scholarly journals Engineering of Tellurite-Resistant Genetic Tools for Single-Copy Chromosomal Analysis of Burkholderia spp. and Characterization of the Burkholderia thailandensis betBA Operon

2009 ◽  
Vol 75 (12) ◽  
pp. 4015-4027 ◽  
Author(s):  
Yun Kang ◽  
Michael H. Norris ◽  
Ashley R. Barrett ◽  
Bruce A. Wilcox ◽  
Tung T. Hoang
Keyword(s):  
Osmotic Stress ◽  
Genetic Manipulation ◽  
Fluorescent Microscopy ◽  
Single Copy ◽  
Burkholderia Cenocepacia ◽  
Helper Plasmid ◽  
Burkholderia Thailandensis ◽  
Genetic Tools ◽  
Fusion Strain ◽  
Semialdehyde Dehydrogenase

ABSTRACT There are few appropriate single-copy genetic tools for most Burkholderia species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents Burkholderia pseudomallei and Burkholderia mallei, necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic selection based on two nonantibiotic selectable markers, tellurite resistance (Telr) and Escherichia coli aspartate-semialdehyde dehydrogenase (asdEc ), were developed to facilitate genetic manipulation in Burkholderia species. These systems include one mariner transposon, a mini-Tn7-derived site-specific transposon, and six FRT reporter fusion vectors based on the lacZ, gfp, and luxCDABE reporter genes. Initially, we showed that the random mariner transposon pBT20-Δbla-Telr-FRT efficiently transposed within Burkholderia cenocepacia, Burkholderia thailandensis, B. pseudomallei, and B. mallei. We then utilized the mini-Tn7-Telr-based transposon vector (mini-Tn7-Telr-betBA) and a transposase-containing helper plasmid (pTNS3-asdEc ) to complement the B. thailandensis ΔbetBA mutation. Next, one of the FRT-lacZ fusion vectors (pFRT1-lacZ-Telr) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp-oriT-asdEc ) to construct the B. thailandensis ΔbetBA::FRT-lacZ-Telr reporter fusion strain. The betBA operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing β-galactosidase assays on the B. thailandensis ΔbetBA::FRT-lacZ-Telr fusion strain. Finally, we engineered B. thailandensis ΔbetBA::FRT-gfp-Telr and ΔbetBA::FRT-lux-Telr fusion strains by utilizing fusion vectors pFRT1-gfp-Telr and pFRT1-lux-Telr, respectively. The induction of the betBA operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.

scholarly journals A MYB-related Transcription Factor from Sheepgrass, LcMYB2, Promotes Seed Germination and Root Growth under Drought Stress

2019 ◽  
Author(s):  
Pincang Zhao ◽  
Shenglin Hou ◽  
xiufang guo ◽  
Junting Jia ◽  
Weiguang Yang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Drought Stress ◽  
Seed Germination ◽  
Osmotic Stress ◽  
Root Growth ◽  
Genetic Manipulation ◽  
Marker Genes ◽  
Plant Responses ◽  
Adverse Conditions ◽  
Related Transcription Factor

Abstract Background Drought is one of the most serious factors limiting plant growth and production. Sheepgrass can adapt well to various adverse conditions, including drought. However, during germination, sheepgrass young seedlings are sensitive to these adverse conditions. Therefore, the adaptability of seedlings is very important for plant survival, especially in plants that inhabit grasslands or the construction of artificial grassland. Results In this study, we found a sheepgrass MYB-related transcription factor, LcMYB2 that is up-regulated by drought stress and returns to a basal level after rewatering. The expression of LcMYB2 was mainly induced by osmotic stress and was localized to the nucleus. Furthermore, we demonstrate that LcMYB2 promoted seed germination and root growth under drought and ABA treatments. Additionally, we confirmed that LcMYB2 can regulate LcDREB2 expression in sheepgrass by binding to its promoter, and it activates the expression of the osmotic stress marker genes AtDREB2A, AtLEA14 and AtP5CS1 by directly binding to their promoters in transgenic Arabidopsis. Conclusions Based on these results, we propose that LcMYB2 improves plant drought stress tolerance by increasing the accumulation of osmoprotectants and promoting root growth. Therefore, LcMYB2 plays pivotal roles in plant responses to drought stress and is an important candidate for genetic manipulation to create drought-resistant crops, especially during seed germination.

