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Gene Cassettes (gene + cassette)
Selected AbstractsSystemic IFN-, drives kidney nephritis in B6.Sle123 miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2008Anna-Marie Fairhurst Abstract The impact of IFN-, secretion on disease progression was assessed by comparing phenotypic changes in the lupus-prone B6.Sle1Sle2Sle3 (B6.Sle123) strain and the parental C57BL/6 (B6) congenic partner using an adenovirus (ADV) expression vector containing a recombinant IFN-, gene cassette (IFN-ADV). A comprehensive comparison of cell lineage composition and activation in young B6 and B6.Sle123 mice revealed a variety of cellular alterations in the presence and absence of systemic IFN-,. Most IFN-,-induced phenotypes were similar in B6 and B6.Sle123 mice; however, B6.Sle123 mice uniquely exhibited increased B1 and plasma cells after IFN-, exposure, although both strains had an overall loss of mature B cells in the bone marrow, spleen and periphery. Although most of the cellular effects of IFN-, were identical in both strains, severe glomerulonephritis occurred only in B6.Sle123 mice. Mice injected with IFN-ADV showed an increase in immune complex deposition in the kidney, together with an unexpected decrease in serum anti-nuclear antibody levels. In summary, the predominant impact of systemic IFN-, in this murine model is an exacerbation of mechanisms mediating end organ damage. [source] Use of a novel nonantibiotic triple marker gene cassette to monitor high survival of Pseudomonas fluorescens SBW25 on winter wheat in the fieldFEMS MICROBIOLOGY ECOLOGY, Issue 2 2008Lotta Jäderlund Abstract Pseudomonas fluorescens SBW25 was tagged with a triple marker gene cassette containing gfp, encoding green fluorescent protein; luxAB, encoding luciferase; and telABkilA, encoding tellurite resistance, and the tagged strain was monitored in the first Swedish field release of a genetically modified microorganism (GMM). The cells were inoculated onto winter wheat seeds and the GMM cells (SBW25,tgl) were monitored in the field from September 2005 to May 2006 using plating, luminometry and microscopic analyses. Cell numbers were high on all sampling occasions and metabolically active cells were detected on all plant parts. Field results were similar to those obtained in a parallel phytotron study, although the amount of SBW25,tgl detected on shoots was significantly higher in the phytotron than in the field. After winter, cell counts were 100-fold higher on the roots and root-associated soil compared with prewinter measurements, although the cells had a lower relative metabolic activity. The wheat seeds were naturally infested with Microdochium nivale, but no treatment resulted in reduction of disease symptoms. No SWB25,tgl cells were ever found in bulk soil or uninoculated plants. The Swedish field trial results complement and contrast with prior field studies performed with the same parent organism in the United Kingdom under different soil, plant and climatic conditions. [source] Population dynamics and gene transfer in genetically modified bacteria in a model microcosmMOLECULAR ECOLOGY, Issue 11 2003A. K. Lilley Abstract The horizontal transfer and effects on host fitness of a neutral gene cassette inserted into three different genomic loci of a plant-colonizing pseudomonad was assessed in a model ecosystem. The KX reporter cassette (kanamycin resistance, aph, and catechol 2, 3, dioxygenase, xylE) was introduced on the disarmed transposon mini-Tn5 into: (I) the chromosome of a spontaneous rifampicin resistant mutant Pseudomonas fluorescens SBW25R; (II) the chromosome of SBW25R in the presence of a naturally occurring lysogenic-phage (phage ,101); and (III) a naturally occurring plasmid pQBR11 (330 kbp, tra+, Hgr) introduced into SBW25R. These bacteria were applied to Stellaria media (chickweed) plants as seed dressings [c. 5 × 104 colony-forming units (cfu)/seed] and the seedlings planted in 16 microcosm chambers containing model plant and animal communities. Gene transfer to pseudomonads in the phyllosphere and rhizosphere was found only in the plasmid treatment (III). Bacteria in the phage treatment (II) initially declined in density and free phage was detected, but populations partly recovered as the plants matured. Surprisingly, bacteria in the chromosome insertion treatment (I) consistently achieved higher population densities than the unmanipulated control and other treatments. Plasmids were acquired from indigenous bacterial populations in the control and chromosome insertion treatments. Plasmid acquisition, plasmid transfer from inocula and selection for plasmid carrying inocula