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- Jonsson, Anders, 1967-
(författare)
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Regulation of Glutamine Synthetase in the Diazotroph Rhodospirillum rubrum
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The bacterial cell needs ammonia for synthesis of glutamine from glutamate. Only one enzyme is able to catalyze this reaction, namely glutamine synthetase (GS). GS can be regulated both transcriptionally and post-translationally and it is present in all kingdoms of life. Our study has been focused on the post-translational regulation of GS in the diazotrophic bacterium Rhodospirillum rubrum. A number of proteins are involved in the covalent regulation of GS, among them are the regulatory PII proteins that depending on growth conditions also like GS are covalently modified. We have purified all proteins involved in GS regulation and developed several in vitro assays with the aim of understanding GS regulation in R. rubrum. Studies on the influence of the small metabolite effectors α-ketoglutarate and glutamine are also included together with the effect of divalent cations. In both R. rubrum and Escherichia coli, one of the enzymes participating in GS regulation is the bifunctional enzyme GlnE. GlnE is responsible for both the attachment and the removal of AMP groups from GS, which basically leads to a more inactive or active enzyme respectively. Apart from examining the above functions of GlnE, we have also found a novel third activity of R. rubrum GlnE, an antioxidant function, which is located in the C-terminal domain. We have examined this novel activity of GlnE in great detail, including site specific mutagenesis. We also generated and analyzed ΔglnE mutants in R. rubrum and the results from these studies show that suppressor mutations can occur within glnA, the gene encoding GS. We assume that the function of these suppressor mutations is to lower the specific activity of GS, which otherwise might be too high in a ΔglnE mutant since they lack the ability to adenylylate GS. In other words, it seems that ΔglnE mutants can not be generated without producing suppressor mutations.
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| 2. |
- Ran, Liang, 1974-
(författare)
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Proteomic profiles and gene expressions in the symbiotically competent cyanobacterium Nostoc sp. PCC 73102
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nostoc PCC 73102 is an evolutionary important cyanobacterium with multiple phenotypic traits and symbiotic capacities. Based on two-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry, a proteomic approach was developed in order to obtain protein profiles of this symbiotically competent cyanobacterium during three trophic types/life stages: free-living photo-autotrophic and diazotrophic growth conditions, dark heterotrophic conditions and hormogonium formation. The proteomic data obtained revealed a total of 82 proteins that could be organized into 12 functional categories. The majority of proteins identified were involved in carbon, nitrogen and energy metabolism. The analysis also indicates that a thioredoxin-dependent redox regulation is vital under photo-autotrophic and diazotrophic growth conditions. The differentiation of vegetative filaments into hormogonia led to distinct morphological as well as drastic changes in C and N metabolism, with levels of Rubisco large subunit and glutamine synthetase clearly down-regulated, while glycogen synthesis (C storage) was enhanced, a metabolic specialisation maintained in symbiosis. RT-PCR and quantitative real-time PCR showed that gene transcription related to surface structures and hormogonium function was affected early during the developmental process: genes encoding proteins involved in motility (pil genes) and gas vesicle formation (gvp genes) were up-regulated, but also genes encoding proteins involved in DNA replication (dnaA) and cell division (ftsZ, ftsA and ftn2) and proteolysis (hetR). Dark heterotrophy, mimicking symbiosis, led to a repression of CO2 fixation, while sugar uptake, the glycolysis pathway and N2-fixation were stimulated. In addition, three proteins involved in light adaptation processes were upregulated. Collectively, these data contribute to our understanding and the definition of ‘symbiotic competence’ and suggest that the cyanobacterium behaves like a cyanobiont even before getting into physical contact with host plants if subject to the appropriate signals.
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