 243-248).

The objective of this study was to evaluate the antibacterial activity of argon fluoride (ArF) excimer laser radiation on clinically important strains of gram-negative bacteria. The antibacterial activity of ArF excimer laser radiation was evaluated on two Acinetobacter baumannii, one Enterobacter cloacae, three Escherichia coli, two Helicobacter pylori, one Klebsiella pneumoniae and two Pseudomonas aeruginosa strains. The strains were isolated from clinical specimens and typed by the usual biochemical procedures. Square agar plates of 12×12 cm were divided into rectangular (2×3 cm) regions and spread with 0.5×10⁴ colony forming units (CFU)/ml of bacterial suspension. The excess liquid was removed and the plates were allowed to dry for 30 min. A total of 96 rectangular (2×3 cm) regions were used for each strain, in order to test an equal number of laser parameters. Each rectangular region was irradiated with different laser parameters, using a 193 nm ArF excimer laser, linked with a simple Galilean afocal system and a rectangular diaphragm of the same dimensions as the original laser beam cross-section, at a distance of 10 cm from the irradiated surface. This system was used in order to keep the laser pulse energy under 80 mJ and to cut-out the non-transverse electromagnetic mode branches of the laser beam. We then studied the bacterial survival ratio versus the number of laser pulses, the repetition frequency and the total laser beam fluence. Our results showed that the total laser beam fluence was the most important parameter to consider in evaluating the bactericidal effect of ArF excimer laser radiation. A critical value of the total fluence was determined for each strain, such that, for laser beam fluences greater than this critical value, no colonies appeared to survive while, for laser fluences less than this critical value, the survival ratio did not exceed 2×10^–7 CFU (2×10^–5%). These critical values were found to vary between 8 J/cm² and 16 J/cm²for the bacterial species studied. Under these conditions, ArF laser irradiation is promising for the sterilisation of hard surfaces and for in situ application.

The effect of osmotic pressure on the membrane fluidity of Saccharomyces cerevisiae at different physiological temperatures by C. Laroche; L. Beney; P. Marechal; P. Gervais (pp. 249-254).

Membrane fluidity in whole cells of Saccharomyces cerevisiae W303-1A was estimated from fluorescence polarization measurements using the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, over a wide range of temperatures (6–35 °C) and at seven levels of osmotic pressure between 1.38 MPa and 133.1 MPa. An increase in phase transition temperatures was observed with increasing osmotic pressure. At 1.38 MPa, a phase transition temperature of 12±2 °C was observed, which increased to 17±4 °C at 43.7 MPa, 21±7 °C at 61.8 MPa, and 24±9 °C at an osmotic pressure of 133.1 MPa. From these results we infer that, with increases in osmotic pressure, the change in phospholipid conformation occurs over a larger temperature range. These results allow the representation of membrane fluidity as a function of temperature and osmotic pressure. Osmotic shocks were applied at two levels of osmotic pressure and at nine temperatures, in order to relate membrane conformation to cell viability.

Introduction of green fluorescent protein gene into phenol-degrading Alcaligenes faecalis cells and their monitoring in phenol-contaminated soil by A. Bastos; M. Cassidy; J. Trevors; H. Lee; A. Rossi (pp. 255-260).

Alcaligenes faecalis (CCT 7145) was isolated from an Amazonian soil sample after an enrichment process to select for phenol-degrading microorganisms. The isolate was labeled with the green fluorescent protein (gfp) gene. The gfp-transformed cells were easily detected using a hand-held UV transilluminator and their taxonomy was confirmed by 16S rRNA sequencing. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the gfp gene was integrated into the chromosome. The addition of the gfp marker did not affect phenol degradation ability compared with the wild-type. Both, wild-type and gfp-marked A. faecalis cells encapsulated in alginate, tolerated 1,700 µg ml^–1 phenol in liquid medium compared with 1,100 µg ml^–1 phenol for free cells. ¹⁴C-Phenol mineralization in soil microcosms was also enhanced by inoculation with encapsulated cells. Survival of gfp-marked cells in phenol-contaminated soil over 22 days was determined from plate counts using an epifluorescence microscope.

A simple mediatorless amperometric method using the cyanobacterium Synechococcus leopoliensis for the detection of phytotoxic pollutants by L. Croisetière; R. Rouillon; R. Carpentier (pp. 261-264).

The unicellular cyanobacterium Synechoccocus leopoliensis is used in a micro-electrochemical cell to generate photocurrents. The photocurrent is dependent on photosynthetic electron transport and is mediated by hydrogen peroxide formation following the reduction of oxygen on the acceptor side of photosystem I. This is the first known application of cyanobacteria in an electrochemical device where no artificial electroactive mediator is needed. The potential for the development of this micro-electrochemical cell for the detection of phytotoxic pollutants, such as herbicides and toxic metal cations, using the photosynthetic system of the cyanobacteria without interference from added electron acceptor is discussed.

Aerobic degradation of mixtures of tetrachloroethylene, trichloroethylene, dichloroethylenes, and vinyl chloride by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1 by H. Shim; D. Ryoo; P. Barbieri; T. Wood (pp. 265-269).

