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Bacterial enhancer binding protein HrpS is regulated by three two-component systems and Lon protease in Erwinia amylovora

Youfu Zhao: University of Illinois at Urbana-Champaign

<div>The bacterial enhancer binding protein HrpS is essential in activating <em>hrpL</em> and the type III secretion system (T3SS) in <em>Erwinia amylovora</em>. However, how the <em>hrpS</em> gene is regulated remains unknown in <em>E. amylovora</em>. In this study, we investigated the role of various two-component systems (TCSs) and Lon in regulating HrpS. Promoter deletion analyses and 5’ RACE showed that the <em>hrpS </em>gene contains two promoters driven by HrpX/HrpY and the Rcs system, respectively. EMSA and qRT-PCR assays demonstrated that integration host factor IHF positively regulates <em>hrpS </em>expression through directly binding the <em>hrpX</em> promoter and positively regulating <em>hrpX/hrpY </em>expression. Western blot analyses showed that mutation in <em>lon </em>directly affected the accumulation and stability of HrpS. Mutation in <em>lon</em> indirectly influenced the expression of <em>hrpS </em>through accumulation of the RcsA/RcsB proteins. Furthermore, the <em>csrA</em> mutant showed significantly reduced transcripts of the <em>hrpX</em>/<em>hrpY</em>, <em>rcsA </em>and <em>rcsB</em> genes, indicating that CsrA is required for full <em>hrpS </em>expression. On the other hand, the <em>csrB </em>mutant exhibited up-regulation of the <em>rcsA</em> and <em>rcsB </em>genes, and <em>hrpS </em>expression was largely diminished in the <em>csrB</em>/<em>rcsB </em>mutant, indicating that the Rcs system is mainly responsible for the increased <em>hrpS </em>expression in the <em>csrB </em>mutant. These findings suggest that <em>E. amylovora</em> recruits multiple TCSs to regulate <em>hrpS </em>at the transcriptional level and HrpS is also regulated at posttranslational level by Lon.</div>