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Applied Microbiology and Biotechnology (v.77, #4)

80th birthday of Prof. Dr. Hans-Jürgen Rehm—congratulations to the founder of ‘Applied Microbiology and Biotechnology’ by Alexander Steinbüchel (pp. 749-750).

Perspectives of biotechnological production of l-tyrosine and its applications by Tina Lütke-Eversloh; Christine Nicole S. Santos; Gregory Stephanopoulos (pp. 751-762).

The aromatic amino acid l-tyrosine is used as a dietary supplement and has promise as a valuable precursor compound for various industrial and pharmaceutical applications. In contrast to chemical production, biotechnological methods can produce l-tyrosine from biomass feedstocks under environmentally friendly and near carbon-free conditions. In this minireview, various strategies for synthesizing l-tyrosine by employing biocatalysts are discussed, including initial approaches as well as more recent advances. Whereas early attempts to engineer l-tyrosine-excreting microbes were based on auxotrophic and antimetabolite-resistant mutants, recombinant deoxyribonucleic acid technology and a vastly increasing knowledge of bacterial physiology allowed recently for more targeted genetic manipulations and strain improvements. As an alternative route, l-tyrosine can also be obtained from the conversion of phenol, pyruvate, and ammonia or phenol and serine in reactions catalyzed by the enzyme tyrosine phenol lyase.

Keywords: Aromatic amino acids; Tyrosine; Metabolic engineering; Tyrosine phenol lyase; Escherichia coli ; Corynebacterium glutamicum

Influence of culture conditions on glutathione production by Saccharomyces cerevisiae by Lucielen Oliveira Santos; Tatiane Araujo Gonzales; Beatriz Torsani Úbeda; Ranulfo Monte Alegre (pp. 763-769).

A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20–30°C), agitation rate (100–300 rpm), initial pH (5.0–7.0), inoculum concentration (5–15%), and glucose concentration (30–70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2^5–2), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2²) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20°C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.

Keywords: Glutathione; Saccharomyces cerevisiae ; Culture optimization; Experimental design

Biotransformation of diosgenin to nuatigenin-type steroid by a newly isolated strain, Streptomyces virginiae IBL-14 by Feng-Qing Wang; Bo Li; Wei Wang; Cheng-Gang Zhang; Dong-Zhi Wei (pp. 771-777).

An actinomycete strain IBL-14 isolated from soil by utilizing diosgenin as the sole carbon and energy source was identified as Streptomyces virginiae. S. virginiae IBL-14 can transform diosgenin to isonuatigenone. To our knowledge, this is the first reported case of producing rare nuatigenin-type spirosteroid (isonuatigenone) from pyrano-spirosteroid (diosgenin) by microbial transformation. From diosgenin to isonuatigenone, the pathway has been confirmed in this study that diosgenin was first converted to diosgenone, and then diosgenone was transformed to isonuatigenone by the C25 tertiary hydroxylation reaction. It appeared to be favorable to accumulate isonuatigenone when diosgenin was added to the onset of the stationary phase of cell growth, and the yield of isonuatigenone was about 28.4% during 48 h from 1.5 mM diosgenin. The C25 tertiary hydroxylation of diosgenone by S. virginiae IBL-14 is a novel and interesting reaction and will be a practical tool in producing natural nuatigenin-type steroids.

Keywords: Diosgenin; Diosgenone; Isonuatigenone; Nuatigenin-type steroid; Tertiary hydroxylation; Streptomyces virginiae

Development of a fed-batch process for the production of a dye-linked formaldehyde dehydrogenase in Hyphomicrobium zavarzinii ZV 580 by Valérie Jérôme; Markus Hermann; Frank Hilbrig; Ruth Freitag (pp. 779-788).