coincided with plant maturation. [source] Widespread distribution of a lexA -regulated DNA damage-inducible multiple gene cassette in the Proteobacteria phylumMOLECULAR MICROBIOLOGY, Issue 1 2004Marc Abella Summary The SOS response comprises a set of cellular functions aimed at preserving bacterial cell viability in front of DNA injuries. The SOS network, negatively regulated by the LexA protein, is found in many bacterial species that have not suffered major reductions in their gene contents, but presents distinctly divergent LexA-binding sites across the Bacteria domain. In this article, we report the identification and characterization of an imported multiple gene cassette in the Gamma Proteobacterium Pseudomonas putida that encodes a LexA protein, an inhibitor of cell division (SulA), an error-prone polymerase (DinP) and the alpha subunit of DNA polymerase III (DnaE). We also demonstrate that these genes constitute a DNA damage-inducible operon that is regulated by its own encoded LexA protein, and we establish that the latter is a direct derivative of the Gram-positive LexA protein. In addition, in silico analyses reveal that this multiple gene cassette is also present in many Proteobacteria families, and that both its gene content and LexA-binding sequence have evolved over time, ultimately giving rise to the lexA lineage of extant Gamma Proteobacteria. [source] Environmental regulation of recA gene expression in Porphyromonas gingivalisMOLECULAR ORAL MICROBIOLOGY, Issue 3 2001Y. Liu The recA gene product in Porphyromonas gingivalis is involved in DNA repair. Further, disruption of this gene can affect the proteolytic activity and expression of other virulence factors in this organism. Since several known environmental factors can influence virulence gene expression in P. gingivalis, we investigated the influence of these signals on the expression of the recA gene in this organism. A heterodiploid strain of P. gingivalis (designated FLL118) containing a transcriptional fusion of the recA promoter region and the promoterless tetracycline-resistant gene [tetA(Q)2] and xylosidase/arabinosidase (xa) gene cassette was constructed. The recA promoter activity was assessed by measurement of xylosidase activity in FLL118. The expression remained relatively constant during different growth phases, at different pH levels and in the presence of DNA-damaging agents. In response to hemin limitation and in the presence of calcium there was a moderate increase in recA promoter activity. Temperature also affected the expression. The highest level of xylosidase activity was observed in cultures at 32°C with a decline of approximately 46% as growth temperature increased to 41°C. Reverse transcriptase polymerase chain reaction analysis revealed that this regulation may be occurring at the transcriptional level. These results suggest that expression of the recA gene in P. gingivalis W83 is responsive to several environmental signals but is not regulated by a DNA damage,inducible SOS-like regulatory system. [source] In vivo Mutational Analysis of the Mupirocin Gene Cluster Reveals Labile Points in the Biosynthetic Pathway: the "Leaky Hosepipe" MechanismCHEMBIOCHEM, Issue 9 2008Ji'en Wu Dr. Abstract A common feature of the mupirocin and other gene clusters of the AT-less polyketide synthase (PKS) family of metabolites is the introduction of carbon branches by a gene cassette that contains a ,-hydroxy-,-methylglutaryl CoA synthase (HMC) homologue and acyl carrier protein (ACP), ketosynthase (KS) and two crotonase superfamily homologues. In vivo studies of Pseudomonas fluorescens strains in which any of these components have been mutated reveal a common phenotype in which the two major isolable metabolites are the truncated hexaketide mupirocin H and the tetraketide mupiric acid. The structure of the latter has been confirmed by stereoselective synthesis. Mupiric acid is also the major metabolite arising from inactivation of the ketoreductase (KR) domain of module 4 of the modular PKS. A number of other mutations in the tailoring region of the mupirocin gene cluster also result in production of both mupirocin H and mupiric acid. To explain this common phenotype we propose a mechanistic rationale in which both mupirocin H and mupiric acid represent the products of selective and spontaneous release from labile points in the pathway that occur at significant levels when mutations block the pathway either close to or distant from the labile points. [source] Integron-associated gene cassettes in Halifax Harbour: assessment of a mobile gene pool in marine sedimentsENVIRONMENTAL MICROBIOLOGY, Issue 4 2008J. E. Koenig Summary The integron/gene