Browsing by Author "Lee, Hui-Ju"

Now showing 1 - 4 of 4

Enhancing ATP-based bacteria and biofilm detection by enzymatic pyrophosphate regeneration
(2010-04-15)
Lee, Hui-Ju
;
Ho, Ming-Rong
;
Bhuwan, Manish
;
Hsu, Ching-Yi
;
Huang, Meng-Shun
The manufacturing processes of many electronic and medical products demand the use of high-quality water. Hence the water supply systems for these processes are required to be examined regularly for the presence of microorganisms and microbial biofilms. Among commonly used bacteria detection approaches, the ATP luminescence assay is a rapid, sensitive, and easy to perform method. The aim of this study is to investigate whether ATP regeneration from inorganic pyrophosphate, a product of the ATP luminescence assay, can stabilize the bioluminescence signals in ATP detection. ADPglc pyrophosphorylase (AGPPase), which catalyzes the synthesis of ATP from PP(i) in the presence of ADPglc, was selected because the system yields much lower luminescence background than the commercially available ATP sulfurylase/adenosine 5'-phosphosulfate (APS) system which was broadly used in pyrosequencing technology. The AGPPase-based assay could be used to measure both PPi and ATP quantitatively and shows 1.5- to 4.0-fold slight increases in a 10-min assay. The method could also be used to stabilize the luminescence signals in detection of Escherichia coli, Pseudo monas aeruginosa, and Bacillus cereus in either broth or biofilm. These findings suggest that the AGPPase-based ATP regeneration system will find many practical applications such as detection of bacterial biofilm in water pipelines. (C) 2009 Elsevier Inc. All rights reserved.
Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria
(2011-11-01)
Lee, Hui-Ju
;
Ho, Min-Rong
;
Tseng, Chih-Sian
;
Hsu, Ching-Yi
;
Huang, Meng-Shun
Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia cob is approximately 10(4) colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP(i) and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP(i)-recycling loop was completed using ATP sulfurylase and adenosine 5' phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water. (C) 2011 Elsevier Inc. All rights reserved.
Histidine-containing Phosphotransfer Protein-B (HptB) Regulates Swarming Motility through Partner-switching System in Pseudomonas aeruginosa PAO1 Strain
(2012-01-13)
Bhuwan, Manish
;
Lee, Hui-Ju
;
Peng, Hwei-Ling
;
Chang, Hwan-You
;
生物科技學系
The histidine-containing phosphotransfer protein-B (HptB; PA3345) is an intermediate protein involved in transferring a phosphoryl group from multiple sensor kinases to the response regulator PA3346 in Pseudomonas aeruginosa PAO1. The objective of this study was to elucidate the biological significance of the HptB-PA3346 interaction and the regulatory mechanisms thereafter. The transcription profiling analysis of an hptB knock-out mutant showed that the expression of a number of motility-related genes was altered consistent with the non-swarming phenotype observed for the mutant. Domain analysis indicated that the PA3346 C-terminal region (PA3346C) exhibits similar to 30% identity with the anti-sigma factor SpoIIAB of Bacillus subtilis. The presence of Ser/Thr protein kinase activity targeting an anti-sigma antagonist, PA3347, at Ser-56 was confirmed in PA3346C using an in vitro phosphorelay assay. Furthermore, PA3346C and the anti-sigma(28) factor FlgM were found to interact with PA3347 individually both in vivo and in vitro. FlgM displaced PA3346C in binding of PA3347 and was then competitively displaced by sigma(28) from the PA3347-FlgM complex, forming a phosphorylation-dependent partner-switching system. The significance of PA3347 phosphorylation in linking the partner-switching system and swarming motility was established by analyzing the swarming phenotype of the PA3347 knock-out mutant and its complement strains.
Identification of amino acid residues important for the phosphomannose isomerase activity of Ps1B in Pseudomonas aeruginosa PAO1
(2008-10-15)
Lee, Hui-Ju
;
Chang, Hwan-You
;
Venkatesan, Nandinin
;
Peng, Hwei-Ling
;
生物科技學院
Phosphomannose isomerase (PMI) plays a pivotal role in biosynthesis of GDP-mannose, an important precursor of many polysaccharides. We demonstrate in this study that Pseudomonas aeruginosa pslB encodes a protein with GDP-mannose pyrophosphorylase/PMI dual activities. The PMI activity is Co2+-dependent and could be inhibited by GDP-mannose in a competitive manner. Furthermore, the activity could be inactivated by 2,3-butanedione suggesting the presence of a catalytic Arg residue. Site-specific mutations at R373, R472, R479, E410, H411, N433 and E458 increase the KM approximately 8-20-fold. The PMI activity of PslB was completely diminished with a R408K or R408A, reflecting the importance of this residue in catalysis. Overall, these results provide a basis for understanding the catalytic mechanism of PMI.