The dye-linked formaldehyde dehydrogenase (dlFalDH) from Hyphomicrobium zavarzinii ZV 580 processes formaldehyde in a highly selective manner and without need for NAD(P). The enzyme thus has considerable potential for technical applications if the difficulties associated with its efficient production can be resolved. In this contribution, a fed-batch bioprocess is developed, which improves both the biomass production of H. zavarzinii ZV 580 (from 0.6 to 2 g l⁻¹ dry mass) and the specific dlFalDH production (from 0.1 to 0.3 units g⁻¹ biomass), resulting in an overall improvement of the productivity by more than an order of magnitude compared to the previously reported process (Klein et al., Biochem J 301:289–295, 1994). In particular, the process uses an automated feeding strategy controlled via the dissolved oxygen concentration. In addition, our results show that the growth of H. zavarzinii ZV 580 is rather sensitive toward increasing salt concentration in the culture medium. Growth is also inhibited by the presence of surfactant-based antifoam reagents. Adjustment of the pH via the addition of methylamine instead of NaOH, on the other hand, leads to an increase in biomass yield.

Keywords: Hyphomicrobium zavarzinii ; Dye-linked formaldehyde dehydrogenase; Fed-batch culture; Fermentation optimization; Process development

Optimization of culture conditions and scale-up to pilot and plant scales for vancomycin production by Amycolatopsis orientalis by Hyung-Moo Jung; Sang-Yong Kim; Hee-Jung Moon; Deok-Kun Oh; Jung-Kul Lee (pp. 789-795).

This report describes the optimization of culture conditions for vancomycin production by Amycolatopsis orientalis KCCM-10836P, an identified high-vancomycin-producing strain (US11/712,494). Among the conditions tested, pH and the dissolved oxygen tension (DOT) were key factors affecting vancomycin production. When the pH and DOT were controlled at 7.0 and 20–30%, respectively, a dry-cell weight (DCW) of 62.0 g l⁻¹ and a vancomycin production of 11.5 g l⁻¹ were obtained after 120 h of batch culture, corresponding to a specific vancomycin content of 185.4 mg g-DCW⁻¹. Vancomycin production was scaled up from a laboratory scale (7-l fermentor) to a pilot scale (300 l) and a plant scale (5,000 l) using the impeller tip velocity (V _tip) as a scale-up parameter. Vancomycin production at the laboratory scale was similar to those at the pilot and plant scales.

Keywords: Amycolatopsis orientalis ; Vancomycin; Optimization of culture conditions; DOT; Scale-up; Impeller tip velocity

Standardized biosynthesis of flavan-3-ols with effects on pancreatic beta-cell insulin secretion by Joseph A. Chemler; Lye T. Lock; Mattheos A. G. Koffas; Emmanuel S. Tzanakakis (pp. 797-807).

Flavan-3-ols, such as green tea catechins represent a major group of phenolic compounds with significant medicinal properties. We describe the construction and optimization of Escherichia coli recombinant strains for the production of mono- and dihydroxylated catechins from their flavanone and phenylpropanoid acid precursors. Use of glucose minimal medium, Fe(II), and control of oxygen availability during shake-flask experiments resulted in production yield increases. Additional production improvement resulted from the use of medium rather than high-copy number plasmids and, in the case of mono-hydroxylated compounds, the addition of extracellular cofactors in the culture medium. The established metabolic engineering approach allowed the biosynthesis of natural catechins at high purity for assessing their possible insulinotropic effects in pancreatic β-cell cultures. We demonstrated that (+)-afzelechin and (+)-catechin modulated the secretion of insulin by pancreatic β-cells. These results indicate the potential of applying metabolic engineering approaches for the synthesis of natural and non-natural catechin analogues as drug candidates in diabetes treatments.

Keywords: Flavonoids; Catechins; Metabolic engineering; Insulin; Diabetes; Flavan-3-ols.

Laccase-initiated cross-linking of lignocellulose fibres using a ultra-filtered lignin isolated from kraft black liquor by G. Elegir; D. Bussini; S. Antonsson; M. E. Lindström; L. Zoia (pp. 809-817).

