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Host Strain (host + strain)
Selected AbstractsA new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxesENVIRONMENTAL MICROBIOLOGY, Issue 4 2006Robin Tecon Summary The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor ,Burkholderia sp. strain RP037 , produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed. [source] Differential binding to and biofilm formation on, HEp-2 cells by Salmonella enterica Serovar Typhimurium is dependent upon allelic variation in the fimH gene of the fim gene clusterMOLECULAR MICROBIOLOGY, Issue 5 2002Jennifer D. Boddicker Summary Type 1 fimbria-mediated adherence to HEp-2 cells by two strains of Salmonella enterica serovar Typhimurium was found to be different. Although both strains exhibited a strong mannose-sensitive haemagglutination reaction with guinea pig erythrocytes, characteristic of the expression of type 1 fimbriae, only one of the strains adhered in large numbers to HEp-2 cells. Characterization of the fimH genes, encoding the fimbrial adhesins, indicated two allelic variants. Using fimH mutants of the two strains it was possible to demonstrate that binding to HEp-2 cells was associated with the presence of one of the alleles regardless of the host strain. Therefore, this differential binding was not a function of the type I fimbrial shaft or the presence of other types of fimbriae produced by one strain but not the other. These observations may explain the differences in HEp-2 binding by type 1 fimbriate strains of Salmonella previously reported by several groups. Also, our studies demonstrate that the FimH adhesin can influence the efficiency of biofilm formation on HEp-2 cells using once-flow-through continuous culture conditions. The formation of biofilms on eukaryotic cells using this procedure is more likely to represent those conditions found in the intestinal tract than conditions using batch culture techniques to investigate adherence and biofilm formation. Indeed, the increased efficiency of biofilm formation in the murine intestinal tract confirmed the role of one of the fimH alleles in this process. [source] Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium,BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007Rebekka Biedendieck Abstract A multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N- and C-terminal fusion of a protein of interest to a Strep II and a His6 -tag is possible. Corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N- or C-terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one-step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry. Biotechnol. Bioeng. 2007;96: 525,537. © 2006 Wiley Periodicals, Inc. [source] Improved production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional cry1C gene in its chromosomeBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2005Chaoyin Yue Abstract A cryIC gene, whose product is active against Spodoptera exigua, was introduced into wildtype Bacillus thuringiensis kurstaki strain YBT1520 using an integrative and thermosensitive vector, pBMB-FLCE, which was developed based on B. thuringiensis transposon Tn4430 harboring a tnpI-tnpA gene. With the mediation of TnpI-TnpA, the cry1C gene was integrated into the chromosome of the host strain. To prevent secondary integration, the integrative vector was eliminated by moving recombinant cultures to 46°C for generations. Two integrative recombinant B. thuringiensis strains BMB1520-E and BMB1520-F were obtained. In recombinant BMB1520-F, the cry1C gene was expressed stably at a significant level and did not reduce the expression of endogenous crystal protein genes. Bioassay results indicated that BMB1520-E and BMB1520-F showed a higher level of activity against S. exigua third-instar larvae than did their parent strains, in addition to the high toxicity to Plutella xylostella third-instar later larvae. © 2005 Wiley Periodicals, Inc. [source] Metabolic characteristics of an isocitrate dehydrogenase defective derivative of escherichia coli BL21(DE3)BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2003Miho Aoshima Abstract A 7.8 kb fragment containing isocitrate dehydrogenase (ICDH) gene and its flanking regions was cloned from Escherichia coli BL21(DE3) and sequenced. Unlike the case of the K-12 strain, the e14 element was not found. The nucleotide divergence between these two strains was about 2%. Using the cloned fragment, ICDH defective mutant strain, MA1935, was generated from