scholarly journals Intracellular survival ofBurkholderia cepaciacomplex in phagocytic cells

2015 ◽  
Vol 61 (9) ◽  
pp. 607-615 ◽  
Author(s):  
Miguel A. Valvano
Keyword(s):  
Burkholderia Cepacia ◽  
Molecular Mechanisms ◽  
Inflammatory Responses ◽  
Genetic Manipulation ◽  
Intracellular Survival ◽  
Burkholderia Cenocepacia ◽  
Burkholderia Cepacia Complex ◽  
Host Responses ◽  
Current View ◽  
Phagocytic Cells

Burkholderia cepacia complex (Bcc) species are a group of Gram-negative opportunistic pathogens that infect the airways of cystic fibrosis patients, and occasionally they infect other immunocompromised patients. Bcc bacteria display high-level multidrug resistance and chronically persist in the infected host while eliciting robust inflammatory responses. Studies using macrophages, neutrophils, and dendritic cells, combined with advances in the genetic manipulation of these bacteria, have increased our understanding of the molecular mechanisms of virulence in these pathogens and the molecular details of cell-host responses triggering inflammation. This article discusses our current view of the intracellular survival of Burkholderia cenocepacia within macrophages.

scholarly journals Overexpression of BvHb2, a Class 2 Non-Symbiotic Hemoglobin from Sugar Beet, Confers Drought-Induced Withering Resistance and Alters Iron Content in Tomato

Agronomy ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1754
Author(s):  
Carmina Gisbert ◽  
Alfonso Timoneda ◽  
Rosa Porcel ◽  
Roc Ros ◽  
José M. Mulet
Keyword(s):  
Abiotic Stress ◽  
Osmotic Stress ◽  
Sugar Beet ◽  
Stress Tolerance ◽  
Iron Content ◽  
Single Copy ◽  
Biotechnological Potential ◽  
Osmotic Stress Tolerance ◽  
Transgenic Lines ◽  
Model Plant Arabidopsis Thaliana

Drought stress is one of the major threats to agriculture and concomitantly to food production. Tomato is one of the most important industrial crops, but its tolerance to water scarcity is very low. Traditional plant breeding has a limited margin to minimize this water requirement. In order to design novel biotechnological approaches to cope with this problem, we have screened a plant cDNA library from the halotolerant crop sugar beet (Beta vulgaris L.) for genes able to confer drought/osmotic stress tolerance to the yeast model system upon overexpression. We have identified the gene that encodes BvHb2, a class 2 non-symbiotic hemoglobin, which is present as a single copy in the sugar beet genome, expressed mainly in leaves and regulated by light and abiotic stress. We have evaluated its biotechnological potential in the model plant Arabidopsis thaliana and found that BvHb2 is able to confer drought and osmotic stress tolerance. We also generated transgenic lines of tomato (Solanum lycopersicum) overexpressing BvHb2 and found that the resulting plants are more resistant to drought-induce withering. In addition, transgenic lines overexpressing BvHb2 exhibit increased levels of iron content in leaves. Here, we show that class 2 non-symbiotic plant hemoglobins are targets to generate novel biotechnological crops tolerant to abiotic stress. The fact that these proteins are conserved in plants opens the possibility for using Non-GMO approaches, such as classical breeding, molecular breeding, or novel breeding techniques to increase drought tolerance using this protein as a target.

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.

