| 1. |
- Thorsen, Michael, 1974, et al.
(författare)
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Glutathione serves an extracellular defence function to decrease arsenite accumulation and toxicity in yeast.
- 2012
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Ingår i: Molecular microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 84:6, s. 1177-88
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Tidskriftsartikel (refereegranskat)abstract
- Arsenic is an environmental toxin and a worldwide health hazard. Since this metalloid is ubiquitous in nature, virtually all living organisms require systems for detoxification and tolerance acquisition. Here, we show that during chronic exposure to arsenite [As(III)], Saccharomyces cerevisiae (budding yeast) exports and accumulates the low-molecular-weight thiol molecule glutathione (GSH) outside of cells. Extracellular accumulation of the arsenite triglutathione complex As(GS)₃ was also detected and direct transport assays demonstrate that As(GS)₃ does not readily enter cells. Yeast cells with increased extracellular GSH levels accumulate less arsenic and display improved growth when challenged with As(III). Conversely, cells defective in export and extracellular accumulation of GSH are As(III) sensitive. Taken together, our data are consistent with a novel detoxification mechanism in which GSH is exported to protect yeast cells from arsenite toxicity by preventing its uptake.
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| 2. |
- Furukawa, Kentaro, et al.
(författare)
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Expression of the yeast aquaporin Aqy2 affects cell surface properties under the control of osmoregulatory and morphogenic signalling pathways.
- 2009
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Ingår i: Molecular microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 74:5, s. 1272-86
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins mediate rapid and selective water transport across biological membranes. The yeast Saccharomyces cerevisiae possesses two aquaporins, Aqy1 and Aqy2. Here, we show that Aqy2 is involved in controlling cell surface properties and that its expression is controlled by osmoregulatory and morphogenic signalling pathways. Deletion of AQY2 results in diminished fluffy colony morphology while overexpression of AQY2 causes strong agar invasion and adherence to plastic surfaces. Hyper-osmotic stress inhibits morphological developments including the above characteristics as well as AQY2 expression through the osmoregulatory Hog1 mitogen-activated protein kinase. Moreover, two pathways known to control morphological developments are involved in regulation of AQY2 expression: the protein kinase A pathway derepresses AQY2 expression through the Sfl1 repressor, and the filamentous growth Kss1 mitogen-activated protein kinase pathway represses AQY2 expression in a Kss1 activity-independent manner. The AQY2 expression pattern resembles in many ways that of MUC1/FLO11, which encodes a cell surface glycoprotein required for morphological developments. Our observations suggest a potential link between aquaporins and cell surface properties, and relate to the proposed role of mammalian aquaporins in tumour cell migration and invasion.
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| 3. |
- Mandal, Goutam, et al.
(författare)
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Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase.
- 2012
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Ingår i: Molecular microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 85:6, s. 1204-1218
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Tidskriftsartikel (refereegranskat)abstract
- Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defence against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen-activated protein kinase, LmjMPK2. Leishmania parasites coexpressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr-197 and this phosphorylation requires LmjMPK2 activity. Lys-42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild-type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. Leishmania mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild-type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a mitogen-activated protein kinase.
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| 4. |
- Zhang, Jie, 1981, et al.
(författare)
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The b-subunits of the Snf1 kinase in Saccharomyces cerevisae, Gal83 and Sip2, but not Sip1 are redundant in glucose derepression and regulation of sterol biosynthesis
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 77:2, s. 371-383
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Tidskriftsartikel (refereegranskat)abstract
- The conserved Snf1/AMP-activated protein kinase family is one of the central components in the nutrient sensing and regulation of the carbon metabolism in eukaryotes. It is also involved in several other processes such as stress resistance, invasive growth and ageing. Snf1 kinase is composed of a catalytic β-subunit Snf1, a regulatory γ-subunit Snf4 and one of three possible β-subunits, Sip1, Sip2 or Gal83. We used a systematic approach to study the role of the three β-subunits by analysing all seven possible combinations of β-subunit deletions together with the reference strain. Previous studies showed that the three β-subunits are redundant for growth on alternative carbon sources. Here we report that the mutant strain with only SIP1 expressed (sip2Δ gal83Δ) could utilize acetate, but neither ethanol nor glycerol, as alternative carbon source. We also showed that Gal83 is the most important isoform not only for the growth on non-fermentable carbon sources, but also for regulation of ergosterol biosynthetic genes, under glucose-limited condition. Furthermore, we found that Sip2, but not Sip1, can take over when Gal83 is deleted, but to a lesser extent. However, Sip1 may be sufficient for some other processes such as regulation of the nitrogen metabolism and meiosis. © 2010 Blackwell Publishing Ltd.
