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Applied Microbiology and Biotechnology (v.78, #4)

Diverse inhibitors of aflatoxin biosynthesis by Robert A. Holmes; Rebecca S. Boston; Gary A. Payne (pp. 559-572).

Pre-harvest and post-harvest contamination of maize, peanuts, cotton, and tree nuts by members of the genus Aspergillus and subsequent contamination with the mycotoxin aflatoxin pose a widespread food safety problem for which effective and inexpensive control strategies are lacking. Since the discovery of aflatoxin as a potently carcinogenic food contaminant, extensive research has been focused on identifying compounds that inhibit its biosynthesis. Numerous diverse compounds and extracts containing activity inhibitory to aflatoxin biosynthesis have been reported. Only recently, however, have tools been available to investigate the molecular mechanisms by which these inhibitors affect aflatoxin biosynthesis. Many inhibitors are plant-derived and a few may be amenable to pathway engineering for tissue-specific expression in susceptible host plants as a defense against aflatoxin contamination. Other compounds show promise as protectants during crop storage. Finally, inhibitors with different modes of action could be used in comparative transcriptional and metabolomic profiling experiments to identify regulatory networks controlling aflatoxin biosynthesis.

Keywords: Aspergillus; Secondary metabolism; Oxidative stress; Host resistance

Development of an online biosensor for in situ monitoring of chlorine dioxide gas disinfection efficacy by Maria del Busto-Ramos; Michael Budzik; Carlos Corvalan; Mark Morgan; Ronald Turco; David Nivens; Bruce Applegate (pp. 573-580).

A prototype bioluminescence-based biosensor was designed and constructed to evaluate the antimicrobial efficacy of chlorine dioxide (ClO₂) gas under various treatment conditions. The biosensor consisted of a bioluminescent bioreporter (Pseudomonas fluorescens 5RL), an optical transducer (photomultiplier tube), and a light-tight chamber housing, the bioreporter and the transducer. The bioluminescent recombinant P. fluorescens 5RL in the biosensor allowed for online monitoring of bioluminescence during ClO₂ gas disinfection. Experiments were performed to evaluate the effects of the two key physical parameters associated with ClO₂ disinfection: relative humidity (40, 60, 80%) and ClO₂ gas concentration (0.5, 1.0, 1.6, 2.1 mg/l) on the bioreporter. Results showed that increasing concentrations of ClO₂ gas corresponded to a faster decrease in luminescence. The rates of luminescence decrease from P. fluorescens 5RL, and the log reduction time (LRT, time required to obtain 1-log reduction in luminescence) were calculated for each treatment tested. The LRT values of luminescence were 103, 78, 53, and 35 s for 0.5, 1.0, 1.6, and 2.1 mg/l of ClO₂ gas treatment, respectively, at 78% relative humidity. The gas concentration which caused a tenfold change in LRT (z value) for luminescence of P. fluorescens 5RL was 3.4 mg/l of ClO₂. The prototype biosensor showed potential for many applications, such as monitoring real-time microbial inactivation and understanding parameters that influence the efficacy of gaseous decontamination procedures.

Keywords: Chlorine dioxide gas; Disinfection efficacy; Online sensor

Environmental optimization for bioconversion of triolein into 7,10-dihydroxy-8(E)-octadecenoic acid by Pseudomonas aeruginosa PR3 by In-Ae Chang; Jae-Han Bae; Min-Jung Suh; In-Hwan Kim; Ching T. Hou; Hak-Ryul Kim (pp. 581-586).

