| 1. |
- Macvanin, Mirjana, et al.
(författare)
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Fusidic acid-resistant EF-G perturbs the accumulation of ppGpp
- 2000
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 37:1, s. 98-107
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Tidskriftsartikel (refereegranskat)abstract
- Reductions in growth rate caused by fusidic acid-resistant EF-G mutants in Salmonella typhimurium correlate strongly with increased mean cell size. This is unusual because growth rate and cell size normally correlate positively. The global transcription regulator molecule ppGpp has a role in co-ordinating growth rate and division, and its basal level normally correlates inversely with cell size at division. We show that fusidic acid-resistant EF-G mutants have perturbed ppGpp basal levels during steady-state growth and perturbed induced levels during starvation. One mutation, fusA1, associated with the slowest growth rate and largest cell size, causes a reduction in the basal level of ppGpp to one-third of that found in the wild-type strain. Other fusA mutants with intermediate or wild-type growth rates and cell sizes have either normal or increased basal levels of ppGpp. There is an inverse relationship between the basal level of ppGpp in vivo and the degree to which translation dependent on mutant EF-G is inhibited by ppGpp in vitro. This enhanced interaction between mutant EF-G and ppGpp correlates with an increased KM for GTP. Our results suggest that mutant EF-G modulates the production of ppGpp by the RelA (PSI) pathway. In conclusion, fusidic acid-resistant EF-G mutations alter the level of ppGpp and break the normal relationship between growth rate and cell size at division. It would not be surprising if other phenotypes associated with these mutants, such as loss of virulence, were also related to perturbations in ppGpp levels effected through altered transcription patterns.
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| 2. |
- Andersson, Dan I., et al.
(författare)
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Biological roles of translesion synthesis DNA polymerases in eubacteria
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 77:3, s. 540-548
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Forskningsöversikt (refereegranskat)abstract
- Biological systems are strongly selected to maintain the integrity of their genomes by prevention and repair of external and internal DNA damages. However, some types of DNA lesions persist and might block the replication apparatus. The universal existence of specialized translesion synthesis DNA polymerases (TLS polymerases) that can bypass such lesions in DNA implies that replication blockage is a general biological problem. We suggest that the primary function for which translesion synthesis polymerases are selected is to rescue cells from replication arrest at lesions in DNA, a situation that, if not amended, is likely to cause an immediate and severe reduction in cell fitness and survival. We will argue that the mutagenesis observed during translesion synthesis is an unavoidable secondary consequence of this primary function and not, as has been suggested, an evolved mechanism to increase mutation rates in response to various stresses. Finally, we will discuss recent data on additional roles for translesion synthesis polymerases in the formation of spontaneous deletions and in transcription-coupled TLS, where the coupling of transcription to TLS is proposed to allow the rescue of the transcription machinery arrested at DNA lesions.
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| 3. |
- Björkman, Johanna, et al.
(författare)
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Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium
- 1999
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Ingår i: Molecular Microbiology. - : BLACKWELL SCIENCE LTD. - 0950-382X .- 1365-2958. ; 31:1, s. 53-58
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Tidskriftsartikel (populärvet., debatt m.m.)abstract
- Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE. Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium. To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium. We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL, being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL. This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity (ram) phenotype.
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| 4. |
- Brandis, Gerrit, 1985-, et al.
(författare)
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Fitness-compensatory mutations in rifampicin-resistant RNA polymerase
- 2012
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Ingår i: Molecular Microbiology. - : Blackwell Publishing. - 0950-382X .- 1365-2958. ; 85:1, s. 142-151
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in rpoB (RNA polymerase β-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (RifR) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized RifR mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a RifR mutant.
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| 5. |
- 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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| 6. |
- Nagaev, Ivan, et al.
(författare)
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Biological cost and compensatory evolution in fusidic acid-resistant Staphylococcus aureus
- 2001
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Ingår i: Molecular Microbiology. - : BLACKWELL SCIENCE LTD. - 0950-382X .- 1365-2958. ; 40:2, s. 433-439
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Tidskriftsartikel (refereegranskat)abstract
- Fusidic acid resistance resulting from mutations in elongation factor G (EF-G) of Staphylococcus aureus is associated with fitness costs during growth in vivo and in vitro. In both environments, these costs can be partly or fully compensated by the acquisition of secondary intragenic mutations. Among clinical isolates of S. aureus, fusidic acid-resistant strains have been identified that carry multiple mutations in EF-G at positions similar to those shown experimentally to cause resistance and fitness compensation. This observation suggests that fitness-compensatory mutations may be an important aspect of the evolution of antibiotic resistance in the clinical environment, and may contribute to a stabilization of the resistant bacteria present in a bacterial population.
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| 7. |
- Tubulekas, Ioannis, et al.