In this work, the effect of Trametes pubescens laccase (TpL) used in combination with a low-molecular-weight ultra-filtered lignin (UFL) to improve mechanical properties of kraft liner pulp and chemi-thermo-mechanical pulp was studied. UFL was isolated by ultra-filtration from the kraft cooking black liquor obtained from softwood pulping. This by-product from the pulp industry contains an oligomeric lignin with almost twice the amount of free phenolic moieties than residual kraft pulp lignin. The reactivity of TpL on UFL and kraft pulp was studied by nuclear magnetic resonance spectroscopy and size exclusion chromatography. Laccase was shown to polymerise UFL and residual kraft pulp lignin in the fibres, seen by the increase in their average molecular weight and in the case of UFL as a decrease in the amount of phenolic hydroxyls. The laccase initiated cross-linking of lignin, mediated by UFL, which gives rise to more than a twofold increase in wet strength of kraft liner pulp handsheets without loosing other critical mechanical properties. Hence, this could be an interesting path to decrease mechano-sorptive creep that has been reported to lessen in extent as wet strength is given to papers. The laccase/2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) mediator system showed a greater increase in wet tensile strength of the resulting pulp sheets than the laccase/UFL system. However, other mechanical properties such as dry tensile strength, compression strength and Scott Bond internal strength were negatively affected by the laccase/ABTS system.

Keywords: Laccase; oxidation; Ultra-filtered lignin; Mediator; Wet strength; Biorefinery; Kraft pulp; Fibre bonding

Reactive blue 19 decolouration by laccase immobilized on silica beads by P.-P. Champagne; J.A. Ramsay (pp. 819-823).

Laccase (31.5 U of activity/g or 4.39 μg of protein/m²) from Trametes versicolor was immobilized on controlled-porosity-carrier silica beads and evaluated for the decolouration of Reactive blue 19, an anthraquinone dye. Although there was an initial, rapid adsorption of the dye to the packed bed in a recirculating reactor, about 97.5% of Reactive blue 19 removal was due to enzymatic degradation. The free enzyme lost 52% of its activity in 48 h. However, the activity of the immobilized laccase was unchanged after 4 months of storage in phosphate buffer under ambient conditions followed by three successive decolourations over 120 h. Treating the laccase immobilized beads with ethanolamine reduced dye adsorption by 40%.

Keywords: Decolouration; Laccase; Dye; Immobilized

Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast Cryptococcus aureus G7a and its nutritive analysis by Lingmei Gao; Zhenming Chi; Jun Sheng; Xiumei Ni; Lin Wang (pp. 825-832).

After crude protein of the marine yeast strains maintained in this laboratory was estimated by the method of Kjehldahl, we found that the G7a strain which was identified to be a strain of Cryptococcus aureus according to the routine identification and molecular methods contained high level of protein and could grow on a wide range of carbon sources. The optimal medium for single-cell protein production was seawater containing 6.0 g of wet weight of Jerusalem artichoke extract per 100 ml of medium and 4.0 g of the hydrolysate of soybean meal per 100 ml of medium, while the optimal conditions for single-cell protein production were pH 5.0 and 28.0°C. After fermentation for 56 h, 10.1 g of cell dry weight per liter of medium and 53.0 g of crude protein per 100 g of cell dry weight (5.4 g/l of medium) were achieved, leaving 0.05 g of reducing sugar per 100 ml of medium and 0.072 g of total sugar per 100 ml of medium total sugar in the fermented medium. The yeast strain only contained 2.1 g of nucleic acid per 100 g of cell dry weight, but its cells contained a large amount of C_16:0 (19.0%), C_18:0 (46.3%), and C_18:1 (33.3%) fatty acids and had a large amount of essential amino acids, especially lysine (12.6%) and leucine (9.1%), and vitamin C (2.2 mg per 100 g of cell dry weight). These results show that the new marine yeast strain was suitable for single-cell protein production.

Keywords: Single-cell protein; Essential amino acids; Cryptococcus aureus ; Marine yeasts

Engineering the phenylacetaldehyde reductase mutant for improved substrate conversion in the presence of concentrated 2-propanol by Yoshihide Makino; Tohru Dairi; Nobuya Itoh (pp. 833-843).