cassette systems identified in bacteria comprise a class of genetic elements that allow adaptation by acquisition of gene cassettes. Integron gene cassettes have been shown to facilitate the spread of drug resistance in human pathogens but their role outside a clinical setting has not been explored extensively. We sequenced 2145 integron gene cassettes from four marine sediment samples taken from the vicinity of Halifax Nova Scotia, Canada, increasing the number of gene cassettes obtained from environmental microbial communities by 10-fold. Sequence analyses reveals that the majority of these cassettes encode novel proteins and that this study is consistent with previous claims of high cassette diversity as we estimate a Chao1 diversity index of ,3000 cassettes from these samples. The functional distribution of environmental cassettes recovered in this study, when compared with that of cassettes from the only other source with significant sampling (Vibrio genomes) suggests that alternate selection regimes might be acting on these two gene pools. The majority of cassettes recovered in this study encode novel, unknown proteins. In instances where we obtained multiple alleles of a novel protein we demonstrate that non-synonymous versus synonymous substitution rates ratios suggest relaxed selection. Cassette-encoded proteins with known homologues represent a variety of functions and prevalent among these are isochorismatases; proteins involved in iron scavenging. Phylogenetic analysis of these isochorismatases as well as of cassette-encoded acetyltransferases reveals a patchy distribution, suggesting multiple sources for the origin of these cassettes. Finally, the two most environmentally similar sample sites considered in this study display the greatest overlap of cassette types, consistent with the hypothesis that cassette genes encode adaptive proteins. [source] Antimicrobial-resistant faecal Escherichia coli in wild mammals in central Europe: multiresistant Escherichia coli producing extended-spectrum beta-lactamases in wild boarsJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2010I. Literak Abstract Aims:, To determine the presence of antibiotic-resistant faecal Escherichia coli in populations of wild mammals in the Czech Republic and Slovakia. Methods and Results:, Rectal swabs or faeces collected during 2006,2008 from wild mammals were spread on MacConkey agar and MacConkey agar containing 2 mg l,1 of cefotaxime. From plates with positive growth, one isolate was recovered and identified as E. coli. Susceptibility to 12 antibiotics was tested using the disk diffusion method. Resistance genes, class 1 and 2 integrons and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). Extended-spectrum beta-lactamases (ESBL) were further characterized by DNA sequencing, macrorestriction profiling and determination of plasmid sizes. Plasmid DNA was subjected to EcoRV digestion, transferability by conjugation and incompatibility grouping by multiplex PCR. The prevalence of resistant isolates was 2% in small terrestrial mammals (rodents and insectivores, nE. coli = 242), 12% in wild ruminants and foxes (nE. coli = 42), while no resistant isolates were detected in brown bears (nE. coli = 16). In wild boars (Sus scrofa) (nE. coli = 290), the prevalence of resistant isolates was 6%. Class 1 and 2 integrons with various gene cassettes were recorded in resistant isolates. From wild boars, five (2%, nrectal smears = 293) multiresistant isolates producing ESBL were recovered: one isolate with blaCTX-M-1 + blaTEM-1, three with blaCTX-M-1 and one with blaTEM-52b. The blaCTX-M-1 genes were carried on approx. 90 kb IncI1 conjugative plasmids. Conclusions:, Antibiotic-resistant E. coli occured in populations of wild mammals in various prevalences. Significance and Impact of the Study:, Wild mammals are reservoirs of antibiotic-resistant E. coli including ESBL-producing strains which were found in wild boars. [source] Characterization of integrons and antimicrobial resistance genes in clinical isolates of Gram-negative bacteria from Palestinian hospitalsMICROBIOLOGY AND IMMUNOLOGY, Issue 11 2009Amjad I. A. Hussein ABSTRACT Sixty Gram-negative bacterial isolates were collected from Palestinian hospitals in 2006. Thirty-two (53.3%) isolates showed multidrug resistance phenotypes. PCR and DNA sequencing were used to characterize integrons and antimicrobial resistance genes. PCR screening showed that 19 (31.7%) and five (8.3%) isolates were positive for class 1 and class 2 integrons, respectively. DNA-sequencing results for the captured antimicrobial resistance gene cassettes within class 1 integrons identified the following genes: dihydrofolate