Hydroxy fatty acids (HFAs), originally found in small amount mainly from plant systems, are well known to have special properties such as higher viscosity and reactivity compared with other normal fatty acids. Recently, various microbial strains were tested to produce HFAs from different unsaturated fatty acids. Among those microbial strains tested, Pseudomonas aeruginosa PR3 are well known to utilize various unsaturated fatty acids to produce mono-, di-, and tri-HFAs. Previously, we reported that strain PR3 could utilize triolein as a substrate for the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) via the induction of lipase activity (Chang et al., Appl Microbiol Biotechnol, 74:301–306, 2007). In this study, we focused on the development of the optimal environmental conditions for DOD production from triolein by PR3. Optimal initial medium pH and incubation temperature were pH 8.0 and 25°C, respectively. Magnesium ion was essentially required for DOD production. Optimal inoculum size, time for substrate addition, and substrate concentration were 1%, 12 to 24 h, and 300 mg, respectively.

Keywords: Pseudomonas aeruginosa ; Hydroxy fatty acid; Triolein; Optimization; Bioconversion

Microscopic examination in vivo and in vitro of natural and cross-linked polyunsaturated mclPHA by M. Bassas; J. Diaz; E. Rodriguez; M. J. Espuny; M. J. Prieto; A. Manresa (pp. 587-596).

Pseudomonas aeruginosa 42A2 produces a polyunsaturated polyhydroxyalkanoates (PHA-L) when grown on linseed oil as a substrate. Its high unsaturation content (36.5%) provides highly reactive PHA-L, generating a cross-linked biopolymer after ultraviolet (UV) irradiation. Both PHAs (PHA-L and uvPHA-L) were characterized by nuclear magnetic resonance, Fourier transform infrared spectroscopy, gel permeation chromatography, gas chromatography–mass spectrometry and differential scanning calorimetry–thermogravimetric analysis. The structural analysis of the new polymer revealed a dramatic decrease in unsaturated monomer content (8.5%), due to the complete disappearance of the polyunsaturated monomers (C_12:2, C_14:2, and C_14:3). The cross-linking reaction was also confirmed by atomic force microscopy (AFM) and transmission electron microscopy. AFM showed morphological changes in bacteria cells with and without PHA granules. The microscope techniques provided us with micrographs of the native and cross-linked polymers, showing the formation of a reticular structure as the consequence of the cross-linking reaction.

Keywords: Polyhydroxyalkanoates; PHA; AFM; Pseudomonas aeruginosa ; Cross-linking reaction

Increased productivity of Clostridium acetobutylicum fermentation of acetone, butanol, and ethanol by pervaporation through supported ionic liquid membrane by Pavel Izák; Katrin Schwarz; Wolfgang Ruth; Hubert Bahl; Udo Kragl (pp. 597-602).

Pervaporation proved to be one of the best methods to remove solvents out of a solvent producing Clostridium acetobutylicum culture. By using an ionic liquid (IL)-polydimethylsiloxane (PDMS) ultrafiltration membrane (pore size 60 nm), we could guarantee high stability and selectivity during all measurements carried out at 37°C. Overall solvent productivity of fermentation connected with continuous product removal by pervaporation was 2.34 g l⁻¹ h⁻¹. The supported ionic liquid membrane (SILM) was impregnated with 15 wt% of a novel ionic liquid (tetrapropylammonium tetracyano-borate) and 85 wt% of polydimethylsiloxane. Pervaporation, accomplished with the optimized SILM, led to stable and efficient removal of the solvents butan-1-ol and acetone out of a C. acetobutylicum culture. By pervaporation through SILM, we removed more butan-1-ol than C. acetobutylicum was able to produce. Therefore, we added an extra dose of butan-1-ol to run fermentation on limiting values where the bacteria would still be able to survive its lethal concentration (15.82 g/l). After pervaporation was switched off, the bacteria died from high concentration of butan-1-ol, which they produced.

Keywords: Clostridium acetobutylicum ; Pervaporation; Supported ionic liquid membrane; Acetone–butanol–ethanol fermentation

Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans by Carolina Peña-Montes; Augusto González; Denise Castro-Ochoa; Amelia Farrés (pp. 603-612).

Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40°C. The enzyme retained activity after incubation at pHs ranging from 8 to11 for 12 h at 37°C and 6 to 8 for 24 h at 37°C. It retained 80% of its activity after incubation at 30 to 70°C for 30 min and lost 50% of its activity after incubation for 15 min at 80°C. Noticeable activation of the enzyme is observed when Fe²⁺ ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu²⁺, Fe³⁺, Hg²⁺, and Zn²⁺ ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.

Keywords: Aspergillus nidulans ; Protease; Esterase; Thermostable; Alkaline

The role of glucosamine-6-phosphate deaminase at the early stages of Aspergillus niger growth in a high-citric-acid-yielding medium by Tina Šolar; Janja Turšič; Matic Legiša (pp. 613-619).

Glucosamine-6-phosphate (GlcN6P) deaminase seems to be the main enzyme in Aspergillus niger cells responsible for rapid glucosamine accumulation during the early stages of growth in a high-citric-acid-yielding medium. By determining basic kinetic parameters on the isolated enzyme, a high affinity toward fructose-6-phosphate (Fru6P) was measured, while in the reverse direction the K _m value for glucosamine-6-phosphate was lower than deaminases from other organisms measured so far. The enzyme characteristics of GlcN6P deaminase suggest it must compete with 6-phosphofructo-1-kinase (PFK1) for the common substrate—Fru6P in A. niger cells. Glucosamine accumulation seems therefore to remove an intermediate from the glycolytic flux, a situation which is reflected in slower citric acid accumulation and a specific growth rate after the germination of spores. When ammonium ions are depleted from the medium, one of the substrates for GlcN6P deaminase becomes limiting and Fru6P can be catabolised by PFK1 which enhances glycolytic flux. Other enzymatic features of GlcN6P deaminase such as pH optima for both aminating and deaminating reactions might play a significant role in rapid glucosamine accumulation during the early phase of fermentation and a slow consumption of aminosugar during the citric-acid-producing phase.

Keywords: Aspergillus niger ; Glucosamine-6-phosphate deaminase; Citric acid; 6-phosphofructo-1-kinase; Glucosamine

Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria by Hiroshi Itaya; Yoshimi Kikuchi (pp. 621-625).

We previously reported on the secretion of Streptomyces mobaraensis transglutaminase by Corynebacterium glutamicum ATCC13869 (formerly classified as Brevibacterium lactofermentum). In the present work, we investigated whether any other coryneform bacteria showed higher productivity than C. glutamicum ATCC13869. We found that most coryneform species secreted pro-transglutaminase efficiently. Moreover, we confirmed that Corynebacterium ammoniagenes ATCC6872 produced about 2.5 g/l pro-transglutaminase over a 71-h period in a jar fermentor. Our findings suggest that some other coryneform bacteria, especially C. ammoniagenes ATCC6872, are potential hosts for industrial scale protein production.

Keywords: Corynebacterium glutamicum ; Corynebacterium ammoniagenes ; Transglutaminase; Protein secretion

A reducing-end-acting chitinase from Vibrio proteolyticus belonging to glycoside hydrolase family 19 by Yuji Honda; Hajime Taniguchi; Motomitsu Kitaoka (pp. 627-634).

A chitinase gene belonging to the glycoside hydrolase family 19 from Vibrio proteolyticus (chi19) was cloned. The recombinant enzyme (Chi19) showed weak activities against polymeric substrates and considerable activities against fully N-acetylated chitooligosaccharides, (GlcNAc) n, whose degree of polymerization was greater than or equal to five. It hydrolyzed (GlcNAc) n at the second linkage position from the reducing ends of the chitooligosaccharides. The hydrolytic products of colloidal chitin were mainly (GlcNAc)₂ from the initial stage of the reaction. The hydrolytic pattern of reduced colloidal chitin clearly suggested that the enzyme hydrolyzed the polymeric substrate from the reducing end.

