scholarly journals New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

2016 ◽  
Vol 82 (8) ◽  
pp. 2240-2246 ◽  
Author(s):  
Alex I. Kanno ◽  
Cibelly Goulart ◽  
Henrique K. Rofatto ◽  
Sergio C. Oliveira ◽  
Luciana C. C. Leite ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Expression Vectors ◽  
Content Type ◽  
Expression Levels ◽  
Wide Range ◽  
Mycobacteriophage L5 ◽  
Green Fluorescent ◽  
Selection Of

ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

2011 ◽  
Vol 77 (22) ◽  
pp. 8193-8196 ◽  
Author(s):  
Lucja M. Jarosz ◽  
Bastiaan P. Krom
Keyword(s):  
Candida Albicans ◽  
Real Time ◽  
Real Time Pcr ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Rapid Screening ◽  
Content Type ◽  
Wide Range ◽  
Hyphal Formation ◽  
Green Fluorescent

ABSTRACTWe propose a screening method for compounds affecting growth and germination inCandida albicansusing a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using PACT1-GFPand PHWP1-GFPreporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

scholarly journals A Propionate-Inducible Expression System for Enteric Bacteria

2005 ◽  
Vol 71 (11) ◽  
pp. 6856-6862 ◽  
Author(s):  
Sung Kuk Lee ◽  
Jay D. Keasling
Keyword(s):  
Fluorescent Protein ◽  
Expression System ◽  
Enteric Bacteria ◽  
Expression Vectors ◽  
Gene Encoding ◽  
Basal Expression ◽  
Expression Of Genes ◽  
Catabolic Genes ◽  
Wide Range ◽  
Green Fluorescent

ABSTRACT A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in Escherichia coli. The pPro vectors contain the prpBCDE promoter (P prpB ) responsible for expression of the propionate catabolic genes (prpBCDE) and prpR encoding the positive regulator of this promoter. The efficiency and regulatory properties of the prpR-P prpB system were measured by placing the gene encoding the green fluorescent protein (gfp) under the control of the inducible P prpB of E. coli. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the prpBCDE promoter has CAP-dependent activation, the prpR-P prpB system exhibited negligible basal expression by addition of glucose to the medium.

scholarly journals Zebrafish as a Novel Vertebrate Model To Dissect Enterococcal Pathogenesis

2013 ◽  
Vol 81 (11) ◽  
pp. 4271-4279 ◽  
Author(s):  
Tomasz K. Prajsnar ◽  
Stephen A. Renshaw ◽  
Nikolay V. Ogryzko ◽  
Simon J. Foster ◽  
Pascale Serror ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Opportunistic Pathogen ◽  
Genetically Engineered ◽  
Intrinsic Resistance ◽  
Content Type ◽  
Wide Range ◽  
Vertebrate Model ◽  
Life Threatening ◽  
Green Fluorescent ◽  
Isogenic Mutants

ABSTRACTEnterococcus faecalisis an opportunistic pathogen responsible for a wide range of life-threatening nosocomial infections, such as septicemia, peritonitis, and endocarditis.E. faecalisinfections are associated with a high mortality and substantial health care costs and cause therapeutic problems due to the intrinsic resistance of this bacterium to antibiotics. Several factors contributing toE. faecalisvirulence have been identified. Due to the variety of infections caused by this organism, numerous animal models have been used to mimicE. faecalisinfections, but none of them is considered ideal for monitoring pathogenesis. Here, we studied for the first timeE. faecalispathogenesis in zebrafish larvae. Using model strains, chosen isogenic mutants, and fluorescent derivatives expressing green fluorescent protein (GFP), we analyzed both lethality and bacterial dissemination in infected larvae. Genetically engineered immunocompromised zebrafish allowed the identification of two critical steps for successful establishment of disease: (i) host phagocytosis evasion mediated by the Epa rhamnopolysaccharide and (ii) tissue damage mediated by the quorum-sensing Fsr regulon. Our results reveal that the zebrafish is a novel, powerful model for studyingE. faecalispathogenesis, enabling us to dissect the mechanism of enterococcal virulence.

scholarly journals Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

2006 ◽  
Vol 17 (7) ◽  
pp. 3009-3020 ◽  
Author(s):  
Johan-Owen De Craene ◽  
Jeff Coleman ◽  
Paula Estrada de Martin ◽  
Marc Pypaert ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Cortex ◽  
Proteomic Screen ◽  
Wide Range ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Hydrophilic Loop ◽  
Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

scholarly journals Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

2011 ◽  
Vol 77 (23) ◽  
pp. 8310-8317 ◽  
Author(s):  
Joshua D. Morris ◽  
Jessica L. Hewitt ◽  
Lawrence G. Wolfe ◽  
Nachiket G. Kamatkar ◽  
Sarah M. Chapman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Nile Red ◽  
Temporal Distribution ◽  
Time Lapse ◽  
Content Type ◽  
Rhamnolipid Production ◽  
Serovar Typhimurium ◽  
Surface Motility ◽  
Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

Selection of transgenic Petunia plants using the green fluorescent protein (GFP)

2011 ◽  
Vol 107 (3) ◽  
pp. 483-492 ◽  
Author(s):  
Viola Mußmann ◽  
Margrethe Serek ◽  
Traud Winkelmann
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
Selection Of ◽  
Transgenic Petunia

scholarly journals Spatial and Developmental Differentiation of Mannitol Dehydrogenase and Mannitol-1-Phosphate Dehydrogenase in Aspergillus niger

