| 1. |
- Pavlov, Michael, et al.
(författare)
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Initiation factor 2 mutants promoting fast joining of ribosomal subunits in the absence of initiator tRNA or GTP
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- We have previously identified several mutations in initiation factor 2 (IF2) located outside the tRNA binding domain IV of IF2 that compensate for lack of initiator tRNA (Met-tRNAi) formylation in vivo. We have also shown that these IF2 mutants promote fast joining of ribosomal subunits even when non-formylated Met-tRNAi or deacylated tRNAi was present in the 30S pre-initiation complex (30S PIC) instead of formylated fMet-tRNAi. We demonstrate here in vitro that these IF2 mutants do not require any tRNA present on the 30S subunit to promote fast subunit joining provided that GTP is present. Moreover, in the presence of fMet-tRNAi the mutants promote fast subunit joining in the presence of only GDP. Thus, A-type IF2 mutants require either GTP or fMet-tRNAi for fast subunit joining. In contrast, fast subunit joining with wild type IF2 requires the presence of both GTP and fMet-tRNAi in the 30S PIC. These results imply that the presence of tRNA on the 30S subunit is per se not required for fast subunit joining but rather for switching the 30S:IF2 complex containing wild type IF2 into its 50S docking conformation. We show also that the rate of subunit joining with A-type IF2 is much less sensitive to the energy level in the reaction mixture than the rate of subunit joining with wild type IF2. We speculate that this insensitivity of initiation to energy levels may result in deteriorated adaptation of formylation-proficient strains harbouring A-type mutations in IF2 to growth under energy-limited conditions.
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| 2. |
- Zorzet, Anna, 1977-, et al.
(författare)
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Compensatory evolution restores fitness to actinonin-resistant Staphylococcus aureus
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- We have studied the emergence of actinonin-resistant mutants in S. aureus. In accordance with earlier studies we identified resistance mutations in the fmt gene that apart from conferring high-level resistance also reduced the growth rate. To study how fitness could be restored we performed compensatory evolution by serial passage of cells in liquid culture. Compensated mutants arose quickly, within 40 generations and sometimes in less than 10 generations. The mutants were fully resistant to actinonin and showed increased growth rates on plates. However, exponential growth rates in liquid media were not higher than for the parental resistant mutants. We sequenced the whole genomes of one slow-growing strain and three compensated and found alterations in three genes. These genes were SAOUHSC_01699 coding for shikimate 5-dehydrogenase, SAOUHSC_00945 coding for a magnesium transporter and SAOUHSC_02264, coding for accessory gene regulator protein C (AgrC). None of these mutants were obvious candidates for compensating lack of formylation. When sequencing these three loci in the remainder of the compensated strains, mutations in agrC were found in 11 of 16 strains. Further studies will show if these mutations are due to general environmental adaption, specific adaptation for slow growth or specific compensation for loss of formylation.
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| 3. |
- Zorzet, Anna, 1977-
(författare)
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Mechanisms of Adaptation to Deformylase Inhibitors
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotic resistance is a growing problem on a global scale. Increasing numbers of bacteria resistant toward one or multiple antibiotics could return us to the high mortality rates for infectious diseases of the pre-antibiotic era. The need for development of new classes of antibiotics is great as is increased understanding of the mechanisms underlying the development of antibiotic resistance. We have investigated the emergence of resistance to peptide deformylase inhibitors, a new class of antibiotics that target bacterial protein synthesis. The fitness of resistant mutants as well as their propensity to acquire secondary compensatory mutations was assessed in order to gain some insight into the potential clinical risk of resistance development. Most of this work was done in the bacterium Salmonella typhimurium, due to the availability of excellent genetic tools to study these phenomena. In addition, we have studied the bacterium Staphylococcus aureus as peptide deformylase inhibitors have been shown to have the greatest effect on Gram-positive organisms. In the course of this work we also examined the mechanistic aspects of translation initiation. Using a cell-free in vitro translation system we studied the effects of various components on translation initiation. These results have been combined with results obtained from resistant and compensated bacterial strains in vivo to gain new insights into the mechanisms of translation initiation.
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