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Hauryliuk, Vasili
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Publications (10 of 28) Show all publications
Kaldalu, N., Maiväli, Ü., Hauryliuk, V. & Tenson, T. (2019). Reanalysis of Proteomics Results Fails To Detect MazF-Mediated Stress Proteins [Letter to the editor]. mBio, 10(3), Article ID e00949-19.
Open this publication in new window or tab >>Reanalysis of Proteomics Results Fails To Detect MazF-Mediated Stress Proteins
2019 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 3, article id e00949-19Article in journal, Letter (Refereed) Published
Place, publisher, year, edition, pages
American Society for Microbiology, 2019
Keywords
endonuclease, proteomics, statistics, toxin/antitoxin systems
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-161744 (URN)10.1128/mBio.00949-19 (DOI)000473596500004 ()31186323 (PubMedID)
Available from: 2019-07-25 Created: 2019-07-25 Last updated: 2019-07-25Bibliographically approved
Kasari, V., Margus, T., Atkinson, G. C., Johansson, M. J. O. & Hauryliuk, V. (2019). Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae. Scientific Reports, 9, Article ID 3037.
Open this publication in new window or tab >>Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 3037Article in journal (Refereed) Published
Abstract [en]

In addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5' part of the open reading frames. We observed no E-site codon-or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-157582 (URN)10.1038/s41598-019-39403-y (DOI)000459891700047 ()30816176 (PubMedID)
Available from: 2019-03-29 Created: 2019-03-29 Last updated: 2019-03-29Bibliographically approved
Van Nerom, K., Tamman, H., Takada, H., Hauryliuk, V. & Garcia-Pino, A. (2019). The Rel stringent factor from Thermus thermophilus: crystallization and X-ray analysis. Acta Crystallographica Section F : Structural Biology Communications, 75, 561-569
Open this publication in new window or tab >>The Rel stringent factor from Thermus thermophilus: crystallization and X-ray analysis
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2019 (English)In: Acta Crystallographica Section F : Structural Biology Communications, ISSN 2053-230X, Vol. 75, p. 561-569Article in journal (Refereed) Published
Abstract [en]

The stringent response, controlled by (p)ppGpp, enables bacteria to trigger a strong phenotypic resetting that is crucial to cope with adverse environmental changes and is required for stress survival and virulence. In the bacterial cell, (p)ppGpp levels are regulated by the concerted opposing activities of RSH (RelA/SpoT homologue) enzymes that can transfer a pyrophosphate group of ATP to the 3′ position of GDP (or GTP) or remove the 3′ pyrophosphate moiety from (p)ppGpp. Bifunctional Rel enzymes are notoriously difficult to crystallize owing to poor stability and a propensity for aggregation, usually leading to a loss of biological activity after purification. Here, the production, biochemical analysis and crystallization of the bifunctional catalytic region of the Rel stringent factor from Thermus thermophilus (RelTtNTD) in the resting state and bound to nucleotides are described. RelTt and RelTtNTD are monomers in solution that are stabilized by the binding of Mn2+ and mellitic acid. RelTtNTD crystallizes in space group P4122, with unit-cell parameters a = b = 88.4, c = 182.7 Å, at 4°C and in space group P41212, with unit-cell parameters a = b = 105.7, c = 241.4 Å, at 20°C.

Place, publisher, year, edition, pages
International Union of Crystallography, 2019
Keywords
stringent response, Rel/RelA/SpoT, (p)ppGpp, bacterial alarmone, Thermus thermophilus
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-162870 (URN)10.1107/S2053230X19010628 (DOI)000480337300007 ()31397328 (PubMedID)
Available from: 2019-09-05 Created: 2019-09-05 Last updated: 2019-09-05Bibliographically approved
Murina, V., Kasari, M., Hauryliuk, V. & Atkinson, G. C. (2018). Antibiotic resistance ABCF proteins reset the peptidyl transferase centre of the ribosome to counter translational arrest. Nucleic Acids Research, 46(7), 3753-3763
Open this publication in new window or tab >>Antibiotic resistance ABCF proteins reset the peptidyl transferase centre of the ribosome to counter translational arrest
2018 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 7, p. 3753-3763Article in journal (Refereed) Published
Abstract [en]

Several ATPases in the ATP-binding cassette F (ABCF) family confer resistance to macrolides, lincosamides and streptogramins (MLS) antibiotics. MLS are structurally distinct classes, but inhibit a common target: the peptidyl transferase (PTC) active site of the ribosome. Antibiotic resistance (ARE) ABCFs have recently been shown to operate through direct ribosomal protection, but the mechanistic details of this resistance mechanism are lacking. Using a reconstituted translational system, we dissect the molecular mechanism of Staphylococcus haemolyticus VgaA(LC) and Enterococcus faecalis LsaA on the ribosome. We demonstrate that VgaA(LC) is an NTPase that operates as a molecular machine strictly requiring NTP hydrolysis (not just NTP binding) for antibiotic protection. Moreover, when bound to the ribosome in the NTP-bound form, hydrolytically inactive EQ(2) ABCF ARE mutants inhibit peptidyl transferase activity, suggesting a direct interaction between the ABCF ARE and the PTC. The likely structural candidate responsible for antibiotic displacement by wild type ABCF AREs, and PTC inhibition by the EQ(2) mutant, is the extended inter-ABC domain linker region. Deletion of the linker region renders wild type VgaA(LC) inactive in antibiotic protection and the EQ(2) mutant inactive in PTC inhibition.

