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Applied Microbiology and Biotechnology (v.58, #2)
Genetic tools for cyanobacteria by O. Koksharova; C. Wolk (pp. 123-137).
Cyanobacteria are oxygenic photosynthetic bacteria that have been used increasingly to study diverse biological processes, including photosynthesis and its regulation; cell differentiation and N2 fixation; metabolism of nitrogen, carbon, and hydrogen; resistance to environmental stresses; and molecular evolution. Many vectors and other genetic tools have been developed for unicellular and filamentous strains of cyanobacteria. Transformation, electroporation, and conjugation are used for gene transfer. Diverse methods of mutagenesis allow the isolation of many sought-for kinds of mutants, including site-directed mutants of specific genes. Reporter genes permit measurement of the level of transcription of particular genes, and assays of transcription within individual colonies or within individual cells in a filament. Complete genomic sequences have been obtained for the unicellular cyanobacterium, Synechocystis sp. strain PCC 6803 and the filamentous, heterocyst-forming cyanobacterium, Anabaena sp. strain PCC 7120. Genomic sequence projects are under way for Nostoc punctiforme strain PCC 73102 (ATCC 29133) and strains of the unicellular genera, Synechococcus, Prochlorococcus, and Gloeobacter. Genomic sequence data provide the opportunity for global monitoring of changes in genetic expression at transcriptional and translational levels in response to variations in environmental conditions. The availability of genomic sequences accelerates the identification, study, modification and comparison of cyanobacterial genes, and facilitates analysis of evolutionary relationships, including the relationship of chloroplasts to ancient cyanobacteria. The many available genetic tools enhance the opportunities for possible biotechnological applications of cyanobacteria.
The elusive roles of bacterial glutathione S-transferases: new lessons from genomes by S. Vuilleumier; M. Pagni (pp. 138-146).
Glutathione S-transferases constitute a large family of enzymes which catalyze the addition of glutathione to endogenous or xenobiotic, often toxic electrophilic chemicals. Eukaryotic glutathione S-transferases usually promote the inactivation, degradation or excretion of a wide range of compounds by formation of the corresponding glutathione conjugates. In bacteria, by contrast, the few glutathione S-transferases for which substrates are known, such as dichloromethane dehalogenase, 1,2-dichloroepoxyethane epoxidase and tetrachlorohydroquinone reductase, are catabolic enzymes with an essential role for growth on recalcitrant chemicals. Glutathione S-transferase genes have also been found in bacterial operons and gene clusters involved in the degradation of aromatic compounds. Information from bacterial genome sequencing projects now suggests that glutathione S-transferases are present in large numbers in proteobacteria. In particular, the genomes of three Pseudomonas species each include at least ten different glutathione S-transferase genes. Several of the corresponding proteins define new classes of the glutathione S-transferase family and may also have novel functions that remain to be elucidated.
Non-conventional yeasts by J. Spencer; A. Ragout de Spencer; C. Laluce (pp. 147-156).
In the beginning there was yeast, and it raised bread, brewed beer, and made wine. After many not days but centuries and even millenia later, it was named Saccharomyces cerevisiae. After more years and centuries there was another yeast, and it was named Schizosaccharomyces pombe; now there were two stars in the yeast heaven. In only a few more years there were other yeasts, and then more, and more, and more. The era of the non-conventional yeasts had begun.
13C-NMR analysis of glucose metabolism during citric acid production by Aspergillus niger by A. Peksel; N. Torres; J. Liu; G. Juneau; C. Kubicek (pp. 157-163).