Keywords: Cellobiohydrolase; Chitinase; GH family 19; Reducing end; Vibrio

Characterization of a lysin from deep-sea thermophilic bacteriophage GVE2 by Ting Ye; Xiaobo Zhang (pp. 635-641).

Thermostable enzymes from thermophiles have attracted extensive studies. However, little is known about thermophilic lysin of bacteriophage obtained from deep-sea hydrothermal vent. In this study, a lysin from deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) was characterized for the first time. It was found that the GVE2 lysin was highly homologous with N-acetylmuramoyl-L-alanine amidases. After expression in Escherichia coli, the recombinant GVE2 lysin was purified. The recombinant lysin was active over a range of temperature from 40 °C to 80 °C, with an optimum at 60 °C. Its optimal pH was 6.0, and it was stable over a wide range of pH from 4.0 to 10.0. The lysin was highly active when some enzyme inhibitors or detergents (phenylmethylsulfonyl fluoride, Tween 20, Triton X-100, and chaps) were used. However, it was strongly inhibited by sodium dodecyl sulfate and ethylene diamine tetraacetic acid. Its enzymatic activity could be slightly stimulated in the presence of Na⁺ and Li⁺. But the metal ions Mg²⁺, Ba²⁺, Zn²⁺, Fe³⁺, Ca²⁺, and Mn²⁺ at concentrations of 1 or 10 mM showed inhibitions to the lysin activity. Our study demonstrated the first characterization of lysin from deep-sea thermophilic bacteriophage.

Keywords: Deep-sea thermophilic bacteriophage; Lysin; Characterization

Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate by Priya Pillai; G. Archana (pp. 643-650).

Keratinases play an important role in biotechnological applications such as improvement of feather meal, enzymatic dehairing and production of amino acids or peptides from high molecular weight substrates. Bacillus subtilis P13, isolated from Vajreshwari hot spring (45–50°C) near Mumbai, India, produces a neutral serine protease and has an optimum temperature of 65°C. This enzyme preparation was keratinolytic in nature and could disintegrate whole chicken feathers, except for the remnants of shafts. The enzyme preparation also exhibited depilation of goat hides with the recovery of intact animal hair. The enzyme preparation could release peptides from ground feathers and bring about their weight reduction; however, similar action on hair was relatively weak. A single major PMSF-sensitive protease band could be detected upon zymogram analysis, indicating that a single enzyme may be responsible for feather degradation and hide depilation. The importance of these findings in the biotechnological application for feather and leather industries is discussed.

Keywords: Keratinase; Bacillus subtilis P13; Feather disintegration; Hide dehairing

Improvement of recombinant baculovirus infection efficiency to express manganese superoxide dismutase in silkworm larvae through dual promoters of Pph and Pp10 by Wan-fu Yue; Xing-hua Li; Wei-cheng Wu; Bhaskar Roy; Guang-li Li; Jian-mei Liu; Xiao-feng Wu; Ji-yong Zhou; Chuan-xi Zhang; Wan Chi Cheong David; Yun-gen Miao (pp. 651-657).

The silkworm, Bombyx mori, has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). There are several problems which will probably be the bottleneck for practical and industrial utilization of silkworm bioreactor. Traditionally, the recombinant virus should infect the larvae through individual dorsal injection by a syringe. This is a time- and labor-consuming procedure. This drawback has become a bottleneck for practical and industrial utilization of baculovirus expression system in the silkworm bioreactor. In this paper, we constructed a dual expression baculovirus to express the renovated polyhedron and target manganese superoxide dismutase (SOD) gene under P10 and polyhedron promoters, respectively, through oral infection. The results showed that the direct injection of recombinant rBacmid/BmNPV/SOD DNA with cellfectin reagent infected the silkworm larvae partially. When next batches of larvae were fed orally with hemolymph, which was collected from first batch of injected and infected larvae, the obvious symptom of infection was found and high target SOD was expressed. These results imply it is feasible to express target genes through combination of recombinant bacmid DNA injection and oral feeding by a dual expression bacmid baculovirus.