Place, publisher, year, edition, pages
Oxford University Press, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-147824 (URN)10.1093/nar/gky050 (DOI)000431137900041 ()29415157 (PubMedID)
Available from: 2018-05-18 Created: 2018-05-18 Last updated: 2018-06-09Bibliographically approved
Brodiazhenko, T., Johansson, M. J. O., Takada, H., Nissan, T., Hauryliuk, V. & Murina, V. (2018). Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems. Frontiers in Microbiology, 9, Article ID 3041.
Open this publication in new window or tab >>Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems
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2018 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 3041Article in journal (Refereed) Published
Abstract [en]

Cell-free translation systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translation system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors – HPF in Bacillus subtilis and Stm1 in Saccharomyces cerevisiae – robustly improve the activities of bacterial and yeast translation systems. Importantly, the elimination of B. subtilis HPF results in a complete loss of 100S ribosomes, which otherwise interfere with disome-based approaches for preparation of stalled ribosomal complexes for cryo-electron microscopy studies.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2018
Keywords
HPF, Stm1, Bacillus subtilis, Saccharomyces cerevisiae, cell-tree translation system
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-155100 (URN)10.3389/fmicb.2018.03041 (DOI)000453653000001 ()
Funder
Swedish Research Council, 2013-4680Swedish Research Council, 2017-04663Ragnar Söderbergs stiftelseMagnus Bergvall Foundation, 2017-02098Åke Wiberg Foundation, M14-0207
Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-01-10Bibliographically approved
Goormaghtigh, F., Fraikin, N., Putrins, M., Hallaert, T., Hauryliuk, V., Garcia-Pino, A., . . . Van Melderen, L. (2018). Reassessing the Role of Type II Toxin-Antitoxin Systems in Formation of Escherichia coli Type II Persister Cells. mBio, 9(3), Article ID e00640-18.
Open this publication in new window or tab >>Reassessing the Role of Type II Toxin-Antitoxin Systems in Formation of Escherichia coli Type II Persister Cells
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2018 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 3, article id e00640-18Article in journal (Refereed) Published
Abstract [en]

Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicillin. Moreover, microfluidic experiments performed with a strain reporting the induction of the yefM-yoeB TA system allowed the observation of a small number of type II persister cells that resume growth after removal of ampicillin. However, we were unable to establish a correlation between high fluorescence and persistence, since the fluorescence of persister cells was comparable to that of the bulk of the population and none of the cells showing high fluorescence were able to resume growth upon removal of the antibiotic. Altogether, these data show that there is no direct link between induction of TA systems and persistence to antibiotics. IMPORTANCE Within a growing bacterial population, a small subpopulation of cells is able to survive antibiotic treatment by entering a transient state of dormancy referred to as persistence. Persistence is thought to be the cause of relapsing bacterial infections and is a major public health concern. Type II toxin-antitoxin systems are small modules composed of a toxic protein and an antitoxin protein counteracting the toxin activity. These systems were thought to be pivotal players in persistence until recent developments in the field. Our results demonstrate that previous influential reports had technical flaws and that there is no direct link between induction of TA systems and persistence to antibiotics.

Place, publisher, year, edition, pages
American Society for Microbiology, 2018
Keywords
RelE, YoeB, ampicillin, single cell
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-155657 (URN)10.1128/mBio.00640-18 (DOI)000454748900022 ()29895634 (PubMedID)
Available from: 2019-01-25 Created: 2019-01-25 Last updated: 2019-01-25Bibliographically approved
Goormaghtigh, F., Fraikin, N., Putrins, M., Hauryliuk, V., Garcia-Pino, A., Udekwu, K., . . . Van Melderen, L. (2018). Reply to Holden and Errington, "Type II Toxin-Antitoxin Systems and Persister Cells" [Letter to the editor]. mBio, 9(5), Article ID e01838-18.
Open this publication in new window or tab >>Reply to Holden and Errington, "Type II Toxin-Antitoxin Systems and Persister Cells"
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2018 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 5, article id e01838-18Article in journal, Letter (Refereed) Published
Place, publisher, year, edition, pages
American Society for Microbiology, 2018
Keywords
E. coli, persisters, toxin-antitoxins
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-153667 (URN)10.1128/mBio.01838-18 (DOI)000449472200034 ()30254127 (PubMedID)
Available from: 2018-11-26 Created: 2018-11-26 Last updated: 2018-11-26Bibliographically approved
Crowe-McAuliffe, C., Graf, M., Huter, P., Takada, H., Abdelshahid, M., Novácek, J., . . . Wilson, D. N. (2018). Structural basis for antibiotic resistance mediated by the Bacillus subtilis ABCF ATPase VmlR. Proceedings of the National Academy of Sciences of the United States of America, 115(36), 8978-8983
Open this publication in new window or tab >>Structural basis for antibiotic resistance mediated by the Bacillus subtilis ABCF ATPase VmlR
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2018 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 36, p. 8978-8983Article in journal (Refereed) Published
Abstract [en]

Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ(2) mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery).

