Date

June 2012

Document Type

Dissertation

Degree Name

Ph.D.

Institution

Oregon Health & Science University

Abstract

Our long-term research goal is to understand how Bacillus subtilis senses and responds to oxygen limitation. B. subtilis can survive anaerobically by opting for nitrate respiration or fermentation. The ResDE signal transduction system has a key role in the transcriptional activation of genes required to support nitrate respiration such as nasDEF (nitrite reductase genes) and hmp (flavohemoglobin gene). However, the full induction of nasDEF and hmp transcription by the ResD response regulator requires NO despite oxygen limitation. Previous study showed that NsrR is responsible for the upregulation of nasD and hmp when cells were treated with exogenous NO. An intriguing question remains as to how NsrR-dependent transcriptional regulation works in response to NO. The overall aim of my research is to understand the mechanism by which NsrR repressor activity is modulated by NO and uncover the realm of NsrR regulation in B. subtilis. NsrR has a [4Fe-4S] cluster and exposure of the purified protein to NO results in iron dinitrosylation in the cluster. Electrophoretic mobility shift assays and in vitro transcription experiments using apo- and holo-NsrR demonstrated that the [4Fe-4S] cluster is essential for its NO-sensitive high-affinity interaction with the nasD promoter. NsrR represses transcription initiation at the nasD promoter by dissociating a preformed nasD-ResD-RNA polymerase complex. This study led to a new finding that two different NsrR-binding sites exist in the nasD promoter, namely class I and class II binding sites. Mutational and deletion analysis of the NsrR-binding regions showed that holo-NsrR recognizes a partial dyad symmetry in the class I site, whereas holo- and apo- equally bind to the A+T-rich class II site with a relaxed sequence specificity. Genome-wide transcriptome analysis revealed many candidate genes for the class II NsrR regulon, which include genes in the Fur and the AbrB/Rok regulons. In vivo transcription assays showed that the NsrR regulon is under complex control exerted by multiple regulators including AbrB, Rok, Fur, and ResD. We addressed NO-sensitive direct interaction of NsrR with class I and II genes in vivo by using ChAP-qPCR (chromatin affinity precipitation followed by qPCR). Both ResD and NsrR do not interact with the promoter of sdpA (encoding sporulation-delaying factor) in the AbrB/Rok regulon, indicating that ResD and NsrR play indirect roles in sdpA transcription. In contrast, NsrR binds to nasD and ykuN (flavodoxin gene) in the Fur regulon in a NO-sensitive manner. ResD also associates with the nasD and ykuN promoters and NsrR inhibits ResD binding to nasD, whereas either NsrR or Fur enhances ResD binding to ykuN. The study presented in this thesis laid the foundation for the further investigation of the detailed mechanism of the interplay among these regulators.

Identifier

doi:10.6083/M4W093XN

Division

Div. of Environmental and Biomolecular Systems

School

School of Medicine

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