The effect of glucose concentration on glycolytic metabolism under conditions of citric acid accumulation by Aspergillus niger was studied with 13C-labelled glucose. The results show that during cultivation at high glucose (14%, w/v), most of the label in citric acid is in C-2/C-4, and is thus due to the pyruvate carboxylase reaction. However, a significant portion is also present in C-1/C-5, whose origin is less clear but most likely due to reconsumption of glycerol and erythritol. Formation of trehalose and mannitol is high during the early phase of fermentation and declines thereafter. The early fermentation phase is further characterized by a high rate of anaplerosis from oxaloacetate to pyruvate, which also decreases with time. At low glucose concentrations (2%, w/v), which lead to a significantly reduced citric acid yield and formation rate, labelling of citrate in C-2/C-4 is decreased and C-1/C-5 labelling increased. Growth on 2% glucose is also characterized by an appreciable scrambling of mannitol and considerable backflux from mannitol to trehalose (indicating tight glycolytic control at the fructose-6-phosphate step) and an increased anaplerotic formation of pyruvate from oxaloacetate. These data indicate that cultivation on high sugar concentrations shifts control of glycolysis from fructose-6-phosphate to the glyceraldehyde-3-phosphate dehydrogenase step.
Polygalacturonase production by Aspergillus awamori on wheat in solid-state fermentation by A. Blandino; T. Iqbalsyah; S. Pandiella; D. Cantero; C. Webb (pp. 164-169).
The production of exo-polygalacturonase (exo-PG) and endo-PG by Aspergillus awamori grown on wheat in solid-state fermentation was studied. Endo- and exo-PG activities were detected after 24 h of inoculation. Glucose released from starch hydrolysis acted as a catabolite repressor for the exo-PG enzyme. In contrast, endo-PG production was not affected by glucose repression. When milled grains were used, the particle-size distribution and the chemical composition of the medium influenced the rate of micro-organism growth and therefore the trend followed by endo- and exo-PG production. However, these two parameters did not affect the maximum production of exo-PG and endo-PG. For one of the milled samples, three different moisture contents were used (50, 55, 60%). Moisture contents of 60% provide a higher yield of pectinases by A. awamori.
Production and biotransformation of 6-pentyl-α-pyrone by Trichoderma harzianum in two-phase culture systems by L. Serrano-Carreón; K. Balderas-Ruíz; E. Galindo; M. Rito-Palomares (pp. 170-174).
The final concentration of 6-pentyl-α-pyrone (6PP) produced in cultures of Trichoderma spp. is limited by the fact that inhibition of biomass growth occurs at 6PP concentrations as low as 100 mg/l. The aim of this work was to evaluate liquid-liquid extractive fermentation systems as an alternative to overcome the toxicity problems and to increase the production of 6PP by this fungus. Two alkanes (n-decane and n-hexadecane) and two dicarboxylic esters (dibutyl phthalate and dioctyl phthalate) were evaluated in shake flask cultures. The highest 6PP production (173 ppm) was achieved when n-hexadecane was used, being 3.5-fold the maximum 6PP concentration of a culture without the solvent. Cultivation of Trichoderma harzianum in a 10-l bioreactor with n-hexadecane yielded 6PP production ninefold higher than that from control cultures. However, 6PP production in the bioreactor (83 ppm) was lower than in shake flasks. Differences in the power drawn to the fluid at each scale could account for such behavior. Even in the presence of the solvent, 6PP content decreased after reaching its maximal concentration.
Effect of shear stress on cultivation of Bacillus thuringiensis for thuringiensin production by W.-T. Wu; Y.-L. Hsu; Y.-F. Ko; L.-L. Yao (pp. 175-177).
Cultivation of Bacillus thuringiensis for thuringiensin production is a mixed-growth-associated system. Cultivation conditions should be different during the cell growth stage and production stage. In this study, agitation speed and aeration rate were varied during the exponential growth phase and stationary phase in order to investigate the effect of shear stress via agitation on cultivation of B. thuringiensis for thuringiensin production. It was found that shear stress had a significant effect on thuringiensin production during the stationary phase. By decreasing the agitation speed during the stationary phase, product formation was increased up to 43%.
Effects of genotypes of maitake (Grifola frondosa) on biological efficiency, quality and crop cycle time by Q. Shen; D. Royse (pp. 178-182).