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| 5. |
- Derouiche, Abderahmane, 1980, et al.
(författare)
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Bacillus subtilisSalA is a phosphorylation-dependent transcription regulator that represses scoC and activates the production of the exoprotease AprE
- 2015
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 97:6, s. 1195-1208
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Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilisMrp family protein SalA has been shown to indirectly promote the production of the exoprotease AprE by inhibiting the expression of scoC, which codes for a repressor of aprE. The exact mechanism by which SalA influences scoC expression has not been clarified previously. We demonstrate that SalA possesses a DNA-binding domain (residues 1-60), which binds to the promoter region of scoC. The binding of SalA to its target DNA depends on the presence of ATP and is stimulated by phosphorylation of SalA at tyrosine 327. The B.subtilis protein-tyrosine kinase PtkA interacts specifically with the C-terminal domain of SalAin vivo and in vitro and is responsible for activating its DNA binding via phosphorylation of tyrosine 327. In vivo, a mutant mimicking phosphorylation of SalA (SalA Y327E) exhibited a strong repression of scoC and consequently overproduction of AprE. By contrast, the non-phosphorylatable SalA Y327F and the ΔptkA exhibited the opposite effect, stronger expression of scoC and lower production of the exoprotease. Interestingly, both SalA and PtkA contain the same ATP-binding Walker domain and have thus presumably arisen from the common ancestral protein. Their regulatory interplay seems to be conserved in other bacteria.
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| 6. |
- Furukawa, Kentaro, et al.
(författare)
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Synthetic biology: lessons from engineering yeast MAPK signalling pathways.
- 2013
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Ingår i: Molecular microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 88:1, s. 5-19
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Tidskriftsartikel (refereegranskat)abstract
- All living cells respond to external stimuli and execute specific physiological responses through signal transduction pathways. Understanding the mechanisms controlling signalling pathways is important for diagnosing and treating diseases and for reprogramming cells with desired functions. Although many of the signalling components in the budding yeast Saccharomyces cerevisiae have been identified by genetic studies, many features concerning the dynamic control of pathway activity, cross-talk, cell-to-cell variability or robustness against perturbation are still incompletely understood. Comparing the behaviour of engineered and natural signalling pathways offers insight complementary to that achievable with standard genetic and molecular studies. Here, we review studies that aim at a deeper understanding of signalling design principles and generation of novel signalling properties by engineering the yeast mitogen-activated protein kinase (MAPK) pathways. The underlying approaches can be applied to other organisms including mammalian cells and offer opportunities for building synthetic pathways and functionalities useful in medicine and biotechnology.
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| 7. |
- Kobir, A., et al.
(författare)
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Phosphorylation of Bacillus subtilis gene regulator AbrB modulates its DNA-binding properties
- 2014
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 92:5, s. 1129-1141
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Tidskriftsartikel (refereegranskat)abstract
- AbrB is a global gene regulator involved in transition phase phenomena in Bacillus subtilis. It participates in a complex regulatory network governing the expression of stationary-phase functions. AbrB was previously found to be phosphorylated on serine 86 located close to its C-terminal oligomerization domain. Here we report that AbrB can be phosphorylated by three B. subtilis serine/threonine kinases expressed during the transition and stationary phase: PrkC, PrkD and YabT. Our in vitro findings suggest that AbrB phosphorylation impedes its DNA binding and abolishes binding cooperativity. In vivo we established that a phospho-mimetic mutation abrB S86D leads to a significant loss of AbrB control over several key target functions: exoprotease production, competence development and sporulation. A wider transcriptome analysis of abrBS86D and S86A mutant strains revealed deregulation of a large number of target genes. We therefore propose that AbrB phosphorylation serves as an additional input for fine-tuning the activity of this ambiactive gene regulator.
