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The ActS-ActR two-component signal transduction system of Sinorhizobium meliloti

Fenner, Beau James (2002) The ActS-ActR two-component signal transduction system of Sinorhizobium meliloti. PhD thesis, Murdoch University.

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Abstract

Sinorhizobium meliloti is the nitrogen-fixing bacterial endosymbiont of the root nodules of Medicago spp. that are agriculturally important as pasture legumes. Establishment of medic pastures on many soils worldwide has been limited by the acid sensitivity of S. meliloti. Among the S. meliloti genes identified as being essential for acid tolerance are actS and actR, which encode the ActS histidine kinase and cognate ActR response regulator. This study was undertaken to further understanding of the role of ActSR in relation to acid tolerance and the general physiology of S. meliloti.

A preliminary search for the transcriptional targets of ActSR using mutagenesis with a promoterless gusA-carrying minitransposon identified eight fusions that were transcriptionally regulated by ActSR. Among the regulated genes were those encoding assimilatory nitrate reductase, cytochrome cbb3, oxidase, glutathione S-transferase, hydantoinase, a PAS two-component receiver protein and the CO2-fixing enzyme ribulose- 1,5-bisphosphate carboxylase/oxygenase.

The fixNOQP genes that encode the symbiotically-essential cytochrome cbb3 terminal oxidase required ActSR for low pH induction and for full expression under microaerobic conditions. The activation of fixNOQP by ActSR did not appear to occur directly, but instead by ActR regulating the transcription of the FixK transcriptional activator, which in turn activates fixNOQP. In addition to the cytochrome cbb3 terminal oxidase, ActR was also found to activate expression of the nifA gene that encodes the NifA nitrogen fixation regulator, and of the cta and qxt operons that encode cytochrome aa3 and a quinol oxidase, respectively, in response to low pH and reduced oxygen tension.

Biochemical and genetic analyses revealed that ActR also coordinated the expression of genes encoding enzymes involved in C1 metabolism. ActR functioned as a transcriptional repressor of the fds operon encoding a NAD+-linked formate dehydrogenase, while this operon was activated independently of ActR by the presence formate. Conversely, ActR activated the cbb operon that encodes enzymes of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway during organoautotrophic growth on formate. The cbb operon was also positively regulated by the CbbR regulator, being essential for the high-level of cbb induction under these conditions.

ActR was overexpressed in E. coli and purified as a His6-tagged recombinant protein. Electrophoretic mobility shift assays revealed that this protein was able to bind specifically to DNA target sequences that are known to be bound by ActR homologues from other species. The binding affinity of His6-ActR was increased several fold by incubating the protein with carbamyl phosphate, which presumably mimics the effect of ActS-mediated phosphorylation of ActR. An analysis of the promoter regions of the identified ActR transcriptional targets revealed the presence of DNA sequences with significant similarity to previously defined binding motifs of ActR homologues from other bacterial species. A DNase footprinting analysis also showed that ActR protected a region from -33 to -10 of the nifA promoter region. The protected region also shared significant sequence similarity to the consensus binding sequence of an ActR homologue from Rhodobacter capsulatus.

The results of this study indicate that ActSR are involved in the transcriptional regulation of a diverse range of physiological processes and clearly play an important role as global regulators of redox-sensitive processes in S. meliloti.

Item Type: Thesis (PhD)
Murdoch Affiliation: Division of Science
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: repository@murdoch.edu.au. Thank you.
Supervisor(s): Dilworth, Michael, Tiwari, Ravi and Glenn, Andrew
URI: http://researchrepository.murdoch.edu.au/id/eprint/51691
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