| 1. |
- Bergman, Jessica, et al.
(författare)
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Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:2, s. e90486-
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 2. |
- Hammarlöf, Disa L., 1980-
(författare)
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EF-Tu and RNase E : Essential and Functionally Connected Proteins
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
- Hammarlöf, Disa L, 1980-, et al.
(författare)
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Extragenic suppressors of RNase E ts mutants
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 4. |
- Hammarlöf, Disa L, et al.
(författare)
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Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu
- 2008
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 70:5, s. 1194-1209
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Hammarlöf, Disa L., et al.
(författare)
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Role of a single noncoding nucleotide in the evolution of an epidemic African clade of Salmonella
- 2018
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:11, s. E2614-E2623
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Hammarlöf, Disa L., et al.
(författare)
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Temperature-sensitive mutants of RNase E in Salmonella enterica
- 2011
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Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 193:23, s. 6639-6650
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
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| 8. |
- Iovino, Federico, et al.
(författare)
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Pneumococcal meningitis is promoted by single cocci expressing pilus adhesin RrgA
- 2016
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Ingår i: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 126:8, s. 2821-2826
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pneumoniae (pneumococcus) is the primary cause of bacterial meningitis. Pneumococcal bacteria penetrates the blood-brain barrier (BBB), but the bacterial factors that enable this process are not known. Here, we determined that expression of pneumococcal pilus-1, which includes the pilus adhesin RrgA, promotes bacterial penetration through the BBB in a mouse model. S. pneumoniae that colonized the respiratory epithelium and grew in the bloodstream were chains of variable lengths; however, the pneumococci that entered the brain were division-competent, spherical, single cocci that expressed adhesive RrgA–containing pili. The cell division protein DivIVA, which is required for an ovoid shape, was localized at the poles and septum of pneumococcal chains of ovoid, nonseparated bacteria, but was absent in spherical, single cocci. In the bloodstream, a small percentage of pneumococci appeared as piliated, RrgA-expressing, DivIVA-negative single cocci, suggesting that only a minority of S. pneumoniae are poised to cross the BBB. Together, our data indicate that small bacterial cell size, which is signified by the absence of DivIVA, and the presence of an adhesive RrgA-containing pilus-1 mediate pneumococcal passage from the bloodstream through the BBB into the brain to cause lethal meningitis.
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| 9. |
- Jones, Allison M., et al.
(författare)
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Genetic Evidence for SecY Translocon-Mediated Import of Two Contact-Dependent Growth Inhibition (CDI) Toxins
- 2021
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Ingår i: mBio. - : American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- The C-terminal (CT) toxin domains of contact-dependent growth inhibition (CDI) CdiA proteins target Gram-negative bacteria and must breach both the outer and inner membranes of target cells to exert growth inhibitory activity. Here, we examine two CdiA-CT toxins that exploit the bacterial general protein secretion machinery after delivery into the periplasm. A Ser281Phe amino acid substitution in transmembrane segment 7 of SecY, the universally conserved channel-forming subunit of the Sec translocon, decreases the cytotoxicity of the membrane depolarizing orphan10 toxin from enterohemorrhagic Escherichia coli EC869. Target cells expressing secY(S281F) and lacking either PpiD or YfgM, two SecY auxiliary factors, are fully protected from CDI-mediated inhibition either by CdiA-CTo10EC869 or by CdiA-CTGN05224, the latter being an EndoU RNase CdiA toxin from Klebsiella aerogenes GN05224 that has a related cytoplasm entry domain. RNase activity of CdiA-CTGN05224 was reduced in secY(S281F) target cells and absent in secY(S281F) Delta ppiD or secY(S281F) Delta yfgM target cells during competition co-cultures. Importantly, an allele-specific mutation in secY (secY(G313W)) renders DppiD or Delta yfgM target cells specifically resistant to CdiA-CTGN05224 but not to CdiA-CTo10EC869, further suggesting a direct interaction between SecY and the CDI toxins. Our results provide genetic evidence of a unique confluence between the primary cellular export route for unfolded polypeptides and the import pathways of two CDI toxins. IMPORTANCE Many bacterial species interact via direct cell-to-cell contact using CDI systems, which provide a mechanism to inject toxins that inhibit bacterial growth into one another. Here, we find that two CDI toxins, one that depolarizes membranes and another that degrades RNA, exploit the universally conserved SecY translocon machinery used to export proteins for target cell entry. Mutations in genes coding for members of the Sec translocon render cells resistant to these CDI toxins by blocking their movement into and through target cell membranes. This work lays the foundation for understanding how CDI toxins interact with the protein export machinery and has direct relevance to development of new antibiotics that can penetrate bacterial cell envelopes.
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| 10. |
- Owen, Sian V., et al.
(författare)
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Characterization of the Prophage Repertoire of African Salmonella Typhimurium ST313 Reveals High Levels of Spontaneous Induction of Novel Phage BTP1
- 2017
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- 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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