| 1. |
- Ghosh, Anirban, et al.
(författare)
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Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations
- 2018
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Ingår i: EMBO Journal. - : WILEY. - 0261-4189 .- 1460-2075. ; 37:9
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Inturi, Raviteja, 1985-, et al.
(författare)
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A splice variant of the human phosphohistidine phosphatase 1 (PHPT1) is degraded by the proteasome
- 2014
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Ingår i: International Journal of Biochemistry and Cell Biology. - : Elsevier. - 1357-2725 .- 1878-5875. ; 57, s. 69-75
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Tidskriftsartikel (refereegranskat)abstract
- Regulation of protein activity by phosphorylation is central in many cellular processes. Phosphorylation of serine, threonine and tyrosine residues is well documented and studied. In addition, other amino acids, like histidine can be phosphorylated, but neither the mechanism nor the function of this modification is well understood. Nevertheless, there is a 14 kDa enzyme with phosphohistidine phosphatase activity, named PHPT1, found in most animals, but not in bacteria, plant or fungi. There are a few splice variant transcripts formed from the human PHPT1 locus and some of them are predicted to form variant proteins, but studies of these proteins are lacking. In order to get insight into the possible function of the variant transcripts encoded at the PHPT1 locus, ectopic expression of PHPT1 transcript variant 6, predicted to be degraded by the non-sense mediated mRNA decay pathway, in HeLa cells was undertaken. In HeLa cells the splice variant protein was degraded by the proteasome, unlike the wild type protein. Using an in silico modeling approach the variant C-terminal end of the proteins were predicted to form different secondary structures that might explain the different properties of the two proteins. The specific degradation of the PHPT1 splice variant indicates that at least for the PHPT1 protein, the quality control and the self-guarding of the cellular system works at two levels, first at the RNA level, aberrant transcripts are degraded by the non-sense mediated mRNA decay pathway, and the small amount of proteins that are formed will be degraded by the proteasome.
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| 3. |
- Maksimov, Vladimir, et al.
(författare)
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Stress sensitivity of a fission yeast strain lacking histidine kinases is rescued by the ectopic expression of Chk1 from Candida albicans
- 2017
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Ingår i: Current Genetics. - : SPRINGER. - 0172-8083 .- 1432-0983. ; 63:2, s. 343-357
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Tidskriftsartikel (refereegranskat)abstract
- The development of new drugs against the pathogenic yeast Candida albicans is compelling and the evolution of relevant bioassays is important to achieve this goal. Promising drug targets are proteins that lack human counterparts which are true for the His-to-Asp phosphorelay signal transduction systems, important for stress sensing in bacteria, fungi, and plants. In the pathogenic yeast, Candida albicans, the CaChk1 histidine kinase is a trigger of the pathway that leads to a switch from yeast to hyphal growth necessary for invasion. Intriguingly, the model yeast Schizosaccharomyces pombe has a similar phosphorelay system, with three histidine kinases named Mak1, Mak2, and Mak3, which are important for the prevention of aberrant mating and sporulation on rich media. This study uncovered distinct functions for the three histidine kinases; Mak1 alone or Mak2 and Mak3 together were sufficient for the repression of the meiotic cycle when nutrients were available. Moreover, strains lacking histidine kinase genes were sensitive to various types of stress conditions in an auxotrophic strain background, while the stress sensitivity was lost in prototrophic strains. Finally, the stress sensitivity of a S. pombe strain that lacks endogenous histidine kinases could be complemented by the ectopic expression of the CaChk1 histidine kinase from C. albicans. This finding opens up for the possibility to perform a drug screen with a biological read-out in S. pombe to find inhibitors of CaChk1.
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| 4. |
- Stårsta, Magnus, 1991-, et al.
(författare)
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Contact-dependent growth inhibition modules contain intracellularly expressed selfish genetic elements
- 2022
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Contact-Dependent growth Inhibition (CDI) confers a fitness advantage forbacteria with the system, by allowing delivery of toxins into cells lacking thecognate immunity protein, resulting in growth arrest. Delivery frequentlyoccurs also between siblings, which could explain how these genes aremaintained within the CDI+ population even in the absence of sensitivecompetitors. Bacteria with CDI systems often contain additional so called“orphan” CDI modules. These modules consist of a toxin not attached tooany delivery mechanism and a cognate immunity protein. How selection forsuch orphan CDI genes or for the normal CDI loci under conditionsprohibiting delivery are upheld, is not known. In this study we find thatfunctional CdiA toxin and immunity is produced from internal promoterswithin the CDI toxin gene, cdiA-CT. This expression protects cells frominhibition by delivered CDI toxins, suggesting that orphan modules could beselected to protect against competing strains with different toxin arsenal.Simultaneously, the intracellular presence of these toxins conferred a smallbut significant reduction in growth rate under nutrient limited conditions,suggesting a role for the toxin-immunity modules in growth regulation. Inaddition, loss of the genetic material was associated with a fitness cost,suggesting that selection for these genes is likely upheld by themfunctioning as selfish genetic elements in the absence of toxin delivery.
