scholarly journals Broad Purpose Vector for Site-Directed Insertional Mutagenesis in Bifidobacterium breve

2021 ◽  
Vol 12 ◽  
Author(s):  
Emily C. Hoedt ◽  
Francesca Bottacini ◽  
Nora Cash ◽  
Roger S. Bongers ◽  
Kees van Limpt ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Genetic Manipulation ◽  
Base Substitution ◽  
Gene Encoding ◽  
Bifidobacterium Breve ◽  
Suicide Vector ◽  
Dehydrogenase Gene ◽  
Restriction Sites ◽  
Target Strain ◽  
Restriction Modification

Members of the genus Bifidobacterium are notoriously recalcitrant to genetic manipulation due to their extensive and variable repertoire of Restriction-Modification (R-M) systems. Non-replicating plasmids are currently employed to achieve insertional mutagenesis in Bifidobacterium. One of the limitations of using such insertion vectors is the presence within their sequence of various restriction sites, making them sensitive to the activity of endogenous restriction endonucleases encoded by the target strain. For this reason, vectors have been developed with the aim of methylating and protecting the vector using a methylase-positive Escherichia coli strain, in some cases containing a cloned bifidobacterial methylase. Here, we present a mutagenesis approach based on a modified and synthetically produced version of the suicide vector pORI28 (named pFREM28), where all known restriction sites targeted by Bifidobacterium breve R-M systems were removed by base substitution (thus preserving the codon usage). After validating the integrity of the erythromycin marker, the vector was successfully employed to target an α-galactosidase gene responsible for raffinose metabolism, an alcohol dehydrogenase gene responsible for mannitol utilization and a gene encoding a priming glycosyltransferase responsible for exopolysaccharides (EPS) production in B. breve. The advantage of using this modified approach is the reduction of the amount of time, effort and resources required to generate site-directed mutants in B. breve and a similar approach may be employed to target other (bifido)bacterial species.

The Optimal Design on Connection Order of Network in Hydraulic Manifold Block

2011 ◽  
Vol 201-203 ◽  
pp. 2190-2194
Author(s):  
Jun Jun Zhang ◽  
Ji Sheng Wang ◽  
Jiang Yong Wang ◽  
Gang Liu ◽  
Jie Wang
Keyword(s):  
Genetic Algorithm ◽  
Optimal Design ◽  
Genetic Manipulation ◽  
Optimal Algorithm ◽  
Single Point ◽  
Adaptive Mutation ◽  
Gene Encoding ◽  
Mutation Strategy ◽  
The Common

As one of the important questions in the design of hydraulic manifold block — connection order of network, give a solution based on genetic algorithm. Genetic algorithm is the common effective intelligent optimal algorithm and suitable for solving a large combinatorial optimal problems. Gene encoding of ordinal representation, single-point crossover strategy and adaptive mutation strategy are used in the design of genetic manipulation.

scholarly journals Conservation of Genomic Localization and Sequence Content of Sau3AI-Like Restriction-Modification Gene Cassettes among Listeria monocytogenes Epidemic Clone I and Selected Strains of Serotype 1/2a

2010 ◽  
Vol 76 (16) ◽  
pp. 5577-5584 ◽  
Author(s):  
Suleyman Yildirim ◽  
Driss Elhanafi ◽  
Wen Lin ◽  
Anthony D. Hitchins ◽  
Robin M. Siletzky ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Gene Cassette ◽  
Conclusive Evidence ◽  
Gene Encoding ◽  
Epidemic Clone ◽  
Food Borne ◽  
Serotype 1 ◽  
Serotype 4B ◽  
Clonal Population Structure ◽  
Restriction Modification

ABSTRACTListeria monocytogenesis a food-borne pathogen with a clonal population structure and apparently limited gene flow between strains of different lineages. Strains of epidemic clone I (ECI) have been responsible for numerous outbreaks and invariably have DNA that is resistant to digestion by Sau3AI, suggesting methylation of cytosine at GATC sites. A putative restriction-modification (RM) gene cassette has been identified in the genome of the ECI strain F2365 and all other tested ECI strains but is absent from other strains of the same serotype (4b). Homologous RM cassettes have not been reported amongL. monocytogenesisolates of other serotypes. Furthermore, conclusive evidence for the involvement of this RM cassette in the Sau3AI resistance phenotype of ECI strains has been lacking. In this study, we describe a highly conserved RM cassette in certain strains of serotypes 1/2a and 4a that have Sau3AI-resistant DNA. In these strains the RM cassette was in the same genomic location as in the ECI reference strain F2365. The cassette included a gene encoding a putative recombinase, suggesting insertion via site-specific recombination. Deletion of the RM cassette in the ECI strain F2365 and the serotype 1/2a strain A7 rendered the DNA of both strains susceptible to Sau3AI digestion, providing conclusive evidence that the cassette includes a gene required for methylation of cytosine at GATC sites in both strains. The findings suggest that, in addition to its presence in ECI strains, this RM cassette and the accompanying genomic DNA methylation is also encountered among selected strains of other lineages.

Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance

2016 ◽  
Vol 116 (12) ◽  
pp. 1022-1031 ◽  
Author(s):  
Yuki Takagi ◽  
Moe Murata ◽  
Toshihiro Kozuka ◽  
Yukiko Nakata ◽  
Ryo Hasebe ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Residual Activity ◽  
Base Substitution ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Missense Mutations ◽  
Wild Type ◽  
Gene Encoding ◽  
Single Base

SummaryAntithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0%, in contrast to values of all variants were maintained at above 80%. The thrombin–AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12%, whereas that of tested variants was 37%–56%. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin–TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin–TM affinity- dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM- mediated anticoagulation.

scholarly journals The Chlamydomonas IDA7 Locus Encodes a 140-kDa Dynein Intermediate Chain Required to Assemble the I1 Inner Arm Complex

1998 ◽  
Vol 9 (12) ◽  
pp. 3351-3365 ◽  
Author(s):  
Catherine A. Perrone ◽  
Pinfen Yang ◽  
Eileen O’Toole ◽  
Winfield S. Sale ◽  
Mary E. Porter
Keyword(s):  
Insertional Mutagenesis ◽  
Gene Transformation ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Radial Spoke ◽  
Important Target ◽  
Dynein Intermediate Chain ◽  
Biochemical Analyses ◽  
Intermediate Chain

To identify new loci that are involved in the assembly and targeting of dynein complexes, we have screened a collection of motility mutants that were generated by insertional mutagenesis. One such mutant, 5B10, lacks the inner arm isoform known as the I1 complex. This isoform is located proximal to the first radial spoke in each 96-nm axoneme repeat and is an important target for the regulation of flagellar motility. Complementation tests reveal that 5B10 represents a new I1 locus, IDA7. Biochemical analyses confirm thatida7 axonemes lack at least five I1 complex subunits. Southern blots probed with a clone containing the gene encoding the 140-kDa intermediate chain (IC) indicate that theida7 mutation is the result of plasmid insertion into the IC140 gene. Transformation with a wild-type copy of the IC140 gene completely rescues the mutant defects. Surprisingly, transformation with a construct of the IC140 gene lacking the first four exons of the coding sequence also rescues the mutant phenotype. These studies indicate that IC140 is essential for assembly of the I1 complex, but unlike other dynein ICs, the N-terminal region is not critical for its activity.

scholarly journals Genetic Manipulation of Transcriptional Regulators Alters Nicotine Biosynthesis in Tobacco

2020 ◽  
Vol 61 (6) ◽  
pp. 1041-1053 ◽  
Author(s):  
Shunya Hayashi ◽  
Mutsumi Watanabe ◽  
Makoto Kobayashi ◽  
Takayuki Tohge ◽  
Takashi Hashimoto ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Genetic Manipulation ◽  
Transcriptional Regulators ◽  
Knockout Mutant ◽  
Gene Encoding ◽  
Helix Loop Helix ◽  
Wide Range ◽  
Nicotine Biosynthesis ◽  
Transient Overexpression ◽  
The Impact

Abstract The toxic alkaloid nicotine is produced in the roots of Nicotiana species and primarily accumulates in leaves as a specialized metabolite. A series of metabolic and transport genes involved in the nicotine pathway are coordinately upregulated by a pair of jasmonate-responsive AP2/ERF-family transcription factors, NtERF189 and NtERF199, in the roots of Nicotiana tabacum (tobacco). In this study, we explored the potential of manipulating the expression of these transcriptional regulators to alter nicotine biosynthesis in tobacco. The transient overexpression of NtERF189 led to alkaloid production in the leaves of Nicotiana benthamiana and Nicotiana alata. This ectopic production was further enhanced by co-overexpressing a gene encoding a basic helix-loop-helix-family MYC2 transcription factor. Constitutive and leaf-specific overexpression of NtERF189 increased the accumulation of foliar alkaloids in transgenic tobacco plants but negatively affected plant growth. By contrast, in a knockout mutant of NtERF189 and NtERF199 obtained through CRISPR/Cas9-based genome editing, alkaloid levels were drastically reduced without causing major growth defects. Metabolite profiling revealed the impact of manipulating the nicotine pathway on a wide range of nitrogen- and carbon-containing metabolites. Our findings provide insights into the biotechnological applications of engineering metabolic pathways by targeting transcription factors.

scholarly journals Calcium- and Calcineurin-Independent Roles for Calmodulin in Cryptococcus neoformans Morphogenesis and High-Temperature Growth

