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  • 1.
    Canals, Rocio
    et al.
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England.
    Larsson, Disa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England.
    Kroger, Carsten
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England; Trinity Coll Dublin, Moyne Inst Prevent Med, Sch Genet & Microbiol, Dept Microbiol, Dublin, Ireland.
    Owen, Sian V.
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England; Harvard Med Sch, Dept Biomed Informat, Boston, MA USA.
    Fong, Wai Yee
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England.
    Lacharme-Lora, Lizeth
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England.
    Zhu, Xiaojun
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England.
    Wenner, Nicolas
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England.
    Carden, Sarah E.
    Stanford Univ, Dept Microbiol & Immunol, Sch Med, Stanford, CA USA.
    Honeycutt, Jared
    Stanford Univ, Dept Microbiol & Immunol, Sch Med, Stanford, CA USA.
    Monack, Denise M.
    Stanford Univ, Dept Microbiol & Immunol, Sch Med, Stanford, CA USA.
    Kingsley, Robert A.
    Quadram Inst Biosci, Norwich Res Pk, Norwich, Norfolk, England.
    Brownridge, Philip
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England.
    Chaudhuri, Roy R.
    Univ Sheffield, Dept Mol Biol & Biotechnol, Sheffield, S Yorkshire, England.
    Rowe, Will P. M.
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England; STFC Daresbury Lab, Dept Comp Sci, Warrington, Cheshire, England.
    Predeus, Alexander V.
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England.
    Hokamp, Karsten
    Trinity Coll Dublin, Smurfit Inst Genet, Sch Genet & Microbiol, Dept Genet, Dublin, Ireland.
    Gordon, Melita A.
    Univ Liverpool, Inst Infect & Global Hlth, Liverpool, Merseyside, England; Univ Malawi, Coll Med, Malawi Liverpool Wellcome Trust Clin Res Programm, Zomba, Malawi.
    Hinton, Jay C. D.
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England.
    Adding function to the genome of African Salmonella Typhimurium ST313 strain D235802019In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 17, no 1, article id e3000059Article in journal (Refereed)
    Abstract [en]

    Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

  • 2.
    Ghosh, Anirban
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Wäneskog, Marcus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elkhalifa, Dina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Larsson, Disa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations2018In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, no 9, article id UNSP e98026Article in journal (Refereed)
    Abstract [en]

    Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing cells is increased only when there are many cells of the same genotype. This may be one of the mechanisms of how CDI systems increase the fitness of their hosts.

  • 3.
    Hammarlöf, Disa L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    EF-Tu and RNase E: Essential and Functionally Connected Proteins2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The rate and accuracy of protein production is the main determinant of bacterial growth. Elongation Factor Tu (EF-Tu) provides the ribosome with aminoacylated tRNAs, and is central for its activity. In Salmonella enterica serovar Typhimurium, EF-Tu is encoded by the genes tufA and tufB. A bacterial cell depending on tufA499-encoded EF-Tu mutant Gln125Arg grows extremely slowly. We found evidence that this is caused by excessive degradation of mRNA, which is suggested to be the result of transcription-translation decoupling because the leading ribosome is ‘starved’ for amino acids and stalls on the nascent mRNA, which is thus exposed to Riboendonuclease RNase E. The slow-growth phenotype can be reversed by mutations in RNase E that reduce the activity of this enzyme.

    We found that the EF-Tu mutant has increased levels of ppGpp during exponential growth in rich medium. ppGpp is usually produced during starvation, and we propose that Salmonella, depending on mutant EF-Tu, incorrectly senses the resulting situation with ribosomes ‘starving’ for amino acids as a real starvation condition. Thus, RelA produces ppGpp which redirects gene expression from synthesis of ribosomes and favours synthesis of building blocks such as amino acids. When ppGpp levels are reduced, either by over-expression of SpoT or by inactivation of relA, growth of the mutant is improved. We suggest this is because the cell stays in a fast-growth mode.

    RNase E mutants with a conditionally lethal temperature-sensitive (ts) phenotype were used to address the long-debated question of the essential role of RNase E. Suppressor mutations of the ts phenotype were selected and identified, both in RNase E as well as in extragenic loci. The internal mutations restore the wild-type RNase E function to various degrees, but no single defect was identified that alone could account for the ts phenotype. In contrast, identifying three different classes of extragenic suppressors lead us to suggest that the essential role of RNaseIE is to degrade mRNA. One possibility to explain the importance of this function is that in the absence of mRNA degradation by RNase E, the ribosomes become trapped on defective mRNAs, with detrimental consequences for continued cell growth.