Twenty-three genotypes of maitake (Grifola frondosa) from Asia, North America and Europe were compared for biological efficiency (BE), quality and crop cycle time. Significant differences among lines were found for BE and crop cycle time when mushrooms were produced on nutrient-supplemented (15% millet, 10% wheat bran and 0.2% gypsum) oak sawdust substrate. Four isolates (WC828, M036, M037, and M040) were found to have the most consistent and highest BEs (38.5%, 39.5%, 35.8%, and 38.9%, respectively) and quality ratings (1.2, 1.3, 1.4, and 1.2, respectively, where 1 is highest quality and 4 is lowest quality). A commercial line (M039) used in China had the shortest crop cycle time (8 weeks). Lines deemed to have the best commercial potential were of Asian origin.
A rapid fluorescence-based assay for detecting soluble methane monooxygenase by A. Miller; W. Keener; M. Watwood; F. Roberto (pp. 183-188).
A fluorescence-based assay was developed to estimate soluble methane monooxygenase (sMMO) activity in solution. Whole cells of Methylosinus trichosporium OB3b expressing sMMO were used to oxidize various compounds to screen for fluorescent products. Of the 12 compounds tested, only coumarin yielded a fluorescent product. The UV absorbance spectrum of the product matches that of 7-hydroxycoumarin, and this identification was confirmed by 13C-NMR spectroscopy. The dependence of the fluorescent reaction on sMMO activity was investigated by pre-incubation with acetylene, a known inhibitor of sMMO activity. Apparent kinetic parameters for whole cells were determined to be K m(app)=262 µM and V max(app)=821 nmol 7-hydroxycoumarin min–1 mg protein–1. The rate of coumarin oxidation by sMMO correlates well with those of trichloroethylene degradation and naphthalene oxidation. Advantages of the fluorescence-based coumarin oxidation assay over the naphthalene oxidation assay include a more stable product, direct detection of the product without additional reagents, and greater speed and convenience.
Secretion of human interferon alpha 2b by Streptomyces lividans by E. Pimienta; R. Fando; J. Sánchez; C. Vallin (pp. 189-194).
Biologically active human interferon alpha 2b (HuIFNα-2b) was secreted into the culture medium by Streptomyces lividans transformed with recombinant plasmids coding for HuIFNα-2b fused to the Streptomyces exfoliatus M11 lipase A signal sequence. Levels were low, 15 or 100 ng/ml, depending on the plasmid used. Neither processed nor unprocessed HuIFNα-2b was detected in cell lysates of the transformants secreting the recombinant product. However, the secreted recombinant product was found to partially degrade when cultures reached the stationary phase by the action of an, as yet, unidentified mycelium-associated factor. Experimental evidence suggests that the degrading factor is related to mycelium-associated proteolytic activity.
Evaluation of pKD1-based plasmid systems for heterologous protein production in Kluyveromyces lactis by W. Panuwatsuk; N. Da Silva (pp. 195-201).
The stability of pKD1-based vectors was evaluated during the synthesis of intracellular and extracellular gene products in the yeast Kluyveromyces lactis. The Escherichia coli lacZ and MF α 1 leader-BPTI (bovine pancreatic trypsin inhibitor) cassettes were placed under the control of the inducible K. lactis LAC4 promoter and inserted into the pKD1-based plasmids. To induce gene expression while maintaining inducer level, a gratuitous gal1-209 K. lactis strain was employed. Selective medium containing 5 g glucose/l and 0.5 g galactose (inducer)/l allowed optimum expression and secretion of heterologous products without a significant effect on the growth of the recombinant cells. During long-term sequential batch cultures (60 generations), plasmid instability was mainly the result of structural instability. The expression and secretion of BPTI resulted in greater structural instability relative to the intracellular β-galactosidase. For both products, vectors carrying the pKD1 replication origin and the cis-acting stability locus (partial-pKD1 vectors) were more stable than vectors carrying the full pKD1 sequence (full-pKD1 vectors). However, after 55 generations, the β-galactosidase and BPTI activities were still higher with the full-pKD1 vectors. This was due to the significantly higher initial β-galactosidase and BPTI activities for the full-pKD1 vectors (approximately 85% and 47% higher, respectively) relative to the partial-pKD1 vectors. Southern blots confirmed that these increases were due to the higher copy number of the vectors carrying the full pKD1 sequence. In contrast to our previously reported results for the secretion of invertase, full-pKD1 vectors were preferred for the expression/secretion of β-galactosidase and BPTI for at least 55 generations. Due to their structural stability, partial-pKD1 vectors will be advantageous for very long cultivation times.
