| 1. |
- Andresen, Liis, et al.
(författare)
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The Small Toxic Salmonella Protein TimP Targets the Cytoplasmic Membrane and Is Repressed by the Small RNA TimR
- 2020
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Ingår i: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Small proteins are gaining increased attention due to their important functions in major biological processes throughout the domains of life. However, their small size and low sequence conservation make them difficult to identify. It is therefore not surprising that enterobacterial ryfA has escaped identification as a small protein coding gene for nearly 2 decades. Since its identification in 2001, ryfA has been thought to encode a noncoding RNA and has been implicated in biofilm formation in Escherichia coli and pathogenesis in Shigella dysenteriae. Although a recent ribosome profiling study suggested ryfA to be translated, the corresponding protein product was not detected. In this study, we provide evidence that ryfA encodes a small toxic inner membrane protein, TimP, overexpression of which causes cytoplasmic membrane leakage. TimP carries an N-terminal signal sequence, indicating that its membrane localization is Sec-dependent. Expression of TimP is repressed by the small RNA (sRNA) TimR, which base pairs with the timP mRNA to inhibit its translation. In contrast to overexpression, endogenous expression of TimP upon timR deletion permits cell growth, possibly indicating a toxicity-independent function in the bacterial membrane. IMPORTANCE Next-generation sequencing (NGS) has enabled the revelation of a vast number of genomes from organisms spanning all domains of life. To reduce complexity when new genome sequences are annotated, open reading frames (ORFs) shorter than 50 codons in length are generally omitted. However, it has recently become evident that this procedure sorts away ORFs encoding small proteins of high biological significance. For instance, tailored small protein identification approaches have shown that bacteria encode numerous small proteins with important physiological functions. As the number of predicted small ORFs increase, it becomes important to characterize the corresponding proteins. In this study, we discovered a conserved but previously overlooked small enterobacterial protein. We show that this protein, which we dubbed TimP, is a potent toxin that inhibits bacterial growth by targeting the cell membrane. Toxicity is relieved by a small regulatory RNA, which binds the toxin mRNA to inhibit toxin synthesis.
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| 2. |
- Holmqvist, Erik, 1977-, et al.
(författare)
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RNA-binding activity and regulatory functions of the emerging sRNA-binding protein ProQ
- 2020
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Ingår i: Biochimica et Biophysica Acta. Gene Regulatory Mechanisms. - : Elsevier BV. - 1874-9399 .- 1876-4320. ; 1863:9
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Forskningsöversikt (refereegranskat)abstract
- Regulatory small RNAs (sRNAs) ubiquitously impact bacterial physiology through antisense-mediated control of mRNA translation and stability. In Gram negative bacteria, sRNAs often associate with RNA-binding proteins (RBPs), both to gain cellular stability and to enable regulatory efficiency. The Hfq and CsrA proteins were for long the only known global RBPs implicated in sRNA biology. During the last five years, the FinO domain-containing protein ProQ has emerged as another global RBP with a broad spectrum of sRNA and mRNA ligands. This review provides a summary of the current knowledge of enterobacterial ProQ, with a special focus on RNA binding activity, RNA ligand preferences, influence on RNA stability and gene expression, and impact on bacterial physiology. Considering that characterization of ProQ is still in its infancy, we highlight aspects that, when addressed, will provide important clues to the physiological functions and regulatory mechanisms of this globally acting RBP.
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| 3. |
- Rizvanovic, Alisa
(författare)
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Adapting to succeed : Post-transcriptional gene regulation in Salmonella
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Salmonella are zoonotic pathogens of worldwide economic and health importance. Both during life outside and inside the host, these pathogens are subject to continuously changing environmental conditions, such as temperature changes, acid stress, nutrient limitations, and others. In order to thrive and survive, Salmonella must respond to these changes by adapting their physiology and morphology through changes in gene expression. RNA-binding proteins (RBPs) often work in concert with small RNAs (sRNAs) to control gene expression at the post-transcriptional level. Their mode of action includes regulation of RNA translation and/or stability, either positive or negative. Recently, ProQ was discovered to be a global RBP with a large repertoire of mRNA and sRNA targets in Salmonella. However, many details regarding ProQ biology are not fully understood, including the requirements for RNA-binding, mechanisms of gene regulation, and ProQ-dependent phenotypic changes. The main purpose of this doctoral thesis was to characterize the RBP ProQ and its regulatory role in Salmonella.First, we developed a method based on saturation mutagenesis coupled to phenotypic sorting and high-throughput sequencing to chart the functionally important regions in ProQ. Our results reveal that both the N-terminal and C-terminal domains are important for ProQ’s gene regulatory function, but the underlying mechanisms differ. Second, we show that ProQ is important for flagellar-mediated motility in Salmonella. More specifically, we show that ProQ and an associated sRNA promotes flagellar gene expression and motility by affecting translation of the master flagellar regulator FlhDC. Finally, we reveal that ProQ induces persister formation in Salmonella and enables a subpopulation of cells to survive high doses of different types of antibiotics through growth arrest.In conclusion, the findings presented herein provide new insights into the role of ProQ as a global post-transcriptional regulator of gene expression in Salmonella. Together, these findings contribute to our understanding of how Salmonella shapes its lifestyle and induces pathogenesis.
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| 4. |
- Rizvanovic, Alisa, et al.