BL21(DE3). Although MA1935 accumulated citrate, citrate synthase activity was not repressed but was rather high. In addition, isocitrate lyase was not highly induced at the stationary phase. MA1935 was shown to be a good host strain for ICDH gene expression. © 2003 Wiley Periodicals Inc. Biotechnol Bioeng84: 732,737, 2003. [source] Effect of Vitreoscilla hemoglobin on production of a chemotherapeutic enzyme, L -asparaginase, by Pseudomonas aeruginosaBIOTECHNOLOGY JOURNAL, Issue 2 2006Hikmet Geckil Abstract The production of L -asparaginase, an enzyme widely used in cancer chemotherapy, is mainly regulated by carbon catabolite repression and oxygen. This study was carried out to understand how different carbon sources and Vitreoscilla hemoglobin (VHb) affect the production of this enzyme in Pseudomonas aeruginosa and its VHb-expressing recombinant strain (PaJC). Both strains grown with various carbon sources showed a distinct profile of the enzyme activity. Compared to no carbohydrate supplemented medium, glucose caused a slight repression of L -asparaginase in P. aeruginosa, while it stimulated it in the PaJC strain. Glucose, regarded as one of the inhibitory sugars for the production L -asparaginase by other bacteria, was determined to be the favorite carbon source compared to lactose, glycerol and mannitol. Furthermore, contrary to common knowledge of oxygen repression of L -asparaginase in other bacteria, oxygen uptake provided by VHb was determined to even stimulate the L -asparaginase synthesis by P. aeruginosa. This study, for the first time, shows that in P. aeruginosa utilizing a recombinant oxygen uptake system, VHb, L -asparaginase synthesis is stimulated by glucose and other carbohydrate sources compared to the host strain. It is concluded that carbon catabolite and oxygen repression of L -asparaginase in fermentative bacteria is not the case for a respiratory non-fermentative bacterium like P. aeruginosa. [source] Expression of an Aspergillus niger Glucose Oxidase in Saccharomyces cerevisiae and Its Use to Optimize Fructo-oligosaccharides SynthesisBIOTECHNOLOGY PROGRESS, Issue 4 2006Magdalena Valdivieso-Ugarte Fructo-oligosaccharides (FOS) represent the most abundantly supplied and utilized group of nondigestible oligosaccharides as food ingredients. These prebiotics can be produced from sucrose using the transglycosylating activity of ,-fructofuranosidases (EC 3.2.1.26) at high concentrations of the starting material. The main problem during FOS synthesis is that the activity of the enzyme is inhibited by the glucose generated during the reaction, and therefore the maximum FOS content in commercial products reaches up to 60% on a dry substance basis. The glucose oxidase (gox) gene from Aspergillus niger BT18 was cloned and integrated, as part of an expression cassette, into the ribosomal DNA of a Saccharomyces cerevisiae host strain. One of the recombinant strains with a high copy number of the gox gene and showing a high GOX specific activity was used to produce the enzyme. Addition of the extracellular glucose oxidase to the FOS synthesis reaction helped to remove the glucose generated, avoiding the inhibition of the fungal ,-fructofuranosidase. As a result, a final syrup containing up to 90% of FOS was obtained. [source] Identification and Repair of Positive Binding Antibodies Containing Randomly Generated Amber Codons from Synthetic Phage Display LibrariesBIOTECHNOLOGY PROGRESS, Issue 3 2006Warren D. Marcus Phage display technology allows for the rapid isolation and characterization of monoclonal antibodies that have vast potential for therapeutic and diagnostic applications. However, the panning process, which utilizes a host strain that suppresses termination by the amber codon, has an inherent bias toward clones containing randomly generated amber stop codons, complicating identification of positive binding antibodies when the antibody genes are finally expressed in a nonsupressor host. Here, we perform biopanning against a Histone 2A peptide using streptavidin- or anti-biotin-coated beads. After four rounds, a dominant clone is characterized but contains a spurious amber stop codon. A protocol is given that readily corrects the amber codon, allowing for soluble antibody production once the phagemid is transformed into a nonsuppressor bacterial strain. This work also highlights the ability to isolate antibodies against a protein antigen by using only a small peptide (15 amino acids) representing a portion of the antigen. [source] The roles and interactions of reproductive isolation mechanisms in fall armyworm (Lepidoptera: Noctuidae) host strainsECOLOGICAL ENTOMOLOGY, Issue 2010ASTRID T. GROOT 1. The moth Spodoptera frugiperda presents an interesting opportunity to study the evolution of reproductive isolation, because it consists of two host races that may be in the process of speciation. 