Mini-Tn7 vectors as genetic tools for single copy gene cloning in Acinetobacter baumannii

2010 ◽  
Vol 82 (3) ◽  
pp. 296-300 ◽  
Author(s):  
Ayush Kumar ◽  
Courtney Dalton ◽  
Jenny Cortez-Cordova ◽  
Herbert P. Schweizer
Keyword(s):  
Acinetobacter Baumannii ◽  
Gene Cloning ◽  
Single Copy ◽  
Single Copy Gene ◽  
Genetic Tools ◽  
Copy Gene

scholarly journals Molecular ruler of the attachment organelle in Mycoplasma pneumoniae

2021 ◽  
Vol 17 (6) ◽  
pp. e1009621
Author(s):  
Daisuke Nakane ◽  
Kohki Murata ◽  
Tsuyoshi Kenri ◽  
Keigo Shibayama ◽  
Takayuki Nishizaka
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Genetic Manipulation ◽  
Fluorescent Microscopy ◽  
Coiled Coil ◽  
Cell Movement ◽  
Molecular Structures ◽  
Molecular Architecture ◽  
Cell Axis ◽  
Transmission Electron ◽  
Molecular Ruler

Length control is a fundamental requirement for molecular architecture. Even small wall-less bacteria have specially developed macro-molecular structures to support their survival. Mycoplasma pneumoniae, a human pathogen, forms a polar extension called an attachment organelle, which mediates cell division, cytadherence, and cell movement at host cell surface. This characteristic ultrastructure has a constant size of 250–300 nm, but its design principle remains unclear. In this study, we constructed several mutants by genetic manipulation to increase or decrease coiled-coil regions of HMW2, a major component protein of 200 kDa aligned in parallel along the cell axis. HMW2-engineered mutants produced both long and short attachment organelles, which we quantified by transmission electron microscopy and fluorescent microscopy with nano-meter precision. This simple design of HMW2 acting as a molecular ruler for the attachment organelle should provide an insight into bacterial cellular organization and its function for their parasitic lifestyles.

scholarly journals A MYB-related Transcription Factor from Sheepgrass, LcMYB2, Promotes Seed Germination and Root Growth under Drought Stress

2019 ◽  
Author(s):  
xiufang guo ◽  
Pincang Zhao ◽  
Shenglin Hou ◽  
Junting Jia ◽  
Weiguang Yang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Drought Stress ◽  
Seed Germination ◽  
Osmotic Stress ◽  
Root Growth ◽  
Genetic Manipulation ◽  
Marker Genes ◽  
Plant Responses ◽  
Adverse Conditions ◽  
Related Transcription Factor

Abstract Background Drought is one of the most serious factors limiting plant growth and production. Sheepgrass can adapt well to various adverse conditions, including drought. However, during germination, sheepgrass young seedlings are sensitive to these adverse conditions. Therefore, the adaptability of seedlings is very important for plant survival, especially in plants that inhabit grasslands or the construction of artificial grassland. Results In this study, we found a sheepgrass MYB-related transcription factor, LcMYB2 that is up-regulated by drought stress and returns to a basal level after rewatering. The expression of LcMYB2 was mainly induced by osmotic stress and was localized to the nucleus. Furthermore, we demonstrate that LcMYB2 promoted seed germination and root growth under drought and ABA treatments. Additionally, we confirmed that LcMYB2 can regulate LcDREB2 expression in sheepgrass by binding to its promoter, and it activates the expression of the osmotic stress marker genes AtDREB2A, AtLEA14 and AtP5CS1 by directly binding to their promoters in transgenic Arabidopsis. Conclusions Based on these results, we propose that LcMYB2 improves plant drought stress tolerance by increasing the accumulation of osmoprotectants and promoting root growth. Therefore, LcMYB2 plays pivotal roles in plant responses to drought stress and is an important candidate for genetic manipulation to create drought-resistant crops, especially during seed germination.