Phenylacetaldehyde reductase (PAR) from Rhodococcus sp. ST-10 is useful for chiral alcohol production because of its broad substrate specificity and high stereoselectivity. The conversion of ketones into alcohols by PAR requires the coenzyme NADH. PAR can regenerate NADH by oxidizing additional alcohols, especially 2-propanol. However, substrate conversion by wild-type PAR is suppressed in concentrated 2-propanol. Previously, we developed the Sar268 mutant of PAR, which can convert several substrates in the presence of concentrated 2-propanol. In this paper, further mutational engineering of Sar268 was performed to achieve higher process yield. Each of nine amino acid positions that had been examined for generating Sar268 was subjected to saturation mutagenesis. Two novel substitutions at the 42nd amino acid position increased m-chlorophenacyl chloride (m-CPC) conversion. Moreover, several nucleotide substitutions identified from libraries of random mutations around the start codon also improved the PAR activity. E. coli cells harboring plasmid pHAR1, which has the integrated sequence of the top clones from the above selections, provided greater conversion of m-CPC and ethyl 4-chloro-3-oxobutanoate than the Sar268 mutant, with very high optical purity of products. This mutant is a promising novel biocatalyst for efficient chiral alcohol production.

Keywords: Asymmetric reduction; Phenylacetaldehyde reductase; 2-Propanol; Enzyme engineering; Chiral alcohols

Cloning, characterization, and mutational analysis of a highly active and stable l-arabinitol 4-dehydrogenase from Neurospora crassa by Ryan Sullivan; Huimin Zhao (pp. 845-852).

An NAD⁺-dependent l-arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12) from Neurospora crassa was cloned and expressed in Escherichia coli and purified to homogeneity. The enzyme was a homotetramer and contained two Zn²⁺ ions per subunit, displaying similar characteristics to medium-chain sorbitol dehydrogenases (SDHs). High enzymatic activity was observed for substrates l-arabinitol, adonitol, and xylitol and no activity for d-mannitol, d-arabinitol, or d-sorbitol. The enzyme showed strong preference for NAD⁺ but also displayed a very low yet detectable activity with NADP⁺. Mutational analysis of residue F59, the single different substrate-binding residue between LADs and d-SDHs, failed to confer the enzyme the ability to accept d-sorbitol as a substrate, suggesting that the amino acids flanking the active site cleft may be responsible for the different activity and affinity patterns between LADs and SDHs. This enzyme should be useful for in vivo and in vitro production of xylitol and ethanol from l-arabinose.

Keywords: Arabinose fermentation; Xylitol production; N. crassa genome; Alcohol dehydrogenase; Homology modeling; Ethanol production

Analyses of the acetate-producing pathways in Corynebacterium glutamicum under oxygen-deprived conditions by Kaori Yasuda; Toru Jojima; Masako Suda; Shohei Okino; Masayuki Inui; Hideaki Yukawa (pp. 853-860).

Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetyl-coenzyme A (CoA). Simultaneous disruption of pta, encoding phosphotransacetylase, and ack, encoding acetate kinase, resulted in no measurable change in acetate productivity. A mutant strain with disruptions in pta, ack and as-yet uncharacterized gene (cgR2472) exhibited 65% reduced acetate productivity compared to the parental strain, although a single disruption of cgR2472 exhibited no effect on acetate productivity. The gene cgR2472 was shown to encode a CoA-transferase (CTF) that catalyzes the formation of acetate from acetyl-CoA. These results indicate that PTA-ACK as well as CTF is involved in acetate production in C. glutamicum. This study provided basic information to reduce acetate production under oxygen-deprived conditions.

Keywords: Acetate-producing pathway; Corynebacterium glutamicum ; CoA-transferase

Overexpression of the ICL1 gene changes the product ratio of citric acid production by Yarrowia lipolytica by André Förster; Kordula Jacobs; Thomas Juretzek; Stephan Mauersberger; Gerold Barth (pp. 861-869).