2005 ◽  
Vol 4 (6) ◽  
pp. 1079-1087 ◽  
Author(s):  
Peter R. Kraus ◽  
Connie B. Nichols ◽  
Joseph Heitman
Keyword(s):  
High Temperature ◽  
Cryptococcus Neoformans ◽  
Insertional Mutagenesis ◽  
Pathogenic Fungus ◽  
Critical Factor ◽  
Growth Defect ◽  
Insertion Mutant ◽  
Gene Encoding ◽  
Temperature Growth ◽  
New Genes

ABSTRACT The function of calcium as a signaling molecule is conserved in eukaryotes from fungi to humans. Previous studies have identified the calcium-activated phosphatase calcineurin as a critical factor in governing growth of the human pathogenic fungus Cryptococcus neoformans at mammalian body temperature. Here, we employed insertional mutagenesis to identify new genes required for growth at 37°C. One insertion mutant, cam1-ts, that displayed a growth defect at 37°C and hypersensitivity to the calcineurin inhibitor FK506 at 25°C was isolated. Both phenotypes were linked to the dominant marker in genetic crosses, and molecular analysis revealed that the insertion occurred in the 3′ untranslated region of the gene encoding the calcineurin activator calmodulin (CAM1) and impairs growth at 37°C by significantly reducing calmodulin mRNA abundance. The CAM1 gene was demonstrated to be essential using genetic analysis of a CAM1/cam1Δ diploid strain. In the absence of calcineurin function, the cam1-ts mutant displayed a severe morphological defect with impaired bud formation. Expression of a calmodulin-independent calcineurin mutant did not suppress the growth defect of the cam1-ts mutant at 37°C, indicating that calmodulin promotes growth at high temperature via calcineurin-dependent and -independent pathways. In addition, a Ca2+-binding-defective allele of CAM1 complemented the 37°C growth defect, FK506 hypersensitivity, and morphogenesis defect of the cam1-ts mutant. Our findings reveal that calmodulin performs Ca2+- and calcineurin-independent and -dependent roles in controlling C. neoformans morphogenesis and high-temperature growth.

scholarly journals Glyphosate Resistance as a Novel Select-Agent-Compliant, Non-Antibiotic-Selectable Marker in Chromosomal Mutagenesis of the Essential Genes asd and dapB of Burkholderia pseudomallei

2009 ◽  
Vol 75 (19) ◽  
pp. 6062-6075 ◽  
Author(s):  
Michael H. Norris ◽  
Yun Kang ◽  
Diana Lu ◽  
Bruce A. Wilcox ◽  
Tung T. Hoang
Keyword(s):  
Burkholderia Pseudomallei ◽  
Selectable Marker ◽  
Genetic Manipulation ◽  
Chromosome 1 ◽  
Chromosome 2 ◽  
Gene Encoding ◽  
Selectable Markers ◽  
Genetic Manipulations ◽  
Semialdehyde Dehydrogenase ◽  
Allelic Replacement

ABSTRACT Genetic manipulation of the category B select agents Burkholderia pseudomallei and Burkholderia mallei has been stifled due to the lack of compliant selectable markers. Hence, there is a need for additional select-agent-compliant selectable markers. We engineered a selectable marker based on the gat gene (encoding glyphosate acetyltransferase), which confers resistance to the common herbicide glyphosate (GS). To show the ability of GS to inhibit bacterial growth, we determined the effective concentrations of GS against Escherichia coli and several Burkholderia species. Plasmids based on gat, flanked by unique flip recombination target (FRT) sequences, were constructed for allelic-replacement. Both allelic-replacement approaches, one using the counterselectable marker pheS and the gat-FRT cassette and one using the DNA incubation method with the gat-FRT cassette, were successfully utilized to create deletions in the asd and dapB genes of wild-type B. pseudomallei strains. The asd and dapB genes encode an aspartate-semialdehyde dehydrogenase (BPSS1704, chromosome 2) and dihydrodipicolinate reductase (BPSL2941, chromosome 1), respectively. Mutants unable to grow on media without diaminopimelate (DAP) and other amino acids of this pathway were PCR verified. These mutants displayed cellular morphologies consistent with the inability to cross-link peptidoglycan in the absence of DAP. The B. pseudomallei 1026b Δasd::gat-FRT mutant was complemented with the B. pseudomallei asd gene on a site-specific transposon, mini-Tn7-bar, by selecting for the bar gene (encoding bialaphos/PPT resistance) with PPT. We conclude that the gat gene is one of very few appropriate, effective, and beneficial compliant markers available for Burkholderia select-agent species. Together with the bar gene, the gat cassette will facilitate various genetic manipulations of Burkholderia select-agent species.

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