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- Gynnå, Arvid H., 1988-
(författare)
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Bacterial DNA repair and molecular search
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Surveillance and repair of DNA damage is necessary in all kinds of life. Different types of DNA damage require different repair mechanisms, but these mechanisms are often similar in all domains of life. The most serious type of damage, double stranded DNA breaks, are for example repaired in conceptually similar ways in both bacteria and eukaryotes. When this kind of breaks are repaired by homologous recombination, a homology to the site of the break must be found. Sometimes, this homology can be located far away from the break necessitating a search. Considering the large amount of heterologous DNA present, the complexity of this search is enormous. If and how this search can proceed has been unclear even in simple and well characterized organisms as E. coli.In this thesis, microscopy together with microfluidics are used to show that DNA repair by homologous recombination occurs even between homologies separated by several micrometers. We also see that it finishes well within the time of a cell generation, with the enigmatic search phase being as quick as eight or possibly even three minutes. Since this time is much faster than expected, we present a physical model demonstrating how homology search on this time scale is indeed plausible. Based on these results, we conclude that homologous repair using distantly located templates is likely to be a physiologically relevant mechanism of DNA repair.Microscopy together with image analysis by deep learning also provides a new method of detecting DNA damage in real time. Combined with tracking of cell lineages, it reveals that DNA damage in E. coli is repaired efficiently enough that the resulting fitness cost is close to none. With the same methods we also study the effect of deletions of several DNA repair enzymes, and largely confirms their previous characterizations. Among these, we confirm that the intriguing RecN protein is important but not absolutely necessary in DSB repair, that it acts early, and possibly aids in physically shaping the structure mediating the search.In addition to this, it is shown how DNA transcription and translation modulates the shape of the E. coli nucleoid. We observe how strong a transcription of a gene within a few minutes moves the gene towards the periphery of the cell where the concentration of ribosomes is higher, a movement possibly also aided by protein translation.We also present MINFLUX, a microscope for both nanometer scale localization of single fluorophores as well as in vivo single particle tracking with unprecedented trace length and resolution. Using this, the E. coli small ribosomal subunit could be observed to quickly shift between fast and slow diffusion states which might represent probing and discarding of RNAs suitable for translation.
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| 2. |
- Stårsta, Magnus, et al.
(författare)
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RHS-elements function as type II toxin-antitoxin modules that regulate intra-macrophage replication of Salmonella Typhimurium
- 2020
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Ingår i: PLOS Genetics. - : PUBLIC LIBRARY SCIENCE. - 1553-7390 .- 1553-7404. ; 16:2
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Tidskriftsartikel (refereegranskat)abstract
- RHS elements are components of conserved toxin-delivery systems, wide-spread within the bacterial kingdom and some of the most positively selected genes known. However, very little is known about how Rhs toxins affect bacterial biology. Salmonella Typhimurium contains a full-length rhs gene and an adjacent orphan rhs gene, which lacks the conserved delivery part of the Rhs protein. Here we show that, in addition to the conventional delivery, Rhs toxin-antitoxin pairs encode for functional type-II toxin-antitoxin (TA) loci that regulate S. Typhimurium proliferation within macrophages. Mutant S. Typhimurium cells lacking both Rhs toxins proliferate 2-times better within macrophages, mainly because of an increased growth rate. Thus, in addition to providing strong positive selection for the rhs loci under conditions when there is little or no toxin delivery, internal expression of the toxin-antitoxin system regulates growth in the stressful environment found inside macrophages.
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| 3. |
- Vogel, Carolin, et al.
(författare)
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Rationally designed Spot 42 RNAs with an inhibition/toxicity profile advantageous for engineering E. coli
- 2020
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Ingår i: ENGINEERING REPORTS. - : Wiley. - 2577-8196. ; 2:3
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial regulatory small RNAs (sRNAs) have shown promise for gene knock-down studies and metabolic engineering. However, some mRNAs might be difficult to target due to poor binding by the Hfq chaperone, individual synthetic sRNAs can have off-target effects, potential sRNA toxicities have not been studied globally, and a consensus on optimal sRNA design has yet to emerge. Here, Spot 42 sRNA is validated as an excellent scaffold by showing that its over-expression minimally affects the growth rate of Escherichia coli, and that inhibition is reliably achieved for all eight tested protein targets by designing antisense to target the first few codons. Two related sRNAs that could not be cloned, possibly due to lethality of the encoded sRNAs, became clonable when an eight-nucleotide sequence was inserted directly upstream of the antisense region. Global fitness costs for E. coli of the designer sRNAs were measured and found to be variable but tolerable. Importantly for utility, there was no correlation between target inhibition and cellular toxicity. As a proof of concept for applications, suppression of the UAG stop codon was improved by knock down of translation release factor 1 (RF1).
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