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Title:Molecular mechanisms underlying regulation of type III secretion system upstream of the master regulator HrpL in Erwinia amylovora
Author(s):Lee, Jae Hoon
Advisor(s):Zhao, Youfu
Department / Program:Crop Sciences
Discipline:Crop Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Erwinia anylovora
Type III secretion system
Integration Host Factor (IHF)
Abstract:Erwinia amylovora, the causal agent of fire blight of apples and pears, is a necrogenic bacterium, whose virulence is dependent upon a functional hypersensitive response and pathogenicity (hrp)-type III secretion system (T3SS). It has been previously demonstrated that HrpL, an ECF sigma factor, is the master regulator of hrp-T3SS. Recently, it is reported that expression of hrpL is under the control of sigma 54 complex, including σ54 (RpoN), its modulation protein YhbH and σ54-enhancer binding protein HrpS. In this study, we investigated the role of integration host factors (IHFs) in regulating σ54-dependent hrpL and other T3SS gene expression. IHFs are nucleoid-associated proteins and consist of two subunits, i. e. IHFα and IHFβ. IHFα and IHFβ usually form heterodimers, which could influence nucleoid structure and gene expression via DNA bending. Two single mutants (ihfA and ihfB) were generated and characterized in E. amylovora. Results showed that both ihfA and ihfB mutants failed to colonize and produce necrotic lesions on immature pear fruits. Bacterial growth of both mutants in pear fruits was greatly reduced and expression of hrpL, dspE, hrpA and hrpN was also significantly down-regulated as compared to wild type (WT) strain. In addition, both ihfA and ihfB mutants exhibited slower growth in rich medium and showed hypermotile phenotype as compared to WT strain. Furthermore, results showed that both IHFs positively regulated the expression of small non-coding regulatory RNA rsmB/csrB, which negatively regulates motility as previously reported. These results indicate that IHFs are required for σ54-dependent hrpL and other T3SS gene expression and virulence in E. amylovora. On the other hand, the bacterial enhancer binding protein (bEBP) HrpS plays a central role in regulating T3SS gene expression by activating the transcription of hrpL gene in E. amylovora. Upon binding to upstream activator sequence (UAS) at the hrpL promoter, HrpS interacts with the σ54-RNA polymerase holoenzyme through conserved GAYTGA motif, which allows the initiation of hrpL transcription. However, where HrpS binds to the promoter of hrpL and what is the role of the conserved GAYTGA motif in regulating hrpL and other T3SS gene expression remain elusive. In this study, our goals were to identify the HrpS binding site and to characterize the role of conserved GAYTGA motif of HrpS in transcription activation of hrpL in E. amylovora. First, eight 5’ deletion constructs of hrpL promoter fused to a promoter-less gfp were made, and promoter activities were measured by flow cytometry. The results of promoter screening suggested a potential region for HrpS binding. Second, complementation of hrpL mutant using twelve constructs containing hrpL gene and various lengths of hrpL promoter further delineated the UAS region for HrpS binding. Bioinformatic analysis of this region revealed a dyad symmetry sequence between -141 to -122 nt (AT-N-TGCAA-N4-TTGCA-N-AT), which is characteristic for bEBP binding. Third, site-directed mutation analyses and quantitative real time-PCR (qRT-PCR) assays demonstrated that the complete-dyad symmetry sequence was all required for T3SS gene expression and complementation of hrpL mutant. Finally, electrophoretic mobility shift assay (EMSA) with purified truncated HrpS protein containing its DNA binding domain further verified that HrpS binds to this sequence, indicating that hrpL promoter from -141 to -122 is the HrpS binding site. In addition, results from site-directed mutagenesis analyses of the conserved GAYTGA motif of HrpS showed that Y100F substitution did not affect the function of HrpS, whereas Y100A and Y101A mutations completely abolished HrpS activity. These results suggest that tyrosine and phenylalanine can compensate functionally for each other in the GAYTGA motif of HrpS in E. amylovora.
Issue Date:2014-09-16
Rights Information:Copyright 2014 Jae Hoon Lee
Date Available in IDEALS:2014-09-16
Date Deposited:2014-08

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