2. The two races exhibit habitat isolation through host-plant preference, and two types of behavioural isolation, i.e. differences in sex pheromone composition and timing of mating activity at night. 3. In this paper, we review the selection pressures acting upon these three barriers as well as their genetic bases, to address the question of how divergence of the two strains may have evolved. 4. We also address possible interactions between the three barriers, whether and how they may have evolved in concert, and we view the evolution of these three prezygotic isolation barriers in the light of postzygotic isolation. [source] New insertion sequences of Sulfolobus: functional properties and implications for genome evolution in hyperthermophilic archaeaMOLECULAR MICROBIOLOGY, Issue 1 2005Zachary D. Blount Summary Analyses of complete genomes indicate that insertion sequences (ISs) are abundant and widespread in hyperthermophilic archaea, but few experimental studies have measured their activities in these hosts. As a way to investigate the impact of ISs on Sulfolobus genomes, we identified seven transpositionally active ISs in a widely distributed Sulfolobus species, and measured their functional properties. Six of the seven were found to be distinct from previously described ISs of Sulfolobus, and one of the six could not be assigned to any known IS family. A type II ,Miniature Inverted-repeat Transposable Element' (MITE) related to one of the ISs was also recovered. Rates of transposition of the different ISs into the pyrEF region of their host strains varied over a 250-fold range. The Sulfolobus ISs also differed with respect to target-site selectivity, although several shared an apparent preference for the pyrEF promoter region. Despite the number of distinct ISs assayed and their molecular diversity, only one demonstrated precise excision from the chromosomal target region. The fact that this IS is the only one lacking inverted repeats and target-site duplication suggests that the observed precise excision may be promoted by the IS itself. Sequence searches revealed previously unidentified partial copies of the newly identified ISs in the Sulfolobus tokodaii and Sulfolobus solfataricus genomes. The structures of these fragmentary copies suggest several distinct molecular mechanisms which, in the absence of precise excision, inactivate ISs and gradually eliminate the defective copies from Sulfolobus genomes. [source] Survival of ovarian allografts in an experimental animal modelPEDIATRIC TRANSPLANTATION, Issue 6 2007Roger G. Gosden Abstract: Transplantation of ovarian tissue is a promising strategy for fertility preservation in young cancer patients with premature ovarian failure if they have cryopreserved their own tissue before undergoing gonadotoxic treatment. However, extension of ovary donation to children and adults seeking treatment for hypogonadism is controversial unless the tissue does not provoke an immune reaction or specific tolerance can be safely and effectively achieved. The survival of heterotopic ovarian allografts was tested in a mouse model. Isografts were placed under the kidney capsule of ovariectomized animals differing at the H-2 haplotype (H-2d or H-2k). Within three wk, and in contrast to isografts, the allografts were rejected, although their survival was extended when donor and host strains shared the same haplotype (H-2k). Allograft survival was not improved if the tissue was implanted orthotopically. When monoclonal antibodies to CD4 antigens were administered at doses exceeding those effective for long-term tolerance to cardiac allografts, graft survival was prolonged in one of two strain combinations, but they failed to restore fertility. These results indicate that the ovary is not an immunologically privileged organ, as the older literature suggested, and chronic immunosuppression is likely to be required for ovarian allografts in clinical settings. [source] Adaptation of the Fungal Parasite Zygorhizidium planktonicum During 200 Generations of Growth on Homogeneous and Heterogeneous Populations of Its Host, the