reductases, dfrA1, dfrA5, dfrA7, dfrA12, dfrA17 and dfrA25; aminoglycoside adenyltransferases, aadA1, aadA2, aadA5, aadA12 and aadB; aminoglycoside acetyltransferase, aac(6,)-Ib; and chloramphenicol resistance gene, cmlA1. ESBL were identified in 25 (41.7%) isolates. The identified ESBL were blaCTX-M-15, blaCTX-M-56, blaOXA-1, blaSHV-1, blaSHV-12, blaSHV-32 and blaTEM-1 genes. Moreover, we characterized the plasmid-mediated quinolone resistance genes, aac(6,)-Ib-cr and qnrB2, which were detected in seven (11.7%) and two (3.3%) isolates, respectively. In this study various types of antibiotic resistance genes have been identified in Gram-negative bacteria from Palestinian hospitals, many of which are reported in the Middle East area for the first time. [source] Integron-encoded IntI integrases preferentially recognize the adjacent cognate attI site in recombination with a 59-be siteMOLECULAR MICROBIOLOGY, Issue 5 2002Christina M. Collis Summary Integrons have the capacity to capture small mobile elements known as gene cassettes, and this reaction is catalysed by integron-encoded IntI integrases. IntI integrases form a distinct family within the tyrosine recombinase superfamily and include a characteristic additional domain that is well conserved. Two different IntI enzymes were used to examine their ability to recognize heterologous attI sites in both integration and excision assays. IntI1 and IntI3 are 59% identical and catalyse both integrative and excisive recombination between a cassette-associated 59-be site and the cognate attI1 or attI3 site. Integrative recombination events involving a 59-be and a non-cognate attI site, attI2 and attI3 for IntI1 or attI1 and attI2 for IntI3, were detected extremely rarely. In cassette excision assays, the non-cognate attI3 site was recognized by IntI1, but attI1 was not well recognized by IntI3. The purified IntI1 and IntI3 proteins bound strongly only to their cognate attI site. [source] Discovery and distribution of super-integrons among PseudomonadsMOLECULAR MICROBIOLOGY, Issue 3 2001Romualdas Vaisvila Until recently, integrons (systems for acquisition and expression of new genetic materials) have been associated generally with antibiotic resistance gene cassettes. The discovery of ,super-integrons' in Vibrionaceae suggests a greater impact of this gene acquisition mechanism on bacterial genome evolution than initially believed. Super-integrons may contain more than 100 gene cassettes and may encode other determinants, including biochemical functions or virulence factors. Here, we report the genetic organization of a super-integron from Pseudomonas alcaligenes ATCC 55044. This is the first evidence of a super-integron in a non-pathogenic bacterium, one which is widely distributed in a great number of ecological niches such as soil and aquatic habitats. Here, the sequence composition, open reading frame (ORF) content and organization of In55044 are described and found to have features intermediate between the multidrug-resistant integrons and the Vibrio cholerae super-integron. Similar structures are inferred to be present in several Pseudomonas species, based on polymerase chain reaction (PCR) experiments. [source] In this issue: Biotechnology Journal 12/2009BIOTECHNOLOGY JOURNAL, Issue 12 2009Article first published online: 14 DEC 200 Genome-scale in silico modeling Milne et al., Biotechnol. J. 2009, 4, 1653,1670 Driven by advancements in high-throughput biological technologies and the growing number of sequenced genomes, the construction of in silico models at the genome scale has provided powerful tools to investigate a vast array of biological systems and applications. Nathan Price and colleagues review comprehensively the use of such models in industrial and medical biotechnology, including biofuel generation, food production, and drug development. As such, genome-scale models can provide a basis for rational genome-scale engineering and synthetic biology. Genome-scale in silico models promise to extend their application and analysis scope to become a transformative tool in biotechnology. From metagenomics to metaproteomics Tuffin et al., Biotechnol. J. 2009, 4, 1671,1683 Metagenomics emerged in the late 1990s as a tool for accessing and studying the collective microbial genetic material in the environment and has been widely predicted to reach new dimensions of the protein sequence space. A decade on, researchers from South Africa see that while several novel enzyme activities and protein structures have been identified the greatest advancement has been made in the isolation of novel protein sequences, some of which have no close relatives, form deeply branched