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Regulation of CO2 fixation in Rhodopseudomonas palustris mediated by a unique two-component regulatory system

Joshi, Gauri Suresh
http://rave.ohiolink.edu/etdc/view?acc_num=osu1273605616

Abstract Details

2010, Doctor of Philosophy, Ohio State University, Microbiology.
Rhodopseudomonas palustris assimilates CO2 by the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway. The R. palustris CGA010 cbbI regulon contains the divergently transcribed cbbR gene encoding a LysR regulator, CbbR, and the cbbLS genes encoding the large and small subunits of form I ribulose -1, 5 -bisphosphate (RuBP) carboxylase oxygenase (RubisCO). Unique to this regulon is the presence of genes encoding the CbbRRS proteins, a redox sensing three protein two-component system that influences transcription of the cbbI genes in photoautotrophically grown cells. The distinctive presence of the CbbRRS system within the cbbI region raised questions as to its functional significance. The proteins of the CbbRRS system include a hybrid sensor kinase (CbbSR) and two response regulators (CbbRR1 and CbbRR2), neither of which contain any discernible DNA binding domain. CbbR belongs to the well studied LysR family of regulators (LTTRs) and has been shown to positively regulate cbb operon expression in photosynthetic and chemoautotrophic bacteria. Often, binding of a coinducer molecule to the LTTR is essential for transcriptional activation of its target gene, believed to occur by virtue of changes in the DNA architecture and interactions with RNA polymerase (RNAP). The coinducer is usually a unique metabolite (s) of the pathway regulated by the specific LTTR. This study was initiated to understand the molecular mechanism of CbbR-mediated transcriptional control of the cbbI operon in R. palustris. With the previous establishment of a regulatory role specifically during photolithoautrophic growth, it was speculated that the CbbRRS system influences cbbLS transcription, most likely by protein-protein interactions with the master transcriptional regulator CbbR. The current study confirmed and established direct protein-protein interactions between the response regulators of the CbbRRS system and the master regulator CbbR, using a variety of techniques including in vivo bacterial two hybrid studies, in vitro gel mobility shift assays, protein crosslinking, and surface plasmon resonance (SPR). Both response regulators (CbbRR1 and CbbRR2) played distinct roles in influencing the interaction of CbbR at the cbbI promoter, as revealed by gel mobility shift assays. CbbRR1 increased the binding affinity of CbbR at the cbbI promoter 3-5 fold while CbbRR2 appeared to stabilize CbbR binding. The CbbR-cbbI promoter interactions were probed in greater detail in order to gain further insight into the molecular mechanism of cbbLS transcription. A variety of potential metabolic effectors (coinducers) were found to alter the cbbI promoter binding properties of CbbR, both alone and in tandem with response regulators (CbbRR1 and CbbRR2) of the CbbRRS system. The current in vitro results suggest that the transcriptional control of the cbbI operon in R. palustris involves a complex interplay of protein regulators and metabolic signals all of which are transduced to the transcription machinery via CbbR. The CbbRRS two-component system thus represents additional transcriptional control beyond that provided by CbbR alone, for potential fine tuning cbbLS gene expression in R. palustris.
Robert Tabita, PhD (Advisor)
Birgit Alber, PhD (Committee Member)
Charles Daniels, PhD (Committee Member)
Joseph Krzycki, PhD (Committee Member)
Microbiology

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Citations

  • Joshi, G. S. (2010). Regulation of CO2 fixation in Rhodopseudomonas palustris mediated by a unique two-component regulatory system [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1273605616

    APA Style (7th edition)

  • Joshi, Gauri. Regulation of CO2 fixation in Rhodopseudomonas palustris mediated by a unique two-component regulatory system. 2010. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1273605616.

    MLA Style (8th edition)

  • Joshi, Gauri. "Regulation of CO2 fixation in Rhodopseudomonas palustris mediated by a unique two-component regulatory system." Doctoral dissertation, Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1273605616

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.