Genexpressions- und Protein-DNA-Interaktions-Studien des Aminosäure-Metabolismus und metallabhängiger Prozesse in Halobacterium salinarum R1

Archaea combine bacterial with eukaryotic features to regulate cellular processes. While initiation of transcription resembles the eukaryotic RNA-Polymerase II apparatus, transcriptional regulation is predominantly bacteria-like. In this work the gene expression profile of amino acid metabolism and metal dependent processes in Halobacterium salinarum R1 was elucidated. To gain insights into transcriptional regulatory processes, microarray technology was used as a global approach. Genes encoding certain DNA binding proteins were deleted and/or overexpressed, to compare the expression pattern of the deletion- and overexpression strains, respectively, with the parental strain R1. For a better understanding of metal dependent processes H. salinarum was grown under iron starvation and compared to cells grown under normal conditions. For the investigation of metal dependent processes the DNA binding proteins SirR and TroR were chosen. To determine a possible function of the regulator protein, conclusions were drawn from a comparison of the deletion mutants ∆sirR and ∆troR, respectively, with the parental strain. SirR (staphylococcal iron regulator repressor) was shown to repress the expression of a Fe(II)/Mn(II) dependent ABC-transporter in the presence of iron. In accordance with this data the same transport operon was shown to be induced under iron starvation. Furthermore, TroR (transport related operon) was shown to repress the expression of a Mn(II)-dependent ABC-transporter. In addition, TroR induces the gene expression of the metal dependent regulator gene idr2, which represses together with iron siderophor synthesis genes. To study the amino acid metabolism in H. salinarum Lrp (leucine-responsive regulatory protein) proteins were chosen, because in both archaea and bacteria Lrp is connected to the coordination of amino acid metabolism. To take a closer look on Lrp-homologs further investigation were performed with lrp and lrpA1. Both genes are located next to genes, encoding proteins involved in amino acid metabolism. Possible Lrp target genes were identified by either constructing lrp and lrpA1 deletion mutants or overexpressing the two genes. Microarray analysis revealed that Lrp functions as a global regulator of transcription. Lrp activates the gene expression of the glutamine synthetase gene glnA, regulates the peptide- and phosphate transport, as well as the central intermediary metabolism, and activates the expression of the transcriptional regulator sirR. By the control of sirR gene expression through Lrp correlation between amino acid metabolism and metal dependent processes could be demonstrated. In contrast to Lrp, LrpA1 regulates gene expression of less genes, amongst them the aspartate transaminase gene aspB3, so that further studies were focussed on the gene regulation of aspB3. The second part of this work examines with specific protein-DNA interactions. Prior to interaction studies, RACE-analysis was used to determine 5´UTR and 3´UTR of certain transcripts. To perform protein-DNA binding studies LrpA1 and TroR were recombinantly expressed in Escherichia coli. A DNA-binding assay adapted to halophilic conditions revealed manganese dependent binding of TroR to its own promoter region. LrpA1 was also shown to bind to the lrpA1 promoter region, as well as an aspartate dependent binding to the aspB3 promoter region. CD-spectroscopy experiments could prove that the interaction between L-aspartate and LrpA1 stabilizes the secondary structure of the protein. To gain more insights into the LrpA1 and L-aspartate dependent aspB3 gene expression, northern blot analysis were performed, that showed an induction of the aspB3 transcription in the absence of L- aspartate. This occurs either in a medium lacking aspartate or after aspartate is metabolized in the stationary phase. At the same time, an induction of the lrpA1 gene expression was observed. This can be illustrated in a model that postulates a reciprocal regulation of the lrpA1 and aspB3 gene expression

A small basic protein from the brz-brb operon is involved in regulation of bop transcription in Halobacterium salinarum

<p>Abstract</p> <p>Background</p> <p>The halophilic archaeon <it>Halobacterium salinarum </it>expresses bacteriorhodopsin, a retinal-protein that allows photosynthetic growth. Transcription of the <it>bop </it>(<it>b</it>acterio<it>op</it>sin) gene is controlled by two transcription factors, Bat and Brz that induce <it>bop </it>when cells are grown anaerobically and under light.</p> <p>Results</p> <p>A new gene was identified that is transcribed together with the <it>brz </it>gene that encodes a small basic protein designated as Brb (bacteriorhodopsin-regulating basic protein). The translation activity of the start codon of the <it>brb </it>gene was confirmed by BgaH reporter assays. <it>In vivo </it>site-directed mutagenesis of the <it>brb </it>gene showed that the Brb protein cooperates with Brz in the regulation of <it>bop </it>expression. Using a GFP reporter assay, it was demonstrated that Brb cooperates with both Brz and Bat proteins to activate <it>bop </it>transcription under phototrophic growth conditions.</p> <p>Conclusions</p> <p>The activation of the <it>bop </it>promoter was shown to be dependent not only on two major factors, Bat and Brz, but is also tuned by the small basic protein, Brb.</p

Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1

<p>Abstract</p> <p>Background</p> <p>Archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. <it>Halobacterium salinarum </it>R1 encodes eight leucine-responsive regulatory protein (Lrp)-homologues. The function of two of them, <it>Irp </it>(OE3923F) and <it>lrpA1 </it>(OE2621R), were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays.</p> <p>Results</p> <p>It was shown that Lrp affects the transcription of multiple target genes, including those encoding enzymes involved in amino acid synthesis, central metabolism, transport processes and other regulators of transcription. In contrast, LrpA1 regulates transcription in a more specific manner. The <it>aspB3 </it>gene, coding for an aspartate transaminase, was repressed by LrpA1 in the presence of L-aspartate. Analytical DNA-affinity chromatography was adapted to high salt, and demonstrated binding of LrpA1 to its own promoter, as well as L-aspartate dependent binding to the <it>aspB3 </it>promoter.</p> <p>Conclusion</p> <p>The gene expression profiles of two archaeal Lrp-homologues report in detail their role in <it>H. salinarum </it>R1. LrpA1 and Lrp show similar functions to those already described in bacteria, but in addition they play a key role in regulatory networks, such as controlling the transcription of other regulators. In a more detailed analysis ligand dependent binding of LrpA1 was demonstrated to its target gene <it>aspB3</it>.</p

pH-Induced Modulation of Vibrio fischeri Population Life Cycle

Commonly used as biological chemosensors in toxicity assays, Vibrio fischeri bacteria were systematically characterized using complementary physicochemical and biological techniques to elucidate the evolution of their properties under varying environmental conditions. Changing the pH above or below the optimal pH 7 was used to model the long-term stress that would be experienced by V. fischeri in environmental toxicology assays. The spectral shape of bioluminescence and cell-surface charge during the exponential growth phase were largely unaffected by pH changes. The pH-induced modulation of V. fischeri growth, monitored via the optical density (OD), was moderate. In contrast, the concomitant changes in the time-profiles of their bioluminescence, which is used as the readout in assays, were more significant. Imaging at discrete timepoints by scanning electron microscopy (SEM) and helium-ion microscopy (HIM) revealed that mature V. fischeri cells maintained a rod-shaped morphology with the average length of 2.2 ± 1 µm and diameter of 0.6 ± 0.1 µm. Detailed morphological analysis revealed subpopulations of rods having aspect ratios significantly larger than those of average individuals, suggesting the use of such elongated rods as an indicator of the multigenerational environmental stress. The observed modulation of bioluminescence and morphology supports the suitability of V. fischeri as biological chemosensors for both rapid and long-term assays, including under environmental conditions that can modify the physicochemical properties of novel anthropogenic pollutants, such as nanomaterials and especially stimulus-responsive nanomaterial

pH-induced modulation of Vibrio fischeri population life cycle

Commonly used as biological chemosensors in toxicity assays, Vibrio fischeri bacteria were systematically characterized using complementary physicochemical and biological techniques to elucidate the evolution of their properties under varying environmental conditions. Changing the pH above or below the optimal pH 7 was used to model the long-term stress that would be experienced by V. fischeri in environmental toxicology assays. The spectral shape of bioluminescence and cell-surface charge during the exponential growth phase were largely unaffected by pH changes. The pH-induced modulation of V. fischeri growth, monitored via the optical density (OD), was moderate. In contrast, the concomitant changes in the time-profiles of their bioluminescence, which is used as the readout in assays, were more significant. Imaging at discrete timepoints by scanning electron microscopy (SEM) and helium-ion microscopy (HIM) revealed that mature V. fischeri cells maintained a rod-shaped morphology with the average length of 2.2 ± 1 µm and diameter of 0.6 ± 0.1 µm. Detailed morphological analysis revealed subpopulations of rods having aspect ratios significantly larger than those of average individuals, suggesting the use of such elongated rods as an indicator of the multigenerational environmental stress. The observed modulation of bioluminescence and morphology supports the suitability of V. fischeri as biological chemosensors for both rapid and long-term assays, including under environmental conditions that can modify the physicochemical properties of novel anthropogenic pollutants, such as nanomaterials and especially stimulus-responsive nanomaterials.Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and COMPETE 2020 (POCI‐ 01‐0145‐FEDER‐006684) and BioTecNorte operation (NORTE‐01‐0145‐FEDER‐000004) funded by European Regional Development Fund under the scope of Norte2020 Programa Operacional Re‐ gional do Norte. A.R.S. holds an FCT fellowship SFRH/BD/131905/2017. Measurements using the helium‐ion microscope were supported by the Karlsruhe Nano Micro Facility (KNMF) of the Karls‐ ruhe Institute of Technology (KIT) under projects 2016‐015‐010689 and 2018‐019‐021475info:eu-repo/semantics/publishedVersio

A selection of proteins containing a CPxCG-related zinc finger motif and definition of patterns and motifs to detect them

<p><b>Copyright information:</b></p><p>Taken from "A small protein from the intergenic region of contains a zinc finger motif and regulates and transcription"</p><p></p><p>Molecular Microbiology 2008;67(4):772-780.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253796.</p><p>© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd</p> A large set of proteins shorter than 100 amino acids contain a CPxCG-related zinc finger motif. Several examples from (HS), (AF), (PA) and (TP) are listed. The upper part shows hypothetical and conserved hypothetical proteins which are unrelated to each of them outside the zinc finger patterns. The proteins in the lower part are functionally characterized. The zinc finger patterns are indicated by bold underlining. A definition of patterns and motifs is provided below the sequences. A CPxCG-related zinc finger motif consists of a pair of patterns, separated by 7–40 amino acids. One of the patterns must be a CPxCG-like pattern (grey), of which three forms exist. The other can be a more general Cys/His pattern containing two Cys or His residues separated by two or three intermediate amino acids (white). All eight types of the general Cys/His pattern are shown, the CPxCG-like pattern being a specification of one of them (CxxCx)

A small protein from the intergenic region of contains a zinc finger motif and regulates and transcription-0

<p><b>Copyright information:</b></p><p>Taken from "A small protein from the intergenic region of contains a zinc finger motif and regulates and transcription"</p><p></p><p>Molecular Microbiology 2008;67(4):772-780.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253796.</p><p>© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd</p

A small protein from the intergenic region of contains a zinc finger motif and regulates and transcription-3

<p><b>Copyright information:</b></p><p>Taken from "A small protein from the intergenic region of contains a zinc finger motif and regulates and transcription"</p><p></p><p>Molecular Microbiology 2008;67(4):772-780.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253796.</p><p>© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd</p