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric (CA) and isocitric (ICA) acids, triggered by growth limitation caused by different factors and an excess of carbon source. Depending on the carbon source used, Y. lipolytica strains produce a mixture of CA and ICA in a characteristic ratio. To examine whether the CA/ICA product ratio can be influenced by gene-dose-dependent overexpression or by disruption of the isocitrate lyase (ICL)-encoding gene ICL1, recombinant Y. lipolytica strains were constructed, which harbour multiple ICL1 copies or a defective icl1 allele. The high-level expression of ICL in ICL1 multicopy integrative transformants resulted in a strong shift of the CA/ICA ratio into direction of CA. On glycerol, glucose and sucrose, the ICA proportion decreased from 10–12% to 3–6%, on sunflower oil or hexadecane even from 37–45% to 4–7% without influencing the total amount of acids (CA and ICA) produced. In contrast, the loss of ICL activity in icl1-defective strains resulted in a moderate 2–5% increase in the ICA proportion compared to ICL wild-type strains on glucose or glycerol.

Keywords: Yarrowia lipolytica ; ICL1 overexpression; Isocitrate lyase; Citric acid; Isocitric acid; Product selectivity

Site-directed integration system using a combination of mutant lox sites for Corynebacterium glutamicum by Nobuaki Suzuki; Masayuki Inui; Hideaki Yukawa (pp. 871-878).

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an optimal C. glutamicum genome, a precise site-directed gene integration method was developed by using a pair of mutant lox sites, one a right element (RE) mutant lox site and the other a left element (LE) mutant lox site. Two DNA fragments, 5.7 and 10.2 kb-long, were successfully integrated into the genome. The recombination efficiency of this system compared to that obtained by single crossover by homologous recombination was 2 orders of magnitude higher. Moreover, the integrated DNA remained stably maintained on removal of Cre recombinase. The Cre/mutant lox system thus represents a potentially attractive tool for integration of foreign DNA in the course of the engineering of C. glutamicum traits.

Keywords: Corynebacterium glutamicum ; Cre/loxP ; Integration

Enhanced hydrogen production from glucose by metabolically engineered Escherichia coli by Toshinari Maeda; Viviana Sanchez-Torres; Thomas K. Wood (pp. 879-890).

To utilize fermentative bacteria for producing the alternative fuel hydrogen, we performed successive rounds of P1 transduction from the Keio Escherichia coli K-12 library to introduce multiple, stable mutations into a single bacterium to direct the metabolic flux toward hydrogen production. E. coli cells convert glucose to various organic acids (such as succinate, pyruvate, lactate, formate, and acetate) to synthesize energy and hydrogen from formate by the formate hydrogen-lyase (FHL) system that consists of hydrogenase 3 and formate dehydrogenase-H. We altered the regulation of FHL by inactivating the repressor encoded by hycA and by overexpressing the activator encoded by fhlA, removed hydrogen uptake activity by deleting hyaB (hydrogenase 1) and hybC (hydrogenase 2), redirected glucose metabolism to formate by using the fdnG, fdoG, narG, focA, focB, poxB, and aceE mutations, and inactivated the succinate and lactate synthesis pathways by deleting frdC and ldhA, respectively. The best of the metabolically engineered strains, BW25113 hyaB hybC hycA fdoG frdC ldhA aceE, increased hydrogen production 4.6-fold from glucose and increased the hydrogen yield twofold from 0.65 to 1.3 mol H₂/mol glucose (maximum, 2 mol H₂/mol glucose).

Keywords: Enhanced hydrogen production; Metabolic engineering; P1 transduction; Glucose metabolism; Fermentative hydrogen

Co-ordinate expression of glycine betaine synthesis genes linked by the FMDV 2A region in a single open reading frame in Pichia pastoris by Sanhong Wang; Quanhong Yao; Jianmin Tao; Yushan Qiao; Zhen Zhang (pp. 891-899).