    List of papers
    1. Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu
    Open this publication in new window or tab >>Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu
    2008 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, no 5, p. 1194-1209Article in journal (Refereed) Published
    Abstract [en]

    Salmonella enterica with mutant EF-Tu (Gln125Arg) has a low level of EF-Tu, a reduced rate of protein synthesis and an extremely slow growth rate. Eighty independent suppressor mutations were selected that restored normal growth. In some cases (n = 7) suppression was due to mutations in tufA but, surprisingly, in most cases (n = 73) to mutations in rne, the gene coding for RNase E. These rne mutations alone had only modest effects on growth rate. Fifty different suppressor mutations were isolated in rne, all located in or close to the N-terminal endonucleolytic half of RNase E. Steady state levels of several mRNAs were lower in the mutant tuf strain but restored to wild-type levels in the tuf-rne double mutant. In contrast, the half-lives of mRNAs were unaffected by the tuf mutation. We propose a model where the tuf mutation causes the ribosome following RNA polymerase to pause, possibly in a codon-specific manner, exposing unshielded nascent message to RNase E cleavage. Normal growth rate can be restored by increasing EF-Tu activity or by reducing RNase E activity. Accordingly, RNase E is suggested to act at two distinct stages in the life of mRNA: early, on the nascent transcript; late, on the complete mRNA.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-107026 (URN)10.1111/j.1365-2958.2008.06472.x (DOI)000261070300012 ()18990188 (PubMedID)
    Available from: 2009-07-15 Created: 2009-07-15 Last updated: 2017-12-13Bibliographically approved
    2. Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
    Open this publication in new window or tab >>Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
    2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 2, p. e90486-Article in journal (Other academic) Published
    Abstract [en]

    Salmonella enterica grows extremely slowly when it depends on tufA499 (encoding the Gln125Arg mutant form of EF-Tu) to drive protein synthesis. We screened a plasmid library for multi-copy suppressors of the slow growth phenotype and identified spoT as a candidate. The spoT gene encodes a dual function enzyme with both ppGpp synthetase and hydrolase activities. When spoT was cloned behind an arabinose-inducible promoter the growth rate of the mutant strain increased in response to arabinose addition. We found that the slow-growing mutant strain had a relatively high basal level of ppGpp during exponential growth in rich medium. Overexpression of spoT significantly reduced this level of ppGpp suggesting that inappropriately high ppGpp levels might cause the slow growth rate associated with tufA499. We tested this hypothesis by inactivating relA (codes for RelA, a ribosome-associated ppGpp synthetase) in the mutant strain. This inactivation decreased the level of ppGpp in the mutant strain and increased its growth rate. Based on these data we propose that ribosomes depending on tufA499 for their supply of ternary complex (EF-Tu•GTP•aa-tRNA) experience amino acid starvation and that RelA on these starving ribosomes produces an excess of the alarmone ppGpp. This results in a suboptimal partitioning of transcription activity between genes important for fast growth in rich medium and genes important for growth in a poor medium. Accordingly, mutant bacteria growing in a rich medium act physiologically as though they were growing in a nutrient-poor environment. We propose that this generates a vicious circle and contributes to the extreme slow-growth phenotype associated with mutant EF-Tu. Reducing the level of ppGpp increases the growth rate of the mutant because it breaks this circle and reduces the wasteful misdirection of resources in the cell.

    Keywords
    tufA; ppGpp; RelA; Salmonella enterica; growth regulation
    National Category
    Microbiology
    Research subject
    Microbiology; Molecular Cellbiology
    Identifiers
    urn:nbn:se:uu:diva-159663 (URN)10.1371/journal.pone.0090486 (DOI)000332396200210 ()
    Note

    Jessica M. Bergman and Disa L. Hammarlöf contributed equally to this work.

    Available from: 2011-10-06 Created: 2011-10-05 Last updated: 2017-12-08Bibliographically approved
    3. Temperature-sensitive mutants of RNase E in Salmonella enterica
    Open this publication in new window or tab >>Temperature-sensitive mutants of RNase E in Salmonella enterica
    2011 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, no 23, p. 6639-6650Article in journal (Refereed) Published
    Abstract [en]

    RNase E has an important role in mRNA turnover and stable RNA processing although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (ts) for growth. Four different ts mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys; Ile207→Ser; Ile207→Asn; Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature-sensitivity. The complete set of RNase E mutations (53 primary mutations including the ts mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E using the available 3-D structures. Most single mutations were predicted to destabilize the structure while second-site mutations that reversed the ts phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (ts, and ts plus second-site mutation) were tested for their effects on the degradation, accumulation and processing of mRNA, rRNA and tRNA. The greatest defect was observed on rne mRNA autoregulation and this correlated with ability to rescue the tufA499-associated slow growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.

