TPP riboswitch characterization in Alishewanella tabrizica and Alishewanella aestuarii and comparison with other TPP riboswitches

Previous RNA sequencing has allowed identifying 129 long 5'-UTRs in the L. lactis MG1363 transcriptome. These sequences potentially harbor cis -acting riboswitches. One of the identified extended 5'-UTRs is a putative thiamine pyrophosphate (TPP) riboswitch. It is located immediately upstream of the thiamine transporter gene thiT ( llmg_0334 ). To confirm this assumption, the 5'-UTR sequence was placed upstream of the gene encoding the super folder green fluorescent protein, sfgfp , allowing examining the expression of sfGFP in the presence or absence of thiamine in the medium. The results show that this sequence indeed represents a thiamine-responsive TPP riboswitch. This RNA-based genetic control device was used to successfully restore the mutant phenotype of an L. lactis strain lacking the major autolysin gene acmA . The L. lactis thiT TPP riboswitch (RS thiT ) is a useful molecular genetic tool enabling to gradually downregulate the expression of genes under its control by adjusting thiamine concentration. Importance The capacity of microbes with biotechnological importance to adapt and survive under quickly changing industrial conditions depends on their ability to adequately control gene expression. Riboswitches are important RNA-based elements involved in rapid and precise gene regulation. Here we present the identification of a natural thiamine-responsive riboswitch of Lactococcus lactis , a bacterium used worldwide in the production of dairy products. We used it to restore a genetic defect in an L. lactis mutant and show that it is a valuable addition to the ever-expanding L. lactis genetic toolbox.

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“…A P3a structure is not obvious in the L. lactis THI elements. P4 and P5 bind to the pyrophosphate group of TPP ( 16 , 19 ).…”

Section: Resultsmentioning

confidence: 99%

Riboswitch RS _thiT as a Molecular Tool in Lactococcus lactis

Hernández-Ortega

Meulen

Kuijpers

2022

Appl Environ Microbiol

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“…In contrast, only a few instances of TPP riboswitches were detected experimentally in eukaryotes. Even though well-established protocols 49 have been developed for experimental detection of riboswitches, such methods are still time-consuming and limited by the choice of model species used to search for them. Our analysis shows that computational tools can be effectively used to significantly enhance the speed and scope of discovery of these regulatory elements.…”

Section: Discussionmentioning

confidence: 99%

Phylogenomic and comparative analysis of the distribution and regulatory patterns of TPP riboswitches in fungi

Mukherjee

Retwitzer

Barash

et al. 2018

Sci Rep

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Riboswitches are metabolite or ion sensing cis-regulatory elements that regulate the expression of the associated genes involved in biosynthesis or transport of the corresponding metabolite. Among the nearly 40 different classes of riboswitches discovered in bacteria so far, only the TPP riboswitch has also been found in algae, plants, and in fungi where their presence has been experimentally validated in a few instances. We analyzed all the available complete fungal and related genomes and identified TPP riboswitch-based regulation systems in 138 fungi and 15 oomycetes. We find that TPP riboswitches are most abundant in Ascomycota and Basidiomycota where they regulate TPP biosynthesis and/or transporter genes. Many of these transporter genes were found to contain conserved domains consistent with nucleoside, urea and amino acid transporter gene families. The genomic location of TPP riboswitches when correlated with the intron structure of the regulated genes enabled prediction of the precise regulation mechanism employed by each riboswitch. Our comprehensive analysis of TPP riboswitches in fungi provides insights about the phylogenomic distribution, regulatory patterns and functioning mechanisms of TPP riboswitches across diverse fungal species and provides a useful resource that will enhance the understanding of RNA-based gene regulation in eukaryotes.

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