The genes encoding the two enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) of glycine betaine synthesis in Suaeda salsa were cloned and fused with the 2A region of foot-and-mouth disease virus in a single open reading frame. The fused genes were placed under the control of the alcohol oxidase (AOX1) promoter in pPIC3B and transformed into P. pastoris GS115. The expression of the fused genes in P. pastoris and the ability of recombinant yeasts to tolerate environmental stresses were studied. The results showed that induced with 0.5% methanol for 96 h, the maximal activities of CMO and BADH in the tested recombinant yeasts were 45- and 44-fold higher than those in the control yeast transformed empty vector only, respectively; the content of glycine betaine in the recombinant yeasts was 28- to 35-fold higher than that in the control. The fused genes linked by 2A region of foot-and-mouth disease virus were expressed in P. pastoris successfully and the polyprotein was ‘cleaved’ to each functional protein. The yeasts transformed the fused genes, which were more resistant to salt, methanol, and high temperature stresses than the control as result of glycine betaine synthesis genes introduced.

Keywords: Glycine betaine synthesis genes; 2A region of foot-and-mouth disease virus; Coexpression; Stress tolerance; Pichia pastoris

A heat shock protein gene (hsp22.4) from Chaetomium globosum confers heat and Na₂CO₃ tolerance to yeast by Z. H. Liu; Q. Yang; J. Ma (pp. 901-908).

A small heat shock protein gene (hsp22.4) was cloned from Chaetomium globosum using rapid amplification of cDNA ends (RACE). The 986-bp full-length hsp22.4 cDNA contains a 609-bp open reading frame encoding a 202-amino-acid protein with an estimated molecular mass of 22.4 kDa. The hsp22.4 gene was amplified using specific primers in the 5′ and 3′ untranslated regions of the hsp22.4 cDNA. The temporal expression of hsp22.4 was measured in C. globosum by real-time reverse transcriptase-polymerase chain reaction after exposure to heat, cold, Na₂CO₃, and NaCl. The expression of hsp22.4 was induced by heat and Na₂CO₃ treatment and inhibited by cold and NaCl treatment. The hsp22.4 gene was inserted into pYES2 containing the inducible GAL1 promoter and transferred into yeast (Saccharomyces cerevisiae) for expression. The hsp22.4 transgenic yeast displayed significantly greater resistance to heat and Na₂CO₃ stresses than control (yeast cells transformed with empty pYES2), suggesting that the expression of hsp22.4 gene confers not only heat tolerance but also significant alkali (Na₂CO₃) stress tolerance.

Keywords: Chaetomium globosum ; Heat shock protein; Gene cloning; Stress tolerance; Yeast expression

Improved adaptation to heat, cold, and solvent tolerance in Lactobacillus plantarum by D. Fiocco; V. Capozzi; P. Goffin; P. Hols; Giuseppe Spano (pp. 909-915).

The effect of overproducing each of the three small heat shock proteins (Hsp; Hsp 18.5, Hsp 18.55, and Hsp 19.3) was investigated in Lactobacillus plantarum strain WCFS1. Overproduction of the three genes, hsp 18.5, hsp 18.55, and hsp 19.3, translationally fused to the start codon of the ldhL gene yielded a protein of approximately 19 kDa, as estimated from Tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis in agreement with the predicted molecular weight of small Hsps. Small Hsp overproduction alleviated the reduction in growth rate triggered by exposing exponentially growing cells to heat shock (37 or 40°C) and cold shock (12°C). Moreover, overproduction of Hsp 18.55 and Hsp 19.3 led to an enhanced survival in the presence of butanol (1% v/v) or ethanol (12% v/v) treatment suggesting a potential role of L. plantarum small Hsps in solvent tolerance.

Keywords: Lactobacillus plantarum ; Heat shock; Overexpression plasmids; Solvent tolerance

Genes differentially expressed by Aspergillus flavus strains after loss of aflatoxin production by serial transfers by Perng-Kuang Chang; Jeffery R. Wilkinson; Bruce W. Horn; Jiujiang Yu; Deepak Bhatnagar; Thomas E. Cleveland (pp. 917-925).