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| 8. |
- Saaki, Terrens N.V., et al.
(författare)
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SepF supports the recruitment of the DNA translocase SftA to the Z-ring
- 2022
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 117:5, s. 1263-1274
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Tidskriftsartikel (refereegranskat)abstract
- In many bacteria, cell division begins before the sister chromosomes are fully segregated. Specific DNA translocases ensure that the chromosome is removed from the closing septum, such as the transmembrane protein FtsK in Escherichia coli. Bacillus subtilis contains two FtsK homologues, SpoIIIE and SftA. SftA is active during vegetative growth whereas SpoIIIE is primarily active during sporulation and pumps the chromosome into the spore compartment. FtsK and SpoIIIE contain several transmembrane helices, however, SftA is assumed to be a cytoplasmic protein. It is unknown how SftA is recruited to the cell division site. Here we show that SftA is a peripheral membrane protein, containing an N-terminal amphipathic helix that reversibly anchors the protein to the cell membrane. Using a yeast two-hybrid screen we found that SftA interacts with the conserved cell division protein SepF. Based on extensive genetic analyses and previous data we propose that the septal localization of SftA depends on either SepF or the cell division protein FtsA. Since SftA seems to interfere with the activity of SepF, and since inactivation of SepF mitigates the sensitivity of a ∆sftA mutant for ciprofloxacin, we speculate that SftA might delay septum synthesis when chromosomal DNA is in the vicinity.
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| 9. |
- Schmitt, Andreas, et al.
(författare)
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PrgB promotes aggregation, biofilm formation, and conjugation through DNA binding and compaction
- 2018
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 109:3, s. 291-305
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Tidskriftsartikel (refereegranskat)abstract
- Gram-positive bacteria deploy type IV secretion systems (T4SSs) to facilitate horizontal gene transfer. The T4SSs of Gram-positive bacteria rely on surface adhesins as opposed to conjugative pili to facilitate mating. Enterococcus faecalis PrgB is a surface adhesin that promotes mating pair formation and robust biofilm development in an extracellular DNA (eDNA) dependent manner. Here, we report the structure of the adhesin domain of PrgB. The adhesin domain binds and compacts DNA in vitro. In vivo PrgB deleted of its adhesin domain does not support cellular aggregation, biofilm development and conjugative DNA transfer. PrgB also binds lipoteichoic acid (LTA), which competes with DNA binding. We propose that PrgB binding and compaction of eDNA facilitates cell aggregation and plays an important role in establishment of early biofilms in mono- or polyspecies settings. Within these biofilms, PrgB mediates formation and stabilization of direct cell-cell contacts through alternative binding of cell-bound LTA, which in turn promotes establishment of productive mating junctions and efficient intra- or inter-species T4SS-mediated gene transfer.
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| 10. |
- Talemi, Soheil Rastgou, et al.
(författare)
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Mathematical modelling of arsenic transport, distribution and detoxification processes in yeast.
- 2014
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 92:6, s. 1343-56
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Tidskriftsartikel (refereegranskat)abstract
- Arsenic has a dual role as causative and curative agent of human disease. Therefore, there is considerable interest in elucidating arsenic toxicity and detoxification mechanisms. By an ensemble modelling approach, we identified a best parsimonious mathematical model which recapitulates and predicts intracellular arsenic dynamics for different conditions and mutants, thereby providing novel insights into arsenic toxicity and detoxification mechanisms in yeast, which could partly be confirmed experimentally by dedicated experiments. Specifically, our analyses suggest that: (i) arsenic is mainly protein-bound during short-term (acute) exposure, whereas glutathione-conjugated arsenic dominates during long-term (chronic) exposure, (ii) arsenic is not stably retained, but can leave the vacuole via an export mechanism, and (iii) Fps1 is controlled by Hog1-dependent and Hog1-independent mechanisms during arsenite stress. Our results challenge glutathione depletion as a key mechanism for arsenic toxicity and instead suggest that (iv) increased glutathione biosynthesis protects the proteome against the damaging effects of arsenic and that (v) widespread protein inactivation contributes to the toxicity of this metalloid. Our work in yeast may prove useful to elucidate similar mechanisms in higher eukaryotes and have implications for the use of arsenic in medical therapy.
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