Browsing by Author "Hsu, Jye-Lin"

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Characterization of the histidine-containing phosphotransfer protein B-mediated multistep phosphorelay system in Pseudomonas aeruginosa PAO1
(2008-04-11)
Hsu, Jye-Lin
;
Chen, Hsuan-Cheng
;
Peng, Hwei-Ling
;
Chang, Hwan-You
;
生物科技學系
Certain bacterial two-component sensor kinases possess a histidine-containing phosphotransfer (Hpt) domain to carry out a multistep phosphotransferring reaction to a cognate response regulator. Pseudomonas aeruginosa PAO1 contains three genes that encode proteins with an Hpt domain but lack a kinase domain. To identify the sensor kinase coupled to these Hpt proteins, a phosphorelay profiling assay was performed. Among the 12 recombinant orphan sensor kinases tested, 4 of these sensors (PA1611, PA1976, PA2824, and RetS) transferred the phosphoryl group to HptB (PA3345). The in vivo interaction between HptB and each of the sensors was also confirmed using the bacterial two-hybrid assay. Interestingly, the phosphoryl groups from these sensors all appeared to be transferred via HptB to PA3346, a novel phosphatase consisting of an N-terminal receiver domain and a eukaryotic type Ser/Thr phosphatase domain, and resulted in a significant increase of its phosphatase activity. The subsequent reverse transcription-PCR analysis revealed an operon structure of hptB-PA3346-PA3347, suggesting a coordinate expression of the three genes to carry out a signal transduction. The possibility was supported by the analysis showing PA3347 is able to be phosphorylated on Ser-56, and this phosphoryl group could be removed by PA3346 protein. Finally, analysis of PA3346 and PA3347 gene knock-out mutants revealed that these genes are associated with bacterial swarming activity and biofilm formation. Together, these results disclose a novel multistep phosphorelay system that is essential for P. aeruginosa to respond to a wide spectrum of environmental signals.
Surface Rigidity Change of Escherichia coli after Filamentous Bacteriophage Infection
(2009-04-21)
Chen, Yi-Yang
;
Wu, Chien-Chen
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Hsu, Jye-Lin
;
Peng, Hwei-Ling
;
Chang, Hwan-You
In this study, the feasibility using atomic force microscopy (AFM) to study the interaction between bacteriophages (phages) and bacteria in situ was demonstrated here. Filamentous phage M 13 specifically infects the male Escherichia coli, which expresses F-pili. After infection, E. coli become fragile and grows at a slower rate. AFM provides a powerful tool for investigating these changes in a near-physiological environment. Using high-resolution AFM in phosphate-buffered saline, the damage to the lipopolysaccharide (LPS) layer on the outer membrane of the M13 phage-infected E. coli was observed. The membrane became smoother and more featureless compared to those that were not infected. Besides, the force-distance (f-d) curves were measured to reveal the surface rigidity change in E. coli after M13 phage infection. The effective spring constant and Young's modulus of E. coli decreased after M13 phage infection. Furthermore, the AFM tip was pressed against E. coli to stud), the response of E. coli under load before and after M 13 phage infection. The results showed that after infection E. coli became less rigid and the membrane was also damaged. However, the stiffness changes, including the spring constant and Young's modulus of E. coli, are negligible after M 13 phage infection compared with those in previous reports, which may be one of the reasons that E. coli still can maintain its viability after filamentous phage infection.
The ATP-binding motif in AcoK is required for regulation of acetoin catabolism in Klebsiella pneumoniae CG43
(2008-11-07)
Hsu, Jye-Lin
;
Peng, Hwei-Ling
;
Chang, Hwan-You
;
生物科技學系
;
Department of Biological Science and Technology
Many bacterial species utilize acetoin as a carbon source. In Klebsiella pneumoniae, the utilization of acetoin is catalyzed by an acetoin dehydrogenase complex encoded by the acoABCD operon, which is positively regulated in the presence of acetoin by the transcriptional factor AcoK. AcoK contains a LuxR type DNA-binding domain at the C-terminal region and putative Walker A and B nucleotide-binding motifs in the N-terminal region. The comprehensive deletion and mutation study performed here shows that mutations in the putative Walker A motif result in a significant reduction of ATP hydrolysis and trans-activation by AcoK of acoABCD expression, presumably due to a loss of ATP-binding ability. AcoK was shown to bind specifically to nucleotides -66 to -36 of the acoABCD promoter, though the DNA-binding ability was not affected by the Walker A motif mutation. Thus, this study provides an additional example of how a member of the signal transduction ATPases with numerous domains family activates its target gene expression. (C) 2008 Elsevier Inc. All rights reserved.