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| 5. |
- 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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| 6. |
- Virtanen, Petra, et al.
(författare)
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Class II contact‐dependent growth inhibition (CDI) systems allow for broad‐range cross‐species toxin delivery within the Enterobacteriaceae family
- 2019
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 111:4, s. 1109-1125
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Tidskriftsartikel (refereegranskat)abstract
- Contact‐dependent growth inhibition (CDI) allows bacteria to recognize kin cells in mixed bacterial populations. In Escherichia coli, CDI mediated effector delivery has been shown to be species‐specific, with a preference for the own strain over others. This specificity is achieved through an interaction between a receptor‐binding domain in the CdiA protein and its cognate receptor protein on the target cell. But how conserved this specificity is has not previously been investigated in detail. Here, we show that class II CdiA receptor‐binding domains and their Enterobacter cloacae analog are highly promiscuous, and can allow for efficient effector delivery into several different Enterobacteriaceae species, including Escherichia, Enterobacter, Klebsiella and Salmonella spp. In addition, although we observe a preference for the own receptors over others for two of the receptor‐binding domains, this did not limit cross‐species effector delivery in all experimental conditions. These results suggest that class II CdiA proteins could allow for broad‐range and cross‐species growth inhibition in mixed bacterial populations.
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| 7. |
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| 8. |
- Wäneskog, Marcus, et al.
(författare)
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Escherichia coli EC93 deploys two plasmid- encoded class I contact- dependent growth inhibition systems for antagonistic bacterial interactions
- 2021
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Ingår i: Microbial Genomics. - : MICROBIOLOGY SOC. - 2057-5858. ; 7:3
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Tidskriftsartikel (refereegranskat)abstract
- The phenomenon of contact- dependent growth inhibition (CDI) and the genes required for CDI (cdiBAI) were identified and isolated in 2005 from an Escherichia coli isolate (EC93) from rats. Although the cdiBAIEC93 locus has been the focus of extensive research during the past 15 years, little is known about the EC93 isolate from which it originates. Here we sequenced the EC93 genome and find two complete and functional cdiBAI loci (including the previously identified cdi locus), both carried on a large 127 kb plasmid. These cdiBAI systems are differentially expressed in laboratory media, enabling EC93 to outcompete E. coli cells lacking cognate cdiI immunity genes. The two CDI systems deliver distinct effector peptides that each dissipate the membrane potential of target cells, although the two toxins display different toxic potencies. Despite the differential expression and toxic potencies of these CDI systems, both yielded similar competitive advantages against E. coli cells lacking immunity. This can be explained by the fact that the less expressed cdiBAI system (cdiBAIEC93-2) delivers a more potent toxin than the highly expressed cdiBAIEC93-1 system. Moreover, our results indicate that unlike most sequenced CDI+ bacterial isolates, the two cdi loci of E. coli EC93 are located on a plasmid and are expressed in laboratory media.
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| 9. |
- Wäneskog, Marcus, et al.
(författare)
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Multi-fragment site-directed mutagenic overlap extension polymerase chain reaction as a competitive alternative to the enzymatic assembly method
- 2014
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 444, s. 32-37
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Tidskriftsartikel (refereegranskat)abstract
- Methods for introducing multiple site-directed mutations are important experimental tools in molecular biology. Research areas that use these methods include the investigation of various protein modifications in cellular processes, modifying proteins for efficient recombinant expression, and the stabilization of mRNAs to allow for increased protein expression. Introducing multiple site-directed mutations is also an important tool in the field of synthetic biology. There are two main methods used in the assembling of fragments generated by mutagenic primers: enzymatic assembly and overlap extension polymerase chain reaction (OE-PCR). In this article, we present an improved OE-PCR method that can be used for the generation of large DNA fragments (up to 7.4kb) where at least 13 changes can be introduced using a genomic template. The improved method is faster (due to fewer reaction steps) and more accurate (due to fewer PCR cycles), meaning that it can effectively compete with the enzymatic assembly method. Data presented here show that the site-directed mutations can be introduced anywhere between 50 and 1800bp from each other. The method is highly reliable and predicted to be applicable to most DNA engineering when the introduction of multiple changes in a DNA sequence is required.
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| 10. |
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