Place, publisher, year, edition, pages
National Academy of Sciences, 2018
Keywords
ABC ATPase, cryo-EM, ribosome, antibiotic resistance, VmlR
National Category
Microbiology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-151767 (URN)10.1073/pnas.1808535115 (DOI)000443555000057 ()30126986 (PubMedID)
Funder
Swedish Research Council, 2013-4680Swedish Research Council, 2015-04746Ragnar Söderbergs stiftelseCarl Tryggers foundation , CTS 34EU, Horizon 2020, 5966
Available from: 2018-09-21 Created: 2018-09-21 Last updated: 2018-09-21Bibliographically approved
Manav, M. C., Beljantseva, J., Bojer, M. S., Tenson, T., Ingmer, H., Hauryliuk, V. & Brodersen, D. E. (2018). Structural basis for (p)ppGpp synthesis by the Staphylococcus aureus small alarmone synthetase RelP. Journal of Biological Chemistry, 293(9), 3254-3264
Open this publication in new window or tab >>Structural basis for (p)ppGpp synthesis by the Staphylococcus aureus small alarmone synthetase RelP
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2018 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 9, p. 3254-3264Article in journal (Refereed) Published
Abstract [en]

The stringent response is a global reprogramming of bacterial physiology that renders cells more tolerant to antibiotics and induces virulence gene expression in pathogens in response to stress. This process is driven by accumulation of the intracellular alarmone guanosine-5'-di(tri)phosphate-3'-diphosphate ((p)ppGpp), which is produced by enzymes of the RelA SpoT homologue (RSH) family. The Gram-positive Firmicute pathogen, Staphylococcus aureus, encodes three RSH enzymes: a multidomain RSH (Rel) that senses amino acid starvation on the ribosome and two small alarmone synthetase (SAS) enzymes, RelQ (SAS1) and RelP (SAS2). In Bacillus subtilis, RelQ (SAS1) was shown to form a tetramer that is activated by pppGpp and inhibited by single-stranded RNA, but the structural and functional regulation of RelP (SAS2) is unexplored. Here, we present crystal structures of S. aureus RelP in two major functional states, pre-catalytic (bound to GTP and the non-hydrolyzable ATP analogue, adenosine 5'-(alpha,beta-methylene) triphosphate (AMP-CPP)), and post-catalytic (bound to pppGpp). We observed that RelP also forms a tetramer, but unlike RelQ (SAS1), it is strongly inhibited by both pppGpp and ppGpp and is insensitive to inhibition by RNA. We also identified putative metal ion-binding sites at the subunit interfaces that were consistent with the observed activation of the enzyme by Zn2+ ions. The structures reported here reveal the details of the catalytic mechanism of SAS enzymes and provide a molecular basis for understanding differential regulation of SAS enzymes in Firmicute bacteria.

Place, publisher, year, edition, pages
The American Society for Biochemistry and Molecular Biology, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-146148 (URN)10.1074/jbc.RA117.001374 (DOI)000426562800019 ()29326162 (PubMedID)
Available from: 2018-05-03 Created: 2018-05-03 Last updated: 2018-06-09Bibliographically approved
Kudrin, P., Dzhygyr, I., Ishiguro, K., Beljantseva, J., Maksimova, E., Oliveira, S. R., . . . Hauryliuk, V. (2018). The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA. Nucleic Acids Research, 46(4), 1973-1983
Open this publication in new window or tab >>The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA
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2018 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 4, p. 1973-1983Article in journal (Refereed) Published
Abstract [en]

During amino acid starvation the Escherichia coli stringent response factor RelA recognizes deacylated tRNA in the ribosomal A-site. This interaction activates RelA-mediated synthesis of alarmone nucleotides pppGpp and ppGpp, collectively referred to as (p)ppGpp. These two alarmones are synthesized by addition of a pyrophosphate moiety to the 3' position of the abundant cellular nucleotide GTP and less abundant nucleotide GDP, respectively. Using untagged native RelA we show that allosteric activation of RelA by pppGpp increases the efficiency of GDP conversion to achieve the maximum rate of (p) ppGpp production. Using a panel of ribosomal RNA mutants, we show that the A-site finger structural element of 23S rRNA helix 38 is crucial for RelA binding to the ribosome and consequent activation, and deletion of the element severely compromises (p) ppGpp accumulation in E. coli upon amino acid starvation. Through binding assays and enzymology, we show that E. coli RelA does not form a stable complex with, and is not activated by, deacylated tRNA off the ribosome. This indicates that in the cell, RelA first binds the empty A-site and then recruits tRNA rather than first binding tRNA and then binding the ribosome.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-145777 (URN)10.1093/nar/gky023 (DOI)000426293300036 ()29390134 (PubMedID)
Available from: 2018-03-22 Created: 2018-03-22 Last updated: 2018-09-26Bibliographically approved
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