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Transcription regulation and growth phase transition in hyperthermoacidophilic archaea
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Organisms from the domain Archaea are ubiquitously represented on our planet and encompass diverse fascinating organisms. The genus Sulfolobus belonging to the phylum Crenarchaeota including hyperthermoacidophilic strains that grow optimally at 65-85°C and pH 2-3. These organisms have been used as model organisms for thermophiles to investigate archaeal DNA replication, transcription, translation, cell cycle, etc.

The focus of this thesis is on the study of archaeal specific transcription factors (TFs) as well as transcriptome changes during growth phase transition of the hyperthermoacidophilic archaeons Sulfolobus acidocaldarius and Sulfolobus solfataricus, respectively, to expand our knowledge on archaeal transcription regulation and growth phase adaptation.

In paper 1, we studied the genome-wide binding sites of BarR, which is a β-alanine responsive Lrp family TF that activates the expression of β-alanine aminotransferase located in a divergent operon in S. acidocaldarius. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) revealed 21 binding regions, including previously characterized barR/Saci_2137 intergenic region. However, only one additional operon containing two glutamine synthase genes (Saci_2320 and Saci_2321) was found to be under activation of BarR. This operon is a common target of LyM and Sa-Lrp, which indicates a regulatory network between different Lrp-like regulators. In paper 2, we showed that a TetR family transcription repressor FadRSa regulates fatty acid metabolism in S. acidocaldarius. FadRSa rests in a gene cluster, Saci_1103-Saci_1126, that mainly contains lipid degradation and fatty acid metabolism genes. ChIP-seq revealed four binding sites within the gene cluster and RNA-seq further confirmed that the entire gene cluster is repressed by FadRSa. FadRSa binds DNA at a 16-base pair motif with dyad symmetry, and binding of medium- to long-chain acyl-CoA molecules resulted in dissociation of FadRSa from the DNA. Although FadRSa is similar to its bacterial counterparts functionally and structurally, fundamentally different ligand binding mode has been observed.

In paper 3, transcriptome data of S. solfataricus at four time points during growth, including early exponential phase, late exponential phase, early stationary phase and late stationary phase, has been studied and revealed a massive change in gene expressions during growth phase transition. 1067 out of a total of 2978 (35.8%) protein coding genes were identified as differentially expressed, which included 456 induced genes most of which were related to transposase, metabolism and stress response, 464 repressed genes most of them involved in translation, basic transcription, DNA replication, amino acids metabolism and defence mechanisms, and 147 genes with fluctuated profile including transporters, oxidation-reduction process related genes and few metabolic genes.

In summary, the studies of two metabolic related TFs in S. acidocaldarius, BarR and FadRSa, shed light on their function and regulatory mechanisms. In addition, the transcriptome data of S. solfataricus not only reveals genome-wide alteration of gene expression during growth phase transition, but also provide a rich source of information for further studies by the archaea research community.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2018. , p. 51
Keywords [en]
Archaea, Sulfolobus, Transcription regulation, Lrp, TetR, BarR, ChIP-seq, RNA-seq, Transcriptome, growth phase transition
National Category
Natural Sciences
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-153538ISBN: 978-91-7797-189-4 (print)ISBN: 978-91-7797-190-0 (electronic)OAI: oai:DiVA.org:su-153538DiVA, id: diva2:1189041
Public defence
2018-04-27, Högbomsalen, Geovetenskapens Hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Available from: 2018-04-04 Created: 2018-03-09 Last updated: 2018-03-22Bibliographically approved
List of papers
1. The genome-scale DNA-binding profile of BarR, a beta-alanine responsive transcription factor in the archaeon Sulfolobus acidocaldarius
Open this publication in new window or tab >>The genome-scale DNA-binding profile of BarR, a beta-alanine responsive transcription factor in the archaeon Sulfolobus acidocaldarius
2016 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 17, article id 569Article in journal (Refereed) Published
Abstract [en]