Aflatoxins are carcinogenic fungal secondary metabolites produced by Aspergillus flavus and other closely related species. Levels of aflatoxins in agricultural commodities are stringently regulated by many countries because of the health hazard, and thus, aflatoxins are of major concern to both producers and consumers. A cluster of genes responsible for aflatoxin biosynthesis has been identified; however, expression of these genes is a complex and poorly understood phenomenon. To better understand the molecular events that are associated with aflatoxin production, three separate nonaflatoxigenic A. flavus strains were produced through serial transfers of aflatoxigenic parental strains. The three independent aflatoxigenic/nonaflatoxigenic pairs were compared via transcription profiling by microarray analyses. Cross comparisons identified 22 features in common between the aflatoxigenic/nonaflatoxigenic pairs. Physical mapping of the 22 features using the Aspergillus oryzae genome sequence for reference identified 16 unique genes. Aflatoxin biosynthetic and regulatory gene expression levels were not significantly different between the aflatoxigenic/nonaflatoxigenic pairs, which suggests that the inability to produce aflatoxins is not due to decreased expression of known biosynthetic or regulatory genes. Of the 16 in common genes, only one gene homologous to glutathione S-transferase genes showed higher expression in the nonaflatoxigenic progeny relative to the parental strains. This gene, named hcc, was selected for over-expression in an aflatoxigenic A. flavus strain to determine if it was directly responsible for loss of aflatoxin production. Although hcc transformants showed six- to ninefold increase in expression, no discernible changes in colony morphology or aflatoxin production were detected. Possible roles of hcc and other identified genes are discussed in relation to regulation of aflatoxin biosynthesis.

Alkaline iron(III) reduction by a novel alkaliphilic, halotolerant, Bacillus sp. isolated from salt flat sediments of Soap Lake by Jarrod Pollock; Karrie A. Weber; Joe Lack; Laurie A. Achenbach; Melanie R. Mormile; John D. Coates (pp. 927-934).

A halotolerant, alkaliphilic dissimilatory Fe(III)-reducing bacterium, strain SFB, was isolated from salt flat sediments collected from Soap Lake, WA. 16S ribosomal ribonucleic acid gene sequence analysis identified strain SFB as a novel Bacillus sp. most similar to Bacillus agaradhaerens (96.7% similarity). Strain SFB, a fermentative, facultative anaerobe, fermented various hexoses including glucose and fructose. The fructose fermentation products were lactate, acetate, and formate. Under fructose-fermenting conditions in a medium amended with Fe(III), Fe(II) accumulated concomitant with a stoichiometric decrease in lactate and an increase in acetate and CO₂. Strain SFB was also capable of respiratory Fe(III) reduction with some unidentified component(s) of Luria broth as an electron donor. In addition to Fe(III), strain SFB could also utilize nitrate, fumarate, or O₂ as alternative electron acceptors. Optimum growth was observed at 30°C and pH 9. Although the optimal salinity for growth was 0%, strain SFB could grow in a medium with up to 15% NaCl by mass. These studies describe a novel alkaliphilic, halotolerant organism capable of dissimilatory Fe(III) reduction under extreme conditions and demonstrate that Bacillus species can contribute to the microbial reduction of Fe(III) in environments at elevated pH and salinity, such as soda lakes.

Keywords: Dissimilatory Fe(III) reduction; Bacillus ; Extremeophile; Alkaliphile; Halotolerant

The Prestige oil spill: bacterial community dynamics during a field biostimulation assay by Núria Jiménez; Marc Viñas; Josep M. Bayona; Joan Albaiges; Anna M. Solanas (pp. 935-945).