Diatom Asterionella formosa,THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2008ARNOUT DE BRUIN ABSTRACT. We followed adaptation of the chytrid parasite Zygorhizidium planktonicum during 200 generations of growth on its host, the freshwater diatom Asterionella formosa, in a serial passage experiment. Evolution of parasite fitness was assessed both on a homogenous and heterogeneous host population, consisting of respectively a single new and ten different new host strains. These 10 host strains were genetically different and also varied in their initial susceptibility to the parasite. Parasite fitness increased significantly and rapidly on the new, genetically homogenous host population, but remained unaltered during 200 generations of growth on the heterogeneous host population. Enhanced parasite fitness was the result of faster and more efficient transmission, resulting in higher values of R0 (number of secondary infections). Consequently, parasites that evolved within the uniclonal host population infected significantly more of these hosts than did their ancestors. We thus provide experimental evidence for the widely held view that host genetic diversity restricts evolution of parasites and moderates their harmful effects. Genetically uniform host populations are not only at increased risk from fungal epidemics because they all share the same susceptibility, but also because new parasite strains are able to adapt quickly to new host environments and to improve their fitness. [source] Heterologous expression of the biosynthetic gene clusters of coumermycin A1, clorobiocin and caprazamycins in genetically modified Streptomyces coelicolor strainsBIOPOLYMERS, Issue 9 2010Katrin Flinspach Abstract The biosynthetic gene clusters of the aminocoumarin antibiotics clorobiocin and coumermycin A1 and of the liponucleoside antibiotic caprazamycin were stably integrated into the genomes of different host strains derived from Streptomyces coelicolor A3(2). For the heterologous expression of clorobiocin derivatives in a chemically defined medium, inclusion of 0.6% of the siloxylated ethylene oxide/propylene oxide copolymer Q2-5247 into the growth medium proved to result in a 4.8-fold increase of productivity. Presumably, this copolymer acts as an oxygen carrier. The additional inclusion of cobalt chloride (0.2,2 mg l,1) dramatically increased the percentage of the desired compound clorobiocin within the total produced clorobiocin derivatives. This is very likely due to a stimulation of a cobalamin-dependent methylation reaction catalyzed by the enzyme CloN6 of clorobiocin biosynthesis. All three investigated host strains (S. coelicolor M512, M1146 and M1154) gave similar production rates of total clorobiocin derivatives (on average, 158 mg l,1 in the presence of 0.6% Q2-5247 and 0.2 mg l,1 CoCl2). In contrast, heterologous production of caprazamycin derivatives was optimal in strain M1154 (amounts of 152 mg l,1 on average). © 2010 Wiley Periodicals, Inc. Biopolymers 93: 823,832, 2010. [source] The in vivo synthesis of plant sesquiterpenes by Escherichia coliBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2001Vincent J.J. Martin Abstract Three plant genes encoding (+)-,-cadinene, 5- epi -aristolochene, and vetispiradiene cyclases were expressed in Escherichia coli to evaluate the potential of this bacterium to synthesize sesquiterpenes in vivo. Various growth temperatures, carbon sources, and host strains were examined to optimize terpene production. The highest levels of sesquiterpene production occurred when the enzymes were expressed in strain DH5, from the trc promoter (Ptrc) of the high-copy plasmidpTrc99A in M9 medium supplemented with 0.2% (v/v) glycerol at 30°C for 5- epi -aristolochene and vetispiradiene and 37°C for (+)-,-cadinene. The highest concentrations of sesquiterpenes observed were 10.3 ,g of (+)-,-cadinene, 0.24 ,g of 5- epi -aristolochene (measured as (+)-,-cadinene equivalents), and 6.4 ,g of vetispiradiene (measured as (+)-,-cadinene equivalents) per liter of culture. These sesquiterpene production levels are >500-fold lower than carotenoid production, both of which are synthesized from endogenous trans -farnesyl diphosphate (FDP) in E. coli. Based on these results, we conclude that the limiting factor for sesquiterpene synthesis in E. coli is the poor expression of the cyclase enzyme and not supply of the FDP precursor. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 497,503, 2001. [source] | ||||||||||