Background: The Leucine-responsive Regulatory Protein (Lrp) family is a widespread family of regulatory transcription factors in prokaryotes. BarR is an Lrp-like transcription factor in the model archaeon Sulfolobus acidocaldarius that activates the expression of a beta-alanine aminotransferase gene, which is involved in beta-alanine degradation. In contrast to classical Lrp-like transcription factors, BarR is not responsive to any of the alpha-amino acids but interacts specifically with beta-alanine. Besides the juxtaposed beta-alanine aminotransferase gene, other regulatory targets of BarR have not yet been identified although beta-alanine is the precursor of coenzyme A and thus an important central metabolite. The aim of this study is to extend the knowledge of the DNA-binding characteristics of BarR and of its corresponding regulon from a local to a genome-wide perspective. Results: We characterized the genome-wide binding profile of BarR using chromatin immunoprecipation combined with high-throughput sequencing (ChIP-seq). This revealed 21 genomic binding loci. High-enrichment binding regions were validated to interact with purified BarR protein in vitro using electrophoretic mobility shift assays and almost all targets were also shown to harbour a conserved semi-palindromic binding motif. Only a small subset of enriched genomic sites are located in intergenic regions at a relative short distance to a promoter, and qRT-PCR analysis demonstrated that only one additional operon is under activation of BarR, namely the glutamine synthase operon. The latter is also a target of other Lrp-like transcription factors. Detailed inspection of the BarR ChIP-seq profile at the beta-alanine aminotransferase promoter region in combination with binding motif predictions indicate that the operator structure is more complicated than previously anticipated, consisting of multiple (major and auxiliary) operators. Conclusions: BarR has a limited regulon, and includes also glutamine synthase genes besides the previously characterized beta-alanine aminotransferase. Regulation of glutamine synthase is suggestive of a link between beta-alanine and alpha-amino acid metabolism in S. acidocaldarius. Furthermore, this work reveals that the BarR regulon overlaps with that of other Lrp-like regulators.

Keywords
beta-alanine, Sulfolobus, Leucine-responsive regulatory protein, Transcription regulation, ChIP-seq
National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-134437 (URN)10.1186/s12864-016-2890-0 (DOI)000381226100009 ()27502941 (PubMedID)
Available from: 2016-10-11 Created: 2016-10-06 Last updated: 2018-03-09Bibliographically approved
2. A bacterial-like FadR transcription factor regulates fatty acid metabolism in the archaeal model organism Sulfolobus acidocaldarius
Open this publication in new window or tab >>A bacterial-like FadR transcription factor regulates fatty acid metabolism in the archaeal model organism Sulfolobus acidocaldarius
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In accordance with the important biological roles of fatty acids, their metabolism is under complex regulation in bacteria and eukaryotes. In contrast, although certain archaea also appear to metabolize fatty acids, nothing is known about the regulation of the underlying pathways in these organisms. Here we show that the crenarchaeon Sulfolobus acidocaldarius harbors a bacterial-type TetR-family transcriptional regulator FadRSa and that it is involved in regulation of fatty acid metabolism. Functional and structural analyses show that the regulator binds semi-palindromic recognition sites in two distinct operator-dependent binding modes and that binding of fatty acyl-CoA molecules causes dissociation of FadRSa-DNA complexes by inducing conformational changes in the protein. Curiously, despite the similarity in overall structure and mechanisms between FadRSa and bacterial TetR-family FadR regulators, we reveal a fundamentally different acyl-CoA binding mode that suggests convergent evolution. Genome-wide transcriptomic and FadRSa-specific chromatin immunoprecipitation analyses further demonstrate that the transcription factor acts as a local repressor of a gene cluster comprising 23 open reading frames that encode lipases, beta-oxidation enzymes and acetyl-CoA acetyltransferases. We conclude that lipid degradation and fatty acid metabolism in S. acidocaldarius is subject to an acyl-CoA responsive transcriptional repression by a homolog of bacterial TetR-family FadR proteins of which the regulatory mechanism suggests that the regulated gene cluster minimally has a catabolic function.

Keywords
Archaea, crenarchaeon, Sulfolobus acidocaldarius, TetR-family transcriptional regulator, fatty acid metabolism
National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-153317 (URN)
Available from: 2018-03-03 Created: 2018-03-03 Last updated: 2018-03-20Bibliographically approved
3. Genome-wide transcription responses during transition from exponential growth to stationary phase in the Archaeon Sulfolobus solfataricus
Open this publication in new window or tab >>Genome-wide transcription responses during transition from exponential growth to stationary phase in the Archaeon Sulfolobus solfataricus
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Transition from exponential phase to stationary phase has been extensively studied in bacteria, which brings in-depth knowledge about bacterial cell adaptation, stress response, and transcriptional regulation network, such as stationary specific σ factors and (p)ppGpp mediated stringent response, etc. However, little is known for archaeal in this field. To shed light on archaeal phase adaptation and metabolic responses during growth phase transitions, global gene expression during growth of S. solfataricus were studied. Total RNAs from cells that were collected at 4 time points, representing exponential growth, the transition stage, early stationary phase and late stationary phase were extracted and sequenced. RNA-seq analysis identified a total of 1067 differentially expressed genes during phase transition, which included 456 induced genes most of which are related to transposases, stress response, and transcription factors, 464 repressed genes most of them involved in translation, basic transcriptional apparatus, DNA replication, cell division, amino acids metabolism and defense mechanisms, and 147 genes with fluctuated profile including transporters, oxidation-reduction process related genes and few metabolic genes. This study not only provide an overview of gene expression profiles of S. solfataricus during growth phase transition, but also provide a rich repository for further studies by the archaea community.

Keywords
Archaea
National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-153922 (URN)
Available from: 2018-03-08 Created: 2018-03-08 Last updated: 2018-03-20Bibliographically approved

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