    National Category
    Natural Sciences Microbiology
    Identifiers
    urn:nbn:se:uu:diva-159661 (URN)10.1128/JB.05868-11 (DOI)000296795600023 ()21949072 (PubMedID)
    Available from: 2011-10-05 Created: 2011-10-05 Last updated: 2017-12-08Bibliographically approved
    4. Extragenic suppressors of RNase E ts mutants
    Open this publication in new window or tab >>Extragenic suppressors of RNase E ts mutants
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    RNase E is an essential endoribonuclease and plays a central role in regulating mRNA levels and stable RNA activity in the bacterial cell. Previous studies of RNA half-life and processing in strains carrying rne mutations have shown that it is the catalytic half of RNase E that is essential for bacterial growth, but have not identified a specific reason for this essentiality. In this study we have used two ts mutations in the catalytic region of RNase E (rne-6 and rne-9) from Salmonella as tools to select and screen for extragenic suppressors of the temperature-sensitive phenotype. We reasoned that identifying extragenic suppressors might give information on the essential function of RNase E. 15 independent extragenic suppressors were isolated and mapped to three different loci on the Salmonella chromosome: rpsA (encoding ribosomal protein S1); vacB (encoding RNase R); and within and neighbouring the ORFs STM1551/1550, putatively encoding a toxin-antitoxin system similar to RelBE from E. coli. Each suppressor mutation could cross-suppress the ts phenotypes of rne-6 and rne-9 and each suppressor mutation alone was viable in a wild-type background. We discuss a model where at the non-permissive temperature an excess of mRNA (including defective species) may trap ribosomes non-productively, reducing the rate of protein synthesis and growth. Accordingly the rpsA mutation may suppress the ts phenotype by reducing the rate of translation initiation, and by default increasing the probability that residual RNase E activity turns over mRNA. The vacB mutations may expand the substrate range of RNase R allowing it to more efficiently substitute for poorly active RNase E in degrading mRNA. Finally, the mutations in the STM1551 region may increase the amount of RelE-like toxin and thereby increase the rate of mRNA turnover. This model makes predictions which can be experimentally tested.

    Keywords
    rpsA; vacB; RNase R; RelBE; RNase E; mRNA turnover
    National Category
    Microbiology
    Research subject
    Microbiology; Molecular Cellbiology
    Identifiers
    urn:nbn:se:uu:diva-159662 (URN)
    Available from: 2011-10-06 Created: 2011-10-05 Last updated: 2011-11-10
  • 4.
    Hammarlöf, Disa L
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu2008In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, no 5, p. 1194-1209Article in journal (Refereed)
    Abstract [en]

    Salmonella enterica with mutant EF-Tu (Gln125Arg) has a low level of EF-Tu, a reduced rate of protein synthesis and an extremely slow growth rate. Eighty independent suppressor mutations were selected that restored normal growth. In some cases (n = 7) suppression was due to mutations in tufA but, surprisingly, in most cases (n = 73) to mutations in rne, the gene coding for RNase E. These rne mutations alone had only modest effects on growth rate. Fifty different suppressor mutations were isolated in rne, all located in or close to the N-terminal endonucleolytic half of RNase E. Steady state levels of several mRNAs were lower in the mutant tuf strain but restored to wild-type levels in the tuf-rne double mutant. In contrast, the half-lives of mRNAs were unaffected by the tuf mutation. We propose a model where the tuf mutation causes the ribosome following RNA polymerase to pause, possibly in a codon-specific manner, exposing unshielded nascent message to RNase E cleavage. Normal growth rate can be restored by increasing EF-Tu activity or by reducing RNase E activity. Accordingly, RNase E is suggested to act at two distinct stages in the life of mRNA: early, on the nascent transcript; late, on the complete mRNA.