Keywords: Silkworm (Bombyx mori L.); Recombinant baculovirus; Expression; Dual promoters of Pph and Pp10; Bac-to-Bac system

Improvement of natural isolates of Saccharomyces cerevisiae strains for synthesis of a chiral building block using classic genetics by Netta Nir; Moran Bahalul; Roi Feingersch; Tal Katz-Ezov; Yechezkel Kashi; Ayelet Fishman (pp. 659-667).

The asymmetric bio-reduction of 4-chloro-acetoacetic-acid-ethyl-ester to the pharmaceutical building block (S)-4-chloro-3-hydroxybutanoate-ethyl-ester requires the utilization of an enantioselective robust biocatalyst. Some of the natural Saccharomyces cerevisiae strains, isolated from Mount Carmel National Park in Israel, were characterized as resistant to environmental stress. Nevertheless, these strains showed relatively low enantiomeric-excess (ee), while a laboratory strain, Y103, exhibited a selectivity of 98% ee. The enantioselective lab strain was crossed with the multi-stress resistant environmental isolate (93% ee) followed by backcross with Y103, to subsequently obtain a haploid offspring of backcross-1, exhibiting both high multi-stress resistance and high enantioselectivity (98% ee). Introducing osmotic (1 M NaCl), oxidative (0.6 mM H₂O₂) and thermal stress (44°C) to growing cultures of the enantioselective parent, resulted in a decrease of 24–32% in specific activity, while the enantioselectivity of the stress-resistant parent decreased by 4–12% ee. Unlike its original parental strains, the new strain maintained constant specific activity and enantioselectivity when introduced to the various stress factors. This work shows that the classic introgression method, can serve as a viable approach for creating a robust enantioselective biocatalyst, designed for industrial production of chiral compounds.

Keywords: Saccharomyces cerevisiae ; 4-chloro-3-hydroxybutanoic acid ethyl ester; 4-chloro-acetoacetic acid ethyl ester; Backcross; Stress resistance; Biocatalysis; Introgression

Display of avian influenza virus nucleoprotein on Bacillus thuringiensis cell surface using CTC as a fusion partner by Mei Liu; Shuyun Li; Sishun Hu; Changming Zhao; Dingren Bi; Ming Sun (pp. 669-676).

The S-layer protein CTC surface display system of Bacillus thuringiensis was used to test the possibility of displaying avian influenza virus nucleoprotein (NP) on the cell surface of B. thuringiensis. By fusing np with the anchoring motif of ctc, four recombinant plasmids were constructed. They harbored fusion gene ctc-np, csa-ctc-np (csa representing csaAB operon, very important in anchoring S-layer protein on cell surface), ctc-npp (npp representing the part fragment of np), and csa-ctc-npp, respectively. Five recombinant strains were obtained by transferring recombinant plasmids to B. thuringiensis plasmid-free derivative strain BMB171. The vegetative cells of five strains were used as agglutinogens for slide agglutination assays. The assays showed recombinant NP proteins successfully displayed on the cell surface of five strains. After immunization of chickens with spores by oral route, all five strains elicited a humoral response to NP and exhibited immunogenicity as indicated by enzyme-linked immunosorbent assay (ELISA). ELISA also showed that one of five strains, CN (bearing csa-ctc-npp), exhibited the highest immunogenicity among five strains, which suggested that the best way of constructing ctc fusion gene was the csa-ctc-npp. The strategy developed in this study suggests the possibility of generating a heat-stable and oral veterinary vaccine with B. thuringiensis surface display system.

Keywords: Bacillus thuringiensis ; S-layer surface display; Avian influenza virus nucleoprotein; Heat-stable oral vaccine

Construction of a chimeric gene cluster for the biosynthesis of apoemulsan with altered molecular weight by Hanna Dams-Kozlowska; Nina Sainath; David L. Kaplan (pp. 677-683).