A recombinant strain of Escherichia coli (JM109/pBZ1260) expressing constitutively toluene-o-xylene monooxygenase (ToMO) of Pseudomonas stutzeri OX1 degraded binary mixtures (100 µM each) of tetrachloroethylene (PCE) with either trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), or vinyl chloride (VC). PCE degradation was 8–20% for these binary mixtures, while TCE and trans-DCE with PCE were degraded at 19%, 1,1-DCE at 37%, cis-DCE at 97%, and VC at 27%. The host P. stutzeri OX1 was also found to degrade binary mixtures of PCE/TCE, PCE/cis-DCE, and PCE/VC when induced with toluene. Degradation of quaternary mixtures of PCE/TCE/trans-DCE/VC and PCE/TCE/cis-DCE/VC by JM109/pBZ1260 were also investigated as well as mixtures of PCE/TCE/trans-DCE/1,1-DCE/cis-DCE/VC; when all the chlorinated compounds were present, the best degradation occurred with 24–51% removal of each. For these degradation reactions, 39–85% of the stoichiometric chloride expected from complete degradation of the chlorinated ethenes was detected. The time course of PCE/TCE/1,1-DCE degradation was also measured for a mixture of 8, 17, and 6 µM, respectively; initial degradation rates were 0.015, 0.023, and 0.029 nmol/min·mg protein, respectively. This indicates that for the first time an aerobic enzyme can degrade mixtures of all chlorinated ethenes, including the once – so it was believed – completely recalcitrant PCE.

Isolation and characterization of a thermotolerant bacterium Ralstonia sp. strain PHS1 that degrades benzene, toluene, ethylbenzene, and o-xylene by S.-K. Lee; S. Lee (pp. 270-275).

A thermotolerant bacterium, designated as PHS1, was isolated from a hot spring in Pohang, Korea, on the basis of its ability to grow on benzene, toluene, ethylbenzene, and xylenes (BTEX) as a sole carbon source. Strain PHS1 is a gram-negative, rod-shaped aerobe and grows optimally at 42 °C and pH 7.2. According to 16 S rDNA analysis, strain PHS1 showed highest similarity to Ralstonia eutropha (previously named Alcaligenes eutrophus). Unlike its closest known Ralstonia species, however, strain PHS1 was able to utilize toluene, ethylbenzene, o-xylene, and both m- and o-cresol. The degradation of o-xylene by strain PHS1 is particularly important, since o-xylene is a compound of considerable environmental interest, owing to its recalcitrance; and very few microorganisms have been reported to utilize o-xylene as a sole carbon source. It was found that strain PHS1 transformed o-xylene to 2,3-dimethylphenol through direct oxygenation of the aromatic ring. The unique properties of strain PHS1, such as thermotolerance and the ability to degrade o-xylene, may have important implications for the treatment of BTEX-contaminated industrial effluents.

Bioaccumulation of mercury from wastewater by genetically engineered Escherichia coli by X. Deng; D. Wilson (pp. 276-279).

Genetically engineered E. coli, which express both a Hg²⁺ transport system and metallothionein, were tested for their ability to remove mercury from wastewater. The wastewater contained more than ten different ions, including 2.58 mg/l mercury, and its pH was 9.6. Mercury uptake was faster from the wastewater than from distilled water, probably because of the higher ionic strength, as the high pH had little effect on mercury accumulation. EDTA also stimulated mercury uptake rather than inhibiting it. A hollow-fiber bioreactor was used to retain induced cells for continuous mercury uptake. The cells removed more than 99% of the mercury in the wastewater and the final amount of mercury accumulated was 26.8 mg/g cell dry weight, while none of the other ions were removed from the water. These results indicated that the induced cells had a high affinity and specificity for mercury.

Methane fermentation of coastal mud sediment by a two-stage upflow anaerobic sludge blanket (UASB) reactor system by K. Takeno; Y. Nakashimada; T. Kakizono; N. Nishio (pp. 280-285).

The removal of organic matter from a coastal mud sediment was carried out by a methane fermentation process under anaerobic conditions. In a batch acidogenic fermentation, the addition of vitamins containing thiamine, nicotinic acid and biotin dramatically enhanced acetate production from the mud sediment (200 g wet wt l^–1 artificial sea water), yielding 77 mM acetate after 6 days, which corresponded to 77% of the organic matter in the mud sediment, measured on the basis of chemical oxygen demand. Thereafter, the two-fold diluted, post-acidogenic fermentation liquor (PAF liquor) was continuously treated at 2.4× original dilution rate day^–1 for 30 days, using an upflow anaerobic sludge blanket methanogenic reactor containing the acclimated methanogenic sludge from the mud sediment. Acetate, 42 mM in the PAF liquor, was converted to methane at a maximum methane production rate of 96 mmol l^–1 day^–1; and 87.5% of the acetate and 88.7% of the total organic carbon in the PAF liquor were removed. Moreover, an efficient treatment of the mud sediment was carried out by a semi-continuous, two-stage reactor system, where the culture broth was circulated between acidogenic and methanogenic reactors. This two-stage reactor system gave a stable operation at 4-day intervals for one treatment period, yielding 112 mmol methane from the wet mud in the PAF liquor (278 g l^–1).

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Applied Microbiology and Biotechnology (v.56, #1-2)