  • 5.
    Hammarlöf, Disa L.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Univ Liverpool, Inst Integrat Biol, Liverpool L69 7ZB, Merseyside, England.
    Kroger, Carsten
    Univ Liverpool, Inst Integrat Biol, Liverpool L69 7ZB, Merseyside, England.;Trinity Coll Dublin, Sch Genet & Microbiol, Moyne Inst Prevent Med, Dept Microbiol, Dublin 2, Ireland..
    Owen, Sian V.
    Univ Liverpool, Inst Integrat Biol, Liverpool L69 7ZB, Merseyside, England..
    Canals, Rocio
    Univ Liverpool, Inst Integrat Biol, Liverpool L69 7ZB, Merseyside, England..
    Lacharme-Lora, Lizeth
    Univ Liverpool, Inst Integrat Biol, Liverpool L69 7ZB, Merseyside, England..
    Wenner, Nicolas
    Univ Liverpool, Inst Integrat Biol, Liverpool L69 7ZB, Merseyside, England..
    Schager, Anna E.
    Univ Birmingham, Inst Microbiol & Infect, Birmingham B15 2TT, W Midlands, England..
    Wells, Timothy J.
    Univ Birmingham, Inst Microbiol & Infect, Birmingham B15 2TT, W Midlands, England..
    Henderson, Ian R.
    Univ Birmingham, Inst Microbiol & Infect, Birmingham B15 2TT, W Midlands, England..
    Wigley, Paul
    Univ Liverpool, Inst Infect & Global Hlth, Liverpool L69 7ZB, Merseyside, England..
    Hokamp, Karsten
    Univ Dublin, Trinity Coll Dublin, Sch Genet & Microbiol, Dept Genet,Smurfit Inst Genet, Dublin 2, Ireland..
    Feasey, Nicholas A.
    Univ Liverpool Liverpool Sch Trop Med, Dept Clin Sci, Liverpool L3 5QA, Merseyside, England.;Univ Malawi, Coll Med, Malawi Liverpool Wellcome Trust Clin Res Programm, Blantyre 3, Malawi..
    Gordon, Melita A.
    Univ Liverpool, Inst Infect & Global Hlth, Liverpool L69 7ZB, Merseyside, England.;Univ Malawi, Coll Med, Malawi Liverpool Wellcome Trust Clin Res Programm, Blantyre 3, Malawi..
    Hinton, Jay C. D.
    Univ Liverpool, Inst Integrat Biol, Liverpool L69 7ZB, Merseyside, England..
    Role of a single noncoding nucleotide in the evolution of an epidemic African clade of Salmonella2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 11, p. E2614-E2623Article in journal (Refereed)
    Abstract [en]

    Salmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of Salmonella genomes has revealed that ST313 is closely related to the ST19 group of S. Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only similar to 1,000 SNPs. We hypothesized that the phenotypic differences that distinguish African Salmonella from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis of Salmonella. We identified a SNP in the promoter of the pgtE gene that caused high expression of the PgtE virulence factor in African S. Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by African S. Typhimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome.

  • 6.
    Hammarlöf, Disa L.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Liljas, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Temperature-sensitive mutants of RNase E in Salmonella enterica2011In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, no 23, p. 6639-6650Article in journal (Refereed)
    Abstract [en]

    RNase E has an important role in mRNA turnover and stable RNA processing although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (ts) for growth. Four different ts mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys; Ile207→Ser; Ile207→Asn; Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature-sensitivity. The complete set of RNase E mutations (53 primary mutations including the ts mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E using the available 3-D structures. Most single mutations were predicted to destabilize the structure while second-site mutations that reversed the ts phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (ts, and ts plus second-site mutation) were tested for their effects on the degradation, accumulation and processing of mRNA, rRNA and tRNA. The greatest defect was observed on rne mRNA autoregulation and this correlated with ability to rescue the tufA499-associated slow growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.

  • 7.
    Hässler, Signe
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Ramsey, Chris
    Karlsson, Mikael C.I.
    Larsson, Disa
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Rozell, Björn
    Backheden, Magnus
    Peltonen, Leena
    Kämpe, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Winqvist, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Aire deficient mice develop hematopoetic irregularities and marginal zone B cell lymphoma2006In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 108, no 6, p. 1941-1948Article in journal (Refereed)
    Abstract [en]

    Autoimmune polyendocrine syndrome type I (APS I) is an inherited recessive disorder with a progressive immunological destruction of many tissues including the adrenal cortex, the parathyroid glands, and the gonads. APS I is caused by mutations in the AIRE gene (autoimmune regulator), expressed in cells of the thymus and spleen, suggesting a role in central and peripheral tolerance. Aire(-/-) mice replicate the autoimmune features of APS I patients with the presence of multiple autoantibodies and lymphocytic infiltrates in various tissues, but young mice appear clinically healthy. We here report the investigation of 15- to 24-month-old Aire(-/-) mice. We did not observe any endocrinological abnormalities, nor did sera from these mice recognize known APS I autoantigens. Interestingly, however, there was a high frequency of marginal zone B-cell lymphoma in Aire(-/-) mice and liver infiltrates of B cells, suggesting chronic antigen exposure and exaggerated activation. Furthermore, increased numbers of monocytes in blood were identified as well as augmented numbers of metallophilic macrophages in the spleen. We propose that Aire, in addition to its function in the thymus, also has a peripheral regulatory role by controlling the development of antigen-presenting cells (APCs) and marginal zone B-cell activation.