A field bioremediation assay using the oleophilic fertilizer S200 was carried out 12 months after the Prestige heavy fuel-oil spill on a beach on the Cantabrian coast (north Spain). This assay showed that S200-enhanced oil degradation, particularly of high-molecular-weight n-alkanes and alkylated PAHs, suggesting an increase in the microbial bioavailability of these compounds. The bacterial community structure was determined by cultivation-independent analysis of polymerase chain reaction-amplified 16S rDNA by denaturing gradient gel electrophoresis. Bacterial community was mainly composed of α-Proteobacteria (Rhodobacteriaceae and Sphingomonadaceae). Representatives of γ-Proteobacteria (Chromatiales, Moraxellaceae, and Halomonadaceae), Bacteroidetes (Flavobacteriaceae), and Actinobacteria group (Nocardiaceae and Corynebacteriaceae) were also found. The addition of the fertilizer led to the appearance of the bacterium Mesonia algae in the early stages, with a narrow range of growth substrates, which has been associated with the common alga Achrosiphonia sonderi. The presence of Mesonia algae may be attributable to the response of the microbial community to the addition of N and P rather than indicating a role in the biodegradation process. The Rhodococcus group appeared in both assay plots, especially at the end of the experiment. It was also found at another site on the Galician coast that had been affected by the same spill. This genus has been associated with the degradation of n-alkanes up to C₃₆. Taking into account the high content of heavy alkanes in the Prestige fuel, these microorganisms could play a significant role in the degradation of such fuel. A similar bacterial community structure was observed at another site that showed a similar degree of fuel weathering.

Effect of electrical stimulation on human immunodeficiency virus type-1 infectivity by Etsuko Kumagai; Masato Tominaga; Shouichiro Nagaishi; Shinji Harada (pp. 947-953).

We examined the effects of electrical stimulation on HIV-1-adsorbed MAGIC-5 (MAGIC-5/HIV-1) cells and unadsorbed MAGIC-5 (MAGIC-5) cells. When MAGIC-5 cells were stimulated by a constant d.c. potential of 1.0 V (vs Ag/Agcl) immediately after HIV-1_LAI infection, infectivity was more affected by electrical stimulation than by cell membrane damage. In particular, after application of potential at 1.0 V for 5 min, about 1% of the membranes of the MAGIC-5/HIV-1_LAI cells were damaged, but the infectivities of both HIV-1_LAI and HIV-1_NL43-luc cells decreased about 37 and 44%, respectively (p < 0.05). After application of potential at 1.0 V for 5 min, the mean fluorescence intensities (MFIs) of highly reactive oxygen species (hROS) and nitric oxide (NO) in MAGIC-5/HIV-1_NL43-Luc cells were significantly increased compared with that of unstimulated MAGIC-5/HIV-1_NL43-Luc cells (p < 0.01). However, the MFIs of hROS and NO in MAGIC-5 cells were also increased, to the same level, by electrical stimulation for 5 min. These results suggest that HIV-1 adsorbed onto or invading cells is damaged by direct or indirect effects of electrical stimulation, resulting in a decrease in HIV-1 infectivity. It is also suggested that hROS and NO induced by electrical stimulation are important factors for inhibiting HIV-1 infection.

Keywords: HIV-1 infectivity; Electrical stimulation; Reactive oxygen species; Nitric oxide

DNA extraction method affects microbial community profiles from soils and sediment by Cora Carrigg; Olivia Rice; Siobhán Kavanagh; Gavin Collins; Vincent O’Flaherty (pp. 955-964).

To evaluate whether different deoxyribonucleic acid (DNA) extraction procedures can affect estimates of bacterial community composition, based on the 16S ribosomal ribonucleic acid gene denaturing gradient gel electrophoresis (DGGE) profiles, we compared four in situ lysis procedures using three soils and one marine sediment. Analysis of DGGE profiles, generated by polymerase chain reaction of purified DNA extracts, demonstrated that the choice of DNA extraction method significantly influenced the bacterial community profiles generated. This was reflected both in the number of bands or ribotypes detected from each sample and in subsequent principle coordinate analysis and unweighted-pair group method using arithmetic average analyses. The methods also differed significantly in their robustness, i.e. reproducibility across multiple analyses. Two methods, both based on bead beating, were demonstrated to be suitable for comparative studies of a range of soil and sediment types.

Keywords: Soil; Sediment; DNA extraction; DGGE; Lysis efficiency; Microbial diversity

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Applied Microbiology and Biotechnology (v.77, #4)

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Applied Microbiology and Biotechnology (v.77, #4)