Molecular detection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites by J. Widada; H. Nojiri; K. Kasuga; T. Yoshida; H. Habe; T. Omori (pp. 202-209).
Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source. Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection. Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways. The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp. strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates. Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates. Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp. strain U2. However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains. These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria.
The development of low temperature inactive (Lti) baker's yeast by C. Gysler; P. Niederberger (pp. 210-216).
The construction of a novel baker's yeast variety via traditional genetic techniques is described. The phenotype was designated "Lti" ("Low temperature inactive"). Lti mutations with the desired characteristics within a genetically well-defined haploid laboratory strain of Saccharomyces cerevisiae were isolated, and two different approaches were taken to obtain baker's yeast strains, which exhibit reduced fermenting activity at refrigeration temperatures. In a first approach, a chosen Lti strain carrying mutation lti9 was combined with other laboratory strains carrying defined MAL alleles. In a second approach, the same lti mutation was introduced in the genetic background of polyploid commercial baker's yeast strains that harbor important "industrial" properties. Lti strains arising from both approaches were characterized with specifically developed screening procedures. Strains of the "academic" Lti strain family displayed between 85% and 92% of the biomass yield of a commercial reference strain, whereas strains of the "industrial" Lti strain family showed a variation between 60% and 115%. Lti strains from both families varied strongly among each other in their activity in model doughs: at 8°C they displayed activities between 5% and 30%, and at 30°C between 40% and 113% of a commercial reference baker's yeast strain.
A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant by J. Ohnishi; S. Mitsuhashi; M. Hayashi; S. Ando; H. Yokoi; K. Ochiai; M. Ikeda (pp. 217-223).
Classical whole-cell mutagenesis has achieved great success in development of many industrial fermentation strains, but has the serious disadvantage of accumulation of uncharacterized secondary mutations that are detrimental to their performance. In the post-genomic era, a novel methodology which avoids this drawback presents itself. This "genome-based strain reconstruction" involves identifying mutations by comparative genomic analysis, defining mutations beneficial for production, and assembling them in a single wild-type background. Described herein is an initial challenge involving reconstruction of classically derived L-lysine-producing Corynebacterium glutamicum. Comparative genomic analysis for the relevant terminal pathways, the efflux step, and the anaplerotic reactions between the wild-type and production strains identified a Val-59→Ala mutation in the homoserine dehydrogenase gene (hom), a Thr-311→Ile mutation in the aspartokinase gene (lysC), and a Pro-458→Ser mutation in the pyruvate carboxylase gene (pyc). Introduction of the hom and lysC mutations into the wild-type strain by allelic replacement resulted in accumulation of 8 g and 55 g of L-lysine/l, respectively, indicating that both these specific mutations are relevant to production. The two mutations were then reconstituted in the wild-type genome, which led to a synergistic effect on production (75 g/l). Further introduction of the pyc mutation resulted in an additional contribution and accumulation of 80 g/l after only 27 h. This high-speed fermentation achieved the highest productivity (3.0 g l–1 h–1) so far reported for microbes producing L-lysine in fed-batch fermentation.
