scholarly journals Weak Transcription of thecry1AcGene in Nonsporulating Bacillus thuringiensis Cells

2012 ◽  
Vol 78 (18) ◽  
pp. 6466-6474 ◽  
Author(s):  
Hui Yang ◽  
Pinshu Wang ◽  
Qi Peng ◽  
Rong Rong ◽  
Chunxia Liu ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Bacillus Thuringiensis ◽  
Fluorescent Protein ◽  
Cellular Level ◽  
Protein Fusion ◽  
Reporter System ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Green Fluorescent ◽  
Sigma E

ABSTRACTThecry1Acgene ofBacillus thuringiensissubsp.kurstakiHD-73 (B. thuringiensisHD-73) is a typical example of a sporulation-dependent crystal gene and is controlled by sigma E and sigma K during sporulation. To monitor the production and accumulation of Cry1Ac at the cellular level, we developed a green fluorescent protein-based reporter system. The production of Cry1Ac was monitored inspo0A,sigE, andsigKmutants, and these mutants were able to express the Cry1Ac-green fluorescent protein fusion protein. In nonsporulatingB. thuringiensisHD-73 cells, low-level expression ofcry1Acwas also observed. Reverse transcription-PCR and Western blotting results confirmed that thecry1Acpromoter has low activity in nonsporulatingB. thuringiensiscells. A beta-galactosidase assay demonstrated that the transcription of thecry1Acgene during exponential and transition phases is positively regulated by Spo0A. Additional bioassay results indicated thatspo0AandsigEmutants containing thecry1Ac-gfpfusion exhibited insecticidal activity againstPlutella xylostellalarvae.

scholarly journals In VivoSelf-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

2014 ◽  
Vol 80 (10) ◽  
pp. 3062-3071 ◽  
Author(s):  
Mark Venning-Slater ◽  
David O. Hooks ◽  
Bernd H. A. Rehm
Keyword(s):  
Green Fluorescent Protein ◽  
Enzyme Immobilization ◽  
Self Assembly ◽  
Fluorescent Protein ◽  
Specific Binding ◽  
Biomass Conversion ◽  
Protein Fusion ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTBacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously thein vivoself-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable α-amylase fromBacillus licheniformis,N-acetyl-d-neuraminic acid aldolase (NanA) fromEscherichia coli, and organophosphohydrolase (OpdA) fromAgrobacterium radiobacter. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The α-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convertN-acetylmannosamine and pyruvate toN-acetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.

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 Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa

2012 ◽  
Vol 78 (15) ◽  
pp. 5060-5069 ◽  
Author(s):  
Morten T. Rybtke ◽  
Bradley R. Borlee ◽  
Keiji Murakami ◽  
Yasuhiko Irie ◽  
Morten Hentzer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Subsequent Development ◽  
Cellular Level ◽  
Host Immune System ◽  
Chronic Infections ◽  
Content Type ◽  
Genes Encoding ◽  
Green Fluorescent

ABSTRACTThe increased tolerance toward the host immune system and antibiotics displayed by biofilm-formingPseudomonas aeruginosaand other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP inP. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsivecdrApromoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP inP. aeruginosastrains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment ofP. aeruginosawith the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology ofP. aeruginosa.

scholarly journals Developmental-Stage-Specific Assembly of ParB Complexes in Streptomyces coelicolor Hyphae

2005 ◽  
Vol 187 (10) ◽  
pp. 3572-3580 ◽  
Author(s):  
Dagmara Jakimowicz ◽  
Bertolt Gust ◽  
Jolanta Zakrzewska-Czerwinska ◽  
Keith F. Chater
Keyword(s):  
Green Fluorescent Protein ◽  
Developmental Stage ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Streptomyces Coelicolor ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Chromosome Partitioning ◽  
Aerial Hyphae ◽  
Green Fluorescent

ABSTRACT In Streptomyces coelicolor ParB is required for accurate chromosome partitioning during sporulation. Using a functional ParB-enhanced green fluorescent protein fusion, we observed bright tip-associated foci and other weaker, irregular foci in S. coelicolor vegetative hyphae. In contrast, in aerial hyphae regularly spaced bright foci accompanied sporulation-associated chromosome condensation and septation.

Sign in / Sign up

Export Citation Format

Share Document