lineages and even represent novel families. However, there is much room for improvement in the methods employed that need to be addressed in order to access novel biocatalytic activities. Recombinant secondary metabolites Schäfer et al., Biotechnol. J. 2009, 4, 1684,1703 Plants produce a high diversity of natural products or secondary metabolites which have interesting biological properties and quite a number are of medicinal importance. Their functions range from the protection against herbivores and/or microbial pathogens to defend against abiotic stress, e.g. UV-B exposure. Because the production of valuable natural products, such as the anticancer drugs paclitaxel, vinblastine or camptothecin in plants is a costly process, biotechnological alternatives to produce these alkaloids more economically become more and more important. This review provides an overview of the state of art to produce alkaloids in recombinant microorganisms, such as bacteria or yeast. In a longterm perspective, it will probably be possible to generate gene cassettes for complete pathways, which could then be used for the production of valuable natural products in bioreactors or for metabolic engineering of crop plants. [source] Medicinally important secondary metabolites in recombinant microorganisms or plants: Progress in alkaloid biosynthesisBIOTECHNOLOGY JOURNAL, Issue 12 2009Holger Schäfer Abstract Plants produce a high diversity of natural products or secondary metabolites which are important for the communication of plants with other organisms. A prominent function is the protection against herbivores and/or microbial pathogens. Some natural products are also involved in defence against abiotic stress, e.g. UV-B exposure. Many of the secondary metabolites have interesting biological properties and quite a number are of medicinal importance. Because the production of the valuable natural products, such as the anticancer drugs paclitaxel, vinblastine or camptothecin in plants is a costly process, biotechnological alternatives to produce these alkaloids more economically become increasingly important. This review provides an overview of the state of art to produce alkaloids in recombinant microorganisms, such as bacteria or yeast. Some progress has been made in metabolic engineering usually employing a single recombinant alkaloid gene. More importantly, for benzylisoquinoline, monoterpene indole and diterpene alkaloids (taxanes) as well as some terpenoids and phenolics the proof of concept for production of complex alkaloids in recombinant Escherichia coli and yeast has already been achieved. In a long-term perspective, it will probably be possible to generate gene cassettes for complete pathways, which could then be used for production of valuable natural products in bioreactors or for metabolic engineering of crop plants. This will improve their resistance against herbivores and/or microbial pathogens. [source] The emergence and implications of metallo-,-lactamases in Gram-negative bacteriaCLINICAL MICROBIOLOGY AND INFECTION, Issue 2005T. R. Walsh Abstract The increase in Gram-negative broad-spectrum antibiotic resistance is worrisome, particularly as there are few, if any, ,,pipeline'' antimicrobial agents possessing suitable activity against Pseudomonas spp. or Acinetobacter spp. The increase in resistance will be further enhanced by the acquisition of metallo-,-lactamase (MBL) genes that can potentially confer broad-spectrum ,-lactam resistance. These genes encode enzymes that can hydrolyse all classes of ,-lactams and the activity of which cannot be neutralised by ,-lactamase inhibitors. MBL genes are often associated with aminoglycoside resistant genes and thus bacteria that possess MBL genes are often co-resistant to aminoglycosides, further compromising therapeutic regimes. Both types of genes can be found as gene cassettes carried by integrons that in turn are embedded within transposons providing a highly ambulatory genetic element. The dissemination of MBL genes is typified by the spread of blaVIM-2, believed to originate from a Portuguese patient in 1995, and is now present in over 20 counties. The increase in international travel is likely to be a contributory factor for the ascendancy of mobile MBL genes as much as the mobility among individual bacteria. Fitness, acquisition and host dependency are key areas that need to be addressed to enhance our understanding of how antibiotic resistance spreads. There is also a pressing need for new, and hopefully novel, compounds active against pan-resistant Gram-negative bacteria , a growing problem that needs to be addressed by both government and industry. [source] | |||||||||||||