(författare)
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Olfactory Discrimination Ability of Asian Elephants (Elephas maximus) forStructurally Related Odorants
- 2013
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Ingår i: Chemical Senses. - Oxford : Oxford University Press. - 0379-864X .- 1464-3553. ; 38:2, s. 107-118
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Tidskriftsartikel (refereegranskat)abstract
- Using a food-rewarded two-choice instrumental conditioning paradigm, we assessed the ability of Asian elephants, Elephasmaximus, to discriminate between 2 sets of structurally related odorants. We found that the animals successfully discriminatedbetween all 12 odor pairs involving members of homologous series of aliphatic 1-alcohols, n-aldehydes, 2-ketones,and n-carboxylic acids even when the stimuli differed from each other by only 1 carbon. With all 4 chemical classes, the elephantsdisplayed a positive correlation between discrimination performance and structural similarity of odorants in terms ofdifferences in carbon chain length. The animals also successfully discriminated between all 12 enantiomeric odor pairs tested.An analysis of odor structure–activity relationships suggests that a combination of molecular structural properties rather thana single molecular feature may be responsible for the discriminability of enantiomers. Compared with other species testedpreviously on the same sets of odor pairs (or on subsets thereof), the Asian elephants performed at least as well as miceand clearly better than human subjects, squirrel monkeys, pigtail macaques, South African fur seals, and honeybees. Furthercomparisons suggest that neither the relative nor the absolute size of the olfactory bulbs appear to be reliable predictors ofbetween-species differences in olfactory discrimination capabilities. In contrast, we found a positive correlation between thenumber of functional olfactory receptor genes and the proportion of discriminable enantiomeric odor pairs. Taken together,the results of the present study support the notion that the sense of smell may play an important role in regulating thebehavior of Asian elephants.
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| 5. |
- Rizvanovic, Alisa, et al.
(författare)
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ProQ and an associated small RNA promote flagellar gene expression in Salmonella
- 2022
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- ProQ has emerged as a major RNA-binding protein that affects expression of virulence-related genes in Salmonella. Despite comprehensive information about RNA targets and their expression patterns, relating these data to a functional role of ProQ is still in its infancy. Here, we report that ProQ and the associated small RNA (sRNA) FlgO, expressed from within the flagellar regulon, promotes flagellar gene expression and motility in Salmonella. We show that FlgO exerts positive feed-back by stimulating translation of the FlhDC master flagellar regulator. Our data suggest that FlgO binds to the flhD 5’ untranslated region, leading to destabilization of an intramolecular structure that otherwise sequesters the Shine-Dalgarno sequence. ProQ positively impacts the cellular abundance of FlgO, thereby promoting translation activation of FlhD, and ultimately, increased expression of downstream flagellar genes. Together, our data expands the regulatory roles of ProQ and FlgO, and highlights their importance for flagella and motility in Salmonella.
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| 6. |
- Rizvanovic, Alisa, et al.
(författare)
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Saturation mutagenesis charts the functional landscape of Salmonella ProQ and reveals a gene regulatory function of its C-terminal domain
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 49:17, s. 9992-10006
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Tidskriftsartikel (refereegranskat)abstract
- The global RNA-binding protein ProQ has emerged as a central player in post-transcriptional regulatory networks in bacteria. While the N-terminal domain (NTD) of ProQ harbors the major RNA-binding activity, the role of the ProQ C-terminal domain (CTD) has remained unclear. Here, we have applied saturation mutagenesis coupled to phenotypic sorting and long-read sequencing to chart the regulatory capacity of Salmonella ProQ. Parallel monitoring of thousands of ProQ mutants allowed mapping of critical residues in both the NTD and the CTD, while the linker separating these domains was tolerant to mutations. Single amino acid substitutions in the NTD associated with abolished regulatory capacity strongly align with RNA-binding deficiency. An observed cellular instability of ProQ associated with mutations in the NTD suggests that interaction with RNA protects ProQ from degradation. Mutation of conserved CTD residues led to overstabilization of RNA targets and rendered ProQ inert in regulation, without affecting protein stability in vivo. Furthermore, ProQ lacking the CTD, although binding competent, failed to protect an mRNA target from degradation. Together, our data provide a comprehensive overview of residues important for ProQ-dependent regulation and reveal an essential role for the enigmatic ProQ CTD in gene regulation.
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| 7. |
- Rizvanovic, Alisa, et al.
(författare)
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The RNA-binding protein ProQ promotes antibiotic persistence in Salmonella
- 2022
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Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 13:6
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial populations can survive the exposure to antibiotics through transient phenotypic and gene expression changes. These changes can be attributed to a small subpopulation of bacteria, giving rise to antibiotic persistence. Even though this phenomenon has been known for decades, much is still to be learnt about the mechanisms that drive persister formation. The RNA-binding protein ProQ has recently emerged as a global regulator of gene expression. Here, we show that ProQ impacts persister formation in Salmonella. ProQ contributes to growth-arrest in single cells, which are able to survive treatment with high concentrations of different antibiotics. The underlying mechanism for ProQ-dependent persister formation involves activation of the flagellar pathway. Importantly, we show that the ProQ-dependent phenotype is relevant during macrophage infection and allows Salmonella to survive the combined action of host immune defences and antibiotics. Together, our data highlights the importance of ProQ in Salmonella persistence and pathogenesis.
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