Phänotypische Charakterisierung von 6S RNA-Deletionsstämmen in dem nicht domestizierten Bacillus subtilis Wildtypstamm NCIB 3610
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Philipps-Universität Marburg
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Abstract
Bacterial 6S RNAs are highly abundant non-coding RNAs that are able to regulate transcription in a growth-stage dependent manner via binding to the active site of RNA polymerase (RNAP) holoenzymes. In this way 6S RNAs inhibit housekeeping transcription during stationary phase growth, thereby acting as global transcriptional regulators. They are nearly universal within the bacterial kingdom and most bacterial groups encode one single 6S RNA. However, numerous Firmicutes, including Bacillus subtilis, possess two 6S RNA paralogs, named 6S-1 and 6S-2 RNA. The mechanistic function of the 6S-1 RNA on the RNAP holoenzyme is well-investigated, whereas the physiological roles of these paralogs are still unknown. A 6S-1 RNA deletion in a B. subtilis lab strain background reveals some mild phenotypes. In contrast to this, the corresponding 6S-2 RNA deletion strain shows no phenotype at all.
Hypothesizing that the regulatory role of both 6S RNA paralogs might be relevant under natural, constantly changing environmental conditions, a phenotypic characterization of 6S RNA deletion mutants in the undomesticated B. subtilis wildtype strain NCIB 3610 was conducted. For the first time our data disclosed a strong phenotype of the 6S-2 RNA deletion mutant, exhibiting increased biofilm formation on solid media as well as the ability to form surface-attached biofilms in liquid culture. The extend of this biofilm derepression phenotype manifests at lower growth temperatures. Quantitative RT-PCR analysis demonstrate that biofilm marker genes tasA, epsA and bslA are transcriptionally upregulated in this mutant, particularly during transition from exponential to stationary growth phase. Furthermore, the 6S-2 RNA deletion strain reveals retarded swarming behaviour and earlier sporulation. The double knockout mutant, which is deleted for both 6S RNAs, shows a prolonged lag phase growing under several tested stress conditions. In addition, first hints lead to the assumption that the loss of one 6S RNA could influence the B. subtilis subpopulation distribution within a bacterial culture.
Another subproject should clarify whether the 6S RNA system of B. subtilis can also influence synthesis of the two nucleotide-based second messengers guanosine pentaphosphate or -tetraphosphate, collectively named as (p)ppGpp, via putative binding to the (p)ppGpp synthetase SAS1. Such an interaction most likely leads to an inhibition of the protein, hypothesizing that one or both 6S RNAs could function as SAS1 inhibitors. Electrophoretic mobility shift assays reveal binding of 6S-1 RNA as well as 6S-2 RNA to SAS1, in particular when the 6S RNAs is present in a complex with its long product RNAs (pRNAs). However, investigations on SAS1 activity using thin-layer chromatography experiments did not result in a reduced pppGpp synthesis in the presence of the 6S-1 RNA or the 6S-1:pRNA14 complex. Therefore, it was not possible to clearly verify whether the 6S RNA system has a putative biological function on SAS1.
Newly acquired insights from this study open a new perspective on the bacterial 6S RNA system, in which the interplay of both 6S RNAs of B. subtilis have a crucial role in fine tuning the transcriptional adaption particularly under physiological conditions. Fundamentally, the new insights demonstrate how significant the choice of strain background is for the functional study of a microorganism’s genetic equipment. Furthermore, the studies undertaken here clearly reveal that phenotypic characterization must go beyond the consideration of bacteria as unicellular organisms to fulfil the elucidation of physiological roles. Bacteria are complexly organized cell assemblies, for example in the form of biofilms. The 6S RNA system from B. subtilis also appears to exert its physiological function at precisely such superordinated population level. Bacterial 6S RNAs represent highly complex regulatory systems of which not all facets have been deciphered so far.
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Created: 2021Issued: 2022-07-04Updated: 2022-07-05
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Fachbereich Pharmazie
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Philipps-Universität Marburg
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ger
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DoctoralThesis
Keywords
Transkriptionsregulation6S RNABiofilmtranscriptional regulationBacillus subtilis
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570
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Thüring, Marietta: Phänotypische Charakterisierung von 6S RNA-Deletionsstämmen in dem nicht domestizierten Bacillus subtilis Wildtypstamm NCIB 3610. : Philipps-Universität Marburg 2022-07-04. DOI: https://doi.org/10.17192/z2022.0042.
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This item has been published with the following license: In Copyright