2010 ◽  
Vol 9 (9) ◽  
pp. 1398-1402 ◽  
Author(s):  
Guillermo Aguilar-Osorio ◽  
Patricia A. vanKuyk ◽  
Bernhard Seiboth ◽  
Dirk Blom ◽  
Peter S. Solomon ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Fluorescent Protein ◽  
Northern Analysis ◽  
Expression Data ◽  
Mannitol Dehydrogenase ◽  
Content Type ◽  
Green Fluorescent ◽  
Developmental Differentiation ◽  
Encoding Genes ◽  
Mannitol Cycle

ABSTRACT The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of Aspergillus niger. In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of A. niger. Northern analysis demonstrated that mpdA was expressed in both sporulating and nonsporulating mycelia, while expression of mtdA was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of mtdA and mpdA, respectively, demonstrated that expression of mpdA occurs in vegetative hyphae while mtdA expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in A. niger as two of the enzymes of the cycle are not present in the same part of A. niger colonies. The results also demonstrate the existence of spore-specific genes and enzymes in A. niger.

scholarly journals Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS256Tempering of WalKR Expression

2013 ◽  
Vol 57 (7) ◽  
pp. 3240-3249 ◽  
Author(s):  
Christopher R. E. McEvoy ◽  
Brian Tsuji ◽  
Wei Gao ◽  
Torsten Seemann ◽  
Jessica L. Porter ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Infection Model ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Phenotype Analysis ◽  
Dosing Regimens ◽  
Mrsa Strains ◽  
Green Fluorescent

ABSTRACTVancomycin-intermediateStaphylococcus aureus(VISA) strains often arise by mutations in the essential two-component regulatorwalKR; however their impact onwalKRfunction has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptibleS. aureus(VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256insertions in thewalKR5′ untranslated region (5′ UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction inwalKRgene expression in the IS256mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256insertions had replaced two SigA-likewalKRpromoters with weaker, hybrid promoters. Removal of IS256reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation ofssaAbut no change in expression ofsakandsceDin both IS256mutants. Whole-genome sequencing of the two mutants revealed an additional IS256insertion withinagrCfor one mutant, and we confirmed that this mutation abolishedagrfunction. These data provide the first substantial analysis ofwalKRpromoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction inwalKRexpression; however, the mechanisms by which this occurs remain to be determined.

scholarly journals Asc Modulates the Function of NLRC4 in Response to Infection of Macrophages by Legionella pneumophila

mBio ◽  
2011 ◽  
Vol 2 (4) ◽  
Author(s):  
Christopher L. Case ◽  
Craig R. Roy
Keyword(s):  
Cell Death ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Microbial Infection ◽  
Content Type ◽  
Caspase 1 ◽  
Cell Death Pathway ◽  
Dependent Cell ◽  
Green Fluorescent ◽  
In Cells

ABSTRACTNucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) activate caspase-1 in response to a variety of bacterium-derived signals in macrophages. NLR-mediated activation of caspase-1 byLegionella pneumophilaoccurs through both an NLRC4/NAIP5-dependent pathway and a pathway requiring the adapter protein Asc. Both pathways are needed for maximal activation of caspase-1 and for the release of the cytokines interleukin-1β (IL-1β) and IL-18. Asc is not required for caspase-1-dependent pore formation and cell death induced upon infection of macrophages byL. pneumophila. Here, temporal and spatial localization of caspase-1-dependent processes was examined to better define the roles of Asc and NLRC4 during infection. Imaging studies revealed that caspase-1 localized to a single punctate structure in infected cells containing Asc but not in cells lacking this adapter. Both endogenous Asc and ectopically produced NLRC4 tagged with green fluorescent protein (GFP) were found to localize to caspase-1 puncta followingL. pneumophilainfection, suggesting that NLRC4 and Asc coordinate signaling through this complex during caspase-1 activation. Formation of caspase-1-containing puncta correlated with caspase-1 processing, suggesting a role for the Asc/NLRC4/caspase-1 complex in caspase-1 cleavage. In cells deficient for Asc, NLRC4 did not assemble into discrete puncta, and pyroptosis occurred at an accelerated rate. These data indicate that Asc mediates integration of NLR components into caspase-1 processing platforms and that recruitment of NLR components into an Asc complex can dampen pyroptotic responses. Thus, a negative feedback role of complexes containing Asc may be important for regulating caspase-1-mediated responses during microbial infection.IMPORTANCECaspase-1 is a protease activated during infection that is central to the regulation of several innate immune pathways. Studies examining the macromolecular complexes containing this protein, known as inflammasomes, have provided insight into the regulation of this protease. This work demonstrates that the intracellular bacteriumLegionella pneumophilainduces formation of complexes containing caspase-1 by multiple mechanisms and illustrates that an adapter molecule called Asc integrates signals from multiple independent upstream caspase-1 activators in order to assemble a spatially distinct complex in the macrophage. There were caspase-1-associated activities such as cytokine processing and secretion that were controlled by Asc. Importantly, this work uncovered a new role for Asc in dampening a caspase-1-dependent cell death pathway called pyroptosis. These findings suggest that Asc plays a central role in controlling a distinct subset of caspase-1-dependent activities by both assembling complexes that are important for cytokine processing and suppressing processes that mediate pyroptosis.

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