scholarly journals Impairment of Infectivity and Immunoprotective Effect of a LYT1 Null Mutant of Trypanosoma cruzi

2007 ◽  
Vol 76 (1) ◽  
pp. 443-451 ◽  
Author(s):  
M. Paola Zago ◽  
Alejandra B. Barrio ◽  
Rubén M. Cardozo ◽  
Tomás Duffy ◽  
Alejandro G. Schijman ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Genetic Manipulation ◽  
Antibiotic Resistance Genes ◽  
Parasite Burden ◽  
Parasite Load ◽  
Single Copy ◽  
Protein Product ◽  
Host Cells ◽  
Insect Vector ◽  
Null Mutant

ABSTRACT Trypanosoma cruzi infection of host cells is a complex process in which many proteins participate but only a few of these proteins have been identified experimentally. One parasite factor likely to be involved is the protein product of LYT1, a single-copy gene cloned, sequenced, and characterized by Manning-Cela et al. (Infect. Immun. 69:3916-3923, 2001). This gene was potentially associated with infectivity, since the deletion of both LYT1 alleles in the CL Brenner strain (the wild type [WT]) resulted in a null mutant T. cruzi clone (L16) that shows an attenuated phenotype in cell culture models. The aim of this work was to characterize the infective behavior of L16 in the insect vector and murine models. The infection of adult Swiss mice with 103 trypomastigotes of both clones revealed a significant reduction in infective behavior of L16, as shown by direct parasitemia, spleen index, and quantitation of tissue parasite burden, suggesting the loss of virulence in the null mutant clone. Although L16 blood counts were almost undetectable, blood-based PCRs indicated the presence of latent and persistent infection during all of the study period and epimastigotes were reisolated from hemocultures until 12 months postinfection. Nevertheless, virulence was not restored in L16 by serial passages in mice, and reisolated parasites lacking the LYT1 gene and bearing the antibiotic resistance genes revealed the stability of the genetic manipulation. Histopathological studies showed a strong diminution in the muscle inflammatory response triggered by L16 compared to that triggered by the WT group, consistent with a lower tissue parasite load. A strong protection against a virulent challenge in both L16- and WT-infected mice was observed; however, the immunizing infection by the genetically modified parasite was highly attenuated.

scholarly journals Single-copy chromosomal integration systems for Francisella tularensis

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1152-1163 ◽  
Author(s):  
Eric D. LoVullo ◽  
Claudia R. Molins-Schneekloth ◽  
Herbert P. Schweizer ◽  
Martin S. Pavelka
Keyword(s):  
Francisella Tularensis ◽  
High Frequency ◽  
Genetic Manipulation ◽  
Single Copy ◽  
Gene Encoding ◽  
Highly Pathogenic ◽  
Pathogenic Organism ◽  
Allelic Exchange ◽  
Specific Manner ◽  
A Site

Francisella tularensis is a fastidious Gram-negative bacterium responsible for the zoonotic disease tularemia. Investigation of the biology and molecular pathogenesis of F. tularensis has been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of genetic tools. However, recent advances have substantially improved the ability of researchers to genetically manipulate this organism. To expand the molecular toolbox we have developed two systems to stably integrate genetic elements in single-copy into the F. tularensis genome. The first system is based upon the ability of transposon Tn7 to insert in both a site- and orientation-specific manner at high frequency into the attTn7 site located downstream of the highly conserved glmS gene. The second system consists of a sacB-based suicide plasmid used for allelic exchange of unmarked elements with the blaB gene, encoding a β-lactamase, resulting in the replacement of blaB with the element and the loss of ampicillin resistance. To test these new tools we used them to complement a novel d-glutamate auxotroph of F. tularensis LVS, created using an improved sacB-based allelic exchange plasmid. These new systems will be helpful for the genetic manipulation of F. tularensis in studies of tularemia biology, especially where the use of multi-copy plasmids or antibiotic markers may not be suitable.

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