Acinetobacter venetianus RAG-1 produces an extracellular protein/high-molecular-weight (HMW) polysaccharide complex termed emulsan. As an emulsion stabilizer, emulsan has potential industrial applications. To control the molecular weight of the polymer, a stable chromosomal mutant was generated where RAG-1 wza, wzb, wzc genes were replaced by Escherichia coli homologs. The heterologous Wza, Wzb, Wzc proteins restored production of HMW polysaccharide. The polymer produced was of higher molecular weight than from the parent strain and with the cells exhibiting modified hydrophobicity.

The pva operon is located on the megaplasmid of Sphingopyxis sp. strain 113P3 and is constitutively expressed, although expression is enhanced by PVA by Xiaoping Hu; Rie Mamoto; Yoshio Fujioka; Akio Tani; Kazuhide Kimbara; Fusako Kawai (pp. 685-693).

The upstream and downstream regions of the tentative pva operon including genes encoding oxidized polyvinyl alcohol (PVA) hydrolase (oph), PVA dehydrogenase (pvaA) and cytochrome c (cytC) from Sphingopyxis sp. strain 113P3 were sequenced. The resultant 7.9 kb sequence contained orf1 in the upstream region and orf2 and orf3 in the downstream region. Reverse transcription-polymerase chain reaction (PCR) analyses revealed that the intergenic regions between orf1 and oph or between cytC and orf2 were expressed neither in PVA medium nor glucose medium, indicating that the pva operon consists of three genes. A transcription start site was determined by 5′-rapid amplification of cDNA ends to be 428 bp upstream of the start codon of the oph. The stop codon of cytC was followed by a sequence of inverted repeats that could function as a factor-independent transcription terminator. Strain 113P3 had one megaplasmid including the pva operon. Northern blot hybridization for the three genes revealed that mRNA size was approximately 3 to 4 kb and expression was elevated in PVA medium compared to glucose medium.

Microarray analysis of toxicogenomic effects of triclosan on Staphylococcus aureus by Hyeung-Jin Jang; Matthew Wook Chang; Freshteh Toghrol; William E. Bentley (pp. 695-707).

For the first time, a genome-wide transcriptional analysis was performed to elucidate the cellular response of Staphylococcus aureus to triclosan. Our results indicate that the effects of triclosan are widespread on metabolism, affecting many vital cellular processes. Triclosan downregulated the transcription of genes involved in virulence factor and energy metabolism such as amino acid, carbohydrate, lipid transport, and metabolism, while multidrug resistance genes, coenzyme transport, and metabolism and transcription genes were upregulated. Furthermore, triclosan downregulated the transcription of genes encoding major lipid metabolism enzymes such as 3-hydroxyacyl-CoA dehydrogenase, acetyl-CoA acetyltransferase, acetyl-CoA synthetase, and acetyl-CoA carboxylase, which all play essential roles in S. aureus lipid metabolism. It is interesting to note that the expression of the enoyl-ACP reductase gene, fabI, was not changed after exposure of S. aureus with 0.05 μM triclosan at 10 and 60 min in our study. This work also implies that triclosan may kill S. aureus by interfering with its ability to form cell membranes. Another important implication of our result is that S. aureus may generate resistance factors under triclosan stress.

Degradation of 4-fluorophenol by Arthrobacter sp. strain IF1 by Maria Isabel M. Ferreira; Julian R. Marchesi; Dick B. Janssen (pp. 709-717).

