Browsing by Author "Bhuwan, Manish"

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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.
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.