  • 8.
    Larsson Hammarlöf, Disa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bergman, Jessica M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Garmendia, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Turnover of mRNAs is one of the essential functions of RNase E2015In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 98, no 1, p. 34-45Article in journal (Refereed)
    Abstract [en]

    RNase E is an essential bacterial endoribonuclease with a central role in processing tRNAs and rRNA, and turning over mRNAs. Previous studies in strains carrying mutations in the rne structural gene have shown that tRNA processing is likely to be an essential function of RNase E but have not determined whether mRNA turnover is also an essential function. To address this we selected extragenic suppressors of temperature-sensitive mutations in rne that cause a large increase in mRNA half-life at the non-permissive temperature. Fifteen suppressors were mapped to three different loci: relBE (toxin-antitoxin system); vacB (RNase R); and rpsA (ribosomal protein S1). Each suppressor class has the potential to interact with mRNA and each restores wild-type levels of mRNA turnover but does not reverse the minor defects in tRNA and rRNA processing. RelE toxin is especially interesting because its only known activity is to cleave mRNAs in the ribosomal A-site. The relBE suppressor mutations increase transcription of relE, and controlled overexpression of RelE alone was sufficient to suppress the rne ts phenotype. Suppression increased turnover of some major mRNAs (tufA, ompA) but not all mRNAs. We propose that turnover of some mRNAs is one of the essential functions of RNase E.

  • 9.
    Owen, Sian V.
    et al.
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England..
    Wenner, Nicolas
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England..
    Canals, Rocio
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England..
    Makumi, Angela
    Katholieke Univ Leuven, Fac Biosci Engn, Food Microbiol Lab, Dept Microbial & Mol Syst, Leuven, Belgium..
    Hammarlöf, Disa L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gordon, Melita A.
    Univ Liverpool, Inst Infect & Global Hlth, Liverpool, Merseyside, England.;Malawi Liverpool Wellcome Trust Clin Res Programm, Blantyre, Malawi..
    Aertsen, Abram
    Katholieke Univ Leuven, Fac Biosci Engn, Food Microbiol Lab, Dept Microbial & Mol Syst, Leuven, Belgium..
    Feasey, Nicholas A.
    Univ Liverpool Liverpool Sch Trop Med, Liverpool, Merseyside, England..
    Hinton, Jay C. D.
    Univ Liverpool, Inst Integrat Biol, Liverpool, Merseyside, England..
    Characterization of the Prophage Repertoire of African Salmonella Typhimurium ST313 Reveals High Levels of Spontaneous Induction of Novel Phage BTP12017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 235Article in journal (Refereed)
    Abstract [en]

    In the past 30 years, Salmonella bloodstream infections have become a significant health problem in sub-Saharan Africa and are responsible for the deaths of an estimated 390,000 people each year. The disease is predominantly caused by a recently described sequence type of Salmonella Typhimurium: ST313, which has a distinctive set of prophage sequences. We have thoroughly characterized the ST313-associated prophages both genetically and experimentally. ST313 representative strain D23580 contains five full-length prophages: BTP1, Gifsy-2D23580, ST64BD23580, Gifsy-1D23580, and BTP5. We show that common S. Typhimurium prophages Gifsy-2, Gifsy-1, and ST64B are inactivated in ST313 by mutations. Prophage BTP1 was found to be a functional novel phage, and the first isolate of the proposed new species "Salmonella virus BTP1", belonging to the P22virus genus. Surprisingly, similar to 10(9) BTP1 virus particles per ml were detected in the supernatant of non-induced, stationary-phase cultures of strain D23580, representing the highest spontaneously induced phage titer so far reported for a bacterial prophage. High spontaneous induction is shown to be an intrinsic property of prophage BTP1, and indicates the phage-mediated lysis of around 0.2% of the lysogenic population. The fact that BTP1 is highly conserved in ST313 poses interesting questions about the potential fitness costs and benefits of novel prophages in epidemic S. Typhimurium ST313.

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