Acid–base enrichment enhances anaerobic hydrogen production process by Chin-Chao Chen; Chiu-Yue Lin; Min-Cheng Lin (pp. 224-228).
This study offers a novel and quick enrichment technology that can be used as a preliminary method to obtain a hydrogen-producing species from the biological sludge produced by wastewater treatment. The influences of acid–base enrichment (by sludge pH adjustment) on the anaerobic hydrogen-producing micro-organisms were investigated using serum bottle assays. The enrichment pH values were controlled at 3, 4, 5, 7, 10, 11 and 12 with 1 N hydrochloric acid and 1 N sodium hydroxide. For each enrichment pH, the cultivation pH values were controlled at 5, 6 and 7. Based on the experimental results, hydrogen accumulation from sludge with acid or base enrichment is higher than that of the control. The hydrogen-production potential of the sludge with acid or base enrichment is 200 and 333 times enhanced, compared with the control, when the enrichment pH is 10 and 3, respectively. The enhancement is due to a shortening of the micro-organisms' lag-time which occurs at a proper cultivation-pH level.
Cloning, characterization and comparison of the Pseudomonas mendocina polyhydroxyalkanoate synthases PhaC1 and PhaC2 by S. Hein; J. Paletta; A. Steinbüchel (pp. 229-236).
This study describes a comparison of the polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 of Pseudomonas mendocina. The P. mendocina pha gene locus, encoding two PHA synthase genes [phaC1Pm and phaC2Pm flanking a PHA depolymerase gene (phaZ)], was cloned, and the nucleotide sequences of phaC1Pm (1,677 bp), phaZ (1,034 bp), and phaC2Pm (1,680 bp) were determined. The amino acid sequences deduced from phaC1Pm and phaC2Pm showed highest similarities to the corresponding PHA synthases from other pseudomonads sensu stricto. The two PHA synthase genes conferred PHA synthesis to the PHA-negative mutants P. putida GPp104 and Ralstonia eutropha PHB–4. In P. putida GPp104, phaC1Pm and phaC2Pm mediated PHA synthesis of medium-chain-length hydroxyalkanoates (C6–C12) as often reported for other pseudomonads. In contrast, in R. eutropha PHB–4, either PHA synthase gene also led to the incorporation of 3-hydroxybutyrate (3HB) into PHA. Recombinant strains of R. eutropha PHB–4 harboring either P. mendocina phaC gene even accumulated a homopolyester of 3HB during cultivation with gluconate, with poly(3HB) amounting to more than 80% of the cell dry matter if phaC2 was expressed. Interestingly, recombinant cells harboring the phaC1 synthase gene accumulated higher amounts of PHA when cultivated with fatty acids as sole carbon source, whereas recombinant cells harboring PhaC2 synthase accumulated higher amounts when gluconate was used as carbon source in storage experiments in either host. Furthermore, isogenic phaC1 and phaC2 knock-out mutants of P. mendocina provided evidence that PhaC1 is the major enzyme for PHA synthesis in P. mendocina, whereas PhaC2 contributes to the accumulation of PHA in this bacterium to only a minor extent, and then only when cultivated on gluconate.
Thermophilic biodesulfurization of naphthothiophene and 2-ethylnaphthothiophene by a dibenzothiophene-desulfurizing bacterium, Mycobacterium phlei WU-F1 by T. Furuya; K. Kirimura; K. Kino; S. Usami (pp. 237-240).
Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and NTH derivatives can be detected in diesel oil following hydrodesulfurization treatment, in addition to DBT derivatives. Mycobacterium phlei WU-F1, which possesses high desulfurizing ability toward DBT and its derivatives over a wide temperature range (20–50 °C), could also grow at 50°C in a medium with NTH or 2-ethylNTH, an alkylated derivative, as the sole source of sulfur. At 50 °C, the resting cells of WU-F1 degraded 67% and 83% of 0.81 mM NTH and 2-ethylNTH, respectively, within 8 h. By GC-MS analysis, 2-ethylNTH-desulfurized metabolites were identified as 2-ethylNTH sulfoxide, 1-(2′-hydroxynaphthyl)-1-butene and 1-naphthyl-2-hydroxy-1-butene, and it was concluded that WU-F1 desulfurized 2-ethylNTH through a sulfur-specific degradation pathway with the selective cleavage of carbon–sulfur bonds. Therefore, M. phlei WU-F1 can effectively desulfurize asymmetric organosulfur compounds, NTH and 2-ethylNTH, as well as symmetric DBT derivatives under high-temperature conditions, and it may be a useful desulfurizing biocatalyst possessing a broad substrate specificity toward organosulfur compounds.
Fungal contribution to in situ biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) film in soil by B.-I. Sang; K. Hori; Y. Tanji; H. Unno (pp. 241-247).
The contribution of fungi to the microbial degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films in soil was studied. Various traces, cavities, and grooves observed on the dented surface of PHBV films demonstrated that the degradation was a concerted effect of a microbial consortium colonizing the film surface, including fungi, bacteria, and actinomycetes. The succession of microbial consortia in the soil around the PHBV films during the degradation showed a distinctive increase in the fungal population, resulting in its dominance. Comparison of the degradation ability of microbial strains isolated from soil where PHBV films were degraded, revealed that fungi showed the highest contribution to PHBV degradation, growing very rapidly along the film surface with their high degradation ability and then expanding their hyphae in a three-dimensional manner.
Assessment of metabolic properties and kinetic parameters of methanogenic sludge by on-line methane production rate measurements by G. Gonzalez-Gil; R. Kleerebezem; G. Lettinga (pp. 248-254).
This report presents a new approach to studying the metabolic and kinetic properties of anaerobic sludge from single batch experiments. The two main features of the method are that the methane production is measured on-line with a relatively cheap system, and that the methane production data can be plotted as rate vs time curves. The case studies of specific methanogenic activity, biodegradability and toxicity tests here presented show that very accurate kinetic data can be obtained. The method is specifically useful in experiments in which strong changes in methane production occur, and it is proposed as a powerful tool to study methanogenic systems. Furthermore, the method is simple and could be implemented by industry in the routine analysis of sludge.
Pure bacterial isolates that convert p-xylene to terephthalic acid by M. Bramucci; C. McCutchen; M. Singh; S. Thomas; B. Larsen; J. Buckholz; V. Nagarajan (pp. 255-259).
Bacteria that grow on p-xylene, p-toluic acid, and terephthalic acid (TPA) were isolated from a wastewater bioreactor that is used to treat a waste stream that contains all three of these compounds. Although previously described aerobic bacteria degrade p-xylene by initially oxidizing a single methyl group to form p-toluic acid and then cleaving the aromatic ring, some of the bacteria isolated during this study transformed p-xylene by oxidizing both methyl groups to produce TPA.
Formaldehyde removal in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1 by A. Hidalgo; A. Lopategi; M. Prieto; J. Serra; M. Llama (pp. 260-264).
Rhodococcus erythropolis strain UPV-1 is able to grow on phenol as the only carbon and energy source and to remove formaldehyde completely from both synthetic and industrial wastewater. The rate of formaldehyde removal is independent of either initial biomass or formaldehyde concentration. The presence of viable, intact cells is strictly necessary for this removal to take place. Discontinuous and continuous formaldehyde-feed systems were successfully tested with synthetic wastewater in shaken flasks. Once biodegradation was well established in model synthetic wastewater, a real wastewater sample was obtained from a local phenolic and melamine resin-manufacturing company. Incubation of biomass with this wastewater at subtoxic concentrations of formaldehyde resulted in the complete removal of the pollutant. Parameters, such as chemical oxygen demand and toxicity, were assessed as indicators of wastewater cleanup progress.