(författare)
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Growth and translation elongation rate are sensitive to the concentration of EF-Tu
- 1993
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 8:4, s. 761-770
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Tidskriftsartikel (refereegranskat)abstract
- We have used quantitative immunoblotting to estimate the amount of EF-Tu in a variety of S. typhimurium strains with wild-type, mutant, insertionally inactivated or plasmid-borne tuf genes. In the same strains we have measured translation elongation rate, exponential growth rate and the level of nonsense codon readthrough. In the wild-type strain, at moderate to fast growth rates, our data show that EF-Tu makes up 8–9% of total cell protein. Strains with either of the tuf genes insertionally inactivated have 65% of the wild-type EF-Tu level, irrespective of which tuf gene remains active, or whether that gene is wild-type or a kirromycin-resistant mutant. Strains with only one active tuf gene have reduced growth and translation elongation rates. From the magnitude of the reduction in elongation rate relative to the level of EF-Tu we calculate that in glucose minimal medium the in vivo saturation level of wild-type ribosomes by ternary complexes is only 63%. Strains with a ribosome mutation causing a poor interaction with ternary complex are non-viable on minimal medium when the level of EF-Tu is reduced.
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| 8. |
- Tubulekas, Ioannis, et al.
(författare)
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Suppression of rpsL phenotypes by tuf mutations reveals a unique relationship between translation elongation and growth rate
- 1993
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 7:2, s. 275-284
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Tidskriftsartikel (refereegranskat)abstract
- We have found a simple relationship between bacterial growth rate and the translation elongation rate. Thus, for a set of defined ribosomal protein S12 mutations which reduce the efficiency of the ternary complex ribosome interaction (and restrict the frequency of translational errors) there is a linear relationship between growth rate and translation elongation rate. When these mutants are combined with defined EF-Tu mutants (which increase the probability of translational errors) both the elongation rate and growth rate reductions are reversed. The reductions and reversals are described by a unique linear relationship. We interpret this to mean that these two types of mutation exert opposing effects on the same molecular interaction. We suggest that this interaction is in the initial selection of the aminoacyl-tRNA on the ribosome. The slope of the relationship between translation elongation rate and growth rate, defined in per cent of the wild-type rates, is close to 1. Interestingly, the reversal of the elongation and growth phenotypes is Incomplete, suggesting that the ribosomal mutants have an additional defect which is not compensated for by the ternary complex interaction. Our results show that the efficiency of the ternary complex ribosome interaction limits the translation elongation rate, which in turn correlates with changes in exponential growth rate.
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| 9. |
- Brandis, Gerrit, 1985-, et al.
(författare)
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Autoregulation of the tufB operon in Salmonella
- 2016
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Ingår i: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 100:6, s. 1004-1016
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Tidskriftsartikel (refereegranskat)abstract
- In Salmonella enterica and related species, translation elongation factor EF-Tu is encoded by two widely separated but near-identical genes, tufA and tufB. Two thirds of EF-Tu is expressed from tufA with the remaining one third coming from tufB. Inactivation of tufA is partly compensated by a doubling in the amount of EF-TuB but the mechanism of this up-regulation is unknown. By experimental evolution selecting for improved growth rate in a strain with an inactive tufA we selected six different noncoding or synonymous point mutations close to the tufB start codon. Based on these results we constructed a total of 161 different point mutations around the tufB start codon, as well as tufB 3′-truncations, and measured tufB expression using tufB-yfp transcriptional and translational fusions. The expression data support the presence of two competing stem-loop structures that can form in the 5′-end of the tufB mRNA. Formation of the ‘closed’ structure leads to Rho-dependent transcriptional termination of the tufB mRNA. We propose a model in which translational speed is used as a sensor for EF-Tu concentration and where the expression of tufB is post-transcriptionally regulated. This model describes for the first time how expression of the most abundant Salmonella protein is autoregulated.
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| 10. |
- Brandis, Gerrit, 1985-, et al.
(författare)
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Co-evolution with recombination affects the stability of mobile genetic element insertions within gene families of Salmonella
- 2018
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Ingår i: Molecular Microbiology. - : WILEY. - 0950-382X .- 1365-2958. ; 108:6, s. 697-710
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Tidskriftsartikel (refereegranskat)abstract
- Bacteria can have multiple copies of a gene at separate locations on the same chromosome. Some of these gene families, including tuf (translation elongation factor EF-Tu) and rrl (ribosomal RNA), encode functions critically important for bacterial fitness. Genes within these families are known to evolve in concert using homologous recombination to transfer genetic information from one gene to another. This mechanism can counteract the detrimental effects of nucleotide sequence divergence over time. Whether such mechanisms can also protect against the potentially lethal effects of mobile genetic element insertion is not well understood. To address this we constructed two different length insertion cassettes to mimic mobile genetic elements and inserted these into various positions of the tuf and rrl genes. Wemeasured rates of recombinational repair that removed the inserted cassette and studied the underlying mechanism. Our results indicate that homologous recombination can protect the tuf and rrl genes from inactivation by mobile genetic elements, but forinsertions within shorter gene sequences the efficiency of repair is very low. Intriguingly, we found that physical distance separating genes on the chromosome directly affects the rate of recombinational repair suggesting that relative location will influence the ability of homologous recombination to maintain homogeneity.
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