A Gram-positive bacterial strain capable of aerobic biodegradation of 4-fluorophenol (4-FP) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. The organism, designated strain IF1, was identified as a member of the genus Arthrobacter on the basis of 16S ribosomal RNA gene sequence analysis. Arthrobacter strain IF1 was able to mineralize 4-FP up to concentrations of 5 mM in batch culture. Stoichiometric release of fluoride ions was observed, suggesting that there is no formation of halogenated dead-end products during 4-FP metabolism. The degradative pathway of 4-FP was investigated using enzyme assays and identification of intermediates by gas chromatography (GC), GC–mass spectrometry (MS), high-performance liquid chromatography, and liquid chromatography–MS. Cell-free extracts of 4-FP-grown cells contained no activity for catechol 1,2-dioxygenase or catechol 2,3-dioxygenase, which indicates that the pathway does not proceed through a catechol intermediate. Cells grown on 4-FP oxidized 4-FP, hydroquinone, and hydroxyquinol but not 4-fluorocatechol. During 4-FP metabolism, hydroquinone accumulated as a product. Hydroquinone could be converted to hydroxyquinol, which was further transformed into maleylacetic acid and β-ketoadipic acid. These results indicate that the biodegradation of 4-FP starts with a 4-FP monooxygenase reaction that yields benzoquinone, which is reduced to hydroquinone and further metabolized via the β-ketoadipic acid pathway.

Keywords: Biodegradation; 4-Fluorophenol; Hydroquinone; Monooxygenase; Fluoride

Improved sandwich-hybridization assay for an electrical DNA-chip-based monitoring of bioprocess-relevant marker genes by Daniel Pioch; Britta Jürgen; Stefan Evers; Karl-Heinz Maurer; Michael Hecker; Thomas Schweder (pp. 719-728).

Recently, it was shown that electrical DNA-chips in connection with a magnetic bead-based sandwich-hybridization assay can be a suitable alternative for the analysis of gene expression by monitoring the respective mRNA levels. In this study, we established an improved assay which allowed for a significantly shortened but sensitive detection of specific mRNAs. These improvements include the change to a solution-based sandwich-hybridization and the rearrangement of the used oligonucleotide probes. The introduction of a second labeled detection probe further increased the hybridization signals and, in turn, leads to a substantial time reduction of the detection protocol. The presented solution-based sandwich-hybridization protocol for the electrochemical detection of specific mRNAs requires about 60 min and the whole procedure, including sampling, cell disruption, and RNA isolation, approx. 80 min. The assay of this study was verified by nutrient-limited growth experiments and the analysis of selected starvation marker genes of the industrial host Bacillus licheniformis. The expression profiles determined with the electrical chip and the optimized protocol were, in most cases, comparable with the profiles determined by real-time RT-PCR measurements.

Keywords: Bacillus licheniformis ; At-line monitoring; Electrical chip; Bioprocess control

Transformation and gene replacement in the facultatively chemoheterotrophic, unicellular cyanobacterium Synechocystis sp. PCC6714 by electroporation by Alfred Ludwig; Thomas Heimbucher; Wolfgang Gregor; Thomas Czerny; Georg Schmetterer (pp. 729-735).

The unicellular cyanobacterium Synechocystis sp. PCC6714 can grow not only under photoautotrophic conditions, but also under chemoheterotrophic conditions if glucose is added to the medium. This makes it useful for the study of many aspects of bioenergetic mechanisms. In contrast to its closely related strain Synechocystis sp. PCC6803, which cannot grow chemoheterotrophically, Synechocystis PCC6714 is not naturally transformable. To enable gene transfer in this strain, we established a method for the introduction of self-replicating IncQ plasmids and for gene replacement using electroporation.

Keywords: Synechocystis sp. PCC6714; Electroporation; IncQ plasmids; Homologous recombination; Cytochrome M

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. 737-737).

Simultaneous biodegradation of creosote-polycyclic aromatic hydrocarbons by a pyrene-degrading Mycobacterium by Zaira López; Joaquim Vila; José-Julio Ortega-Calvo; Magdalena Grifoll (pp. 739-739).

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Applied Microbiology and Biotechnology (v.78, #4)

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Applied Microbiology and Biotechnology (v.78, #4)