Biology subjects

Thorstensen, T.

Publications and source records attributed to Thorstensen, T..

4 recordsLinked to original sources

bioRxiv2026

Novel insights into the biosynthesis and diversity of leaderless multipeptide bacteriocins

Garvicin KS (GarKS) is a three-peptide, leaderless, broad-spectrum bacteriocin that is active against a wide range of Gram-positive bacteria, including several foodborne pathogens and antibiotic-resistant strains. This bacteriocin is considered a candidate for application in food preservation and medical treatments; however, key knowledge about the producer strain, regulation of production, and the mechanism of action is still lacking for leaderless, multipeptide bacteriocins, including GarKS. A hybrid sequencing strategy was used to obtain a high-quality closed genome assembly of the native GarKS producer Lactococcus garvieae KS1546, which showed that the bacteriocin was encoded on a 50 kb plasmid (pKS50). Comparative analysis with updated sequence databases indicates that the producer strain should be reclassified as Lactococcus petauri. The roles of the biosynthetic genes, putatively encoding a transcriptional regulator (gakR) and immunity protein (gakI), were examined using heterologous expression. Removal of gakR resulted in a 6-fold decrease in GarKS production, and expression of gakI caused a 250-fold decrease in susceptibility to GarKS, with varying degrees of cross-immunity to other multipeptide bacteriocins. Isolation and whole-genome sequencing of spontaneous GarKS mutants in L. lactis showed that resistance levels are low, but that ythA, a PspC-domain-containing protein involved in a phage stress response pathway is involved in the GarKS susceptible phenotype. To decipher conserved features involved in production of these bacteriocins, we finally used genome mining to identify 11 candidates for new multipeptide bacteriocins; four of them were obtained synthetically and confirmed to be bioactive peptides inhibiting important pathogens including Listeria monocytogenes and enterococci.

microbiology↗

bioRxiv2024

CRISPR-Cas9 mediated deletions of FvMYB46 reduces fruit set and biosynthesis of flavonoids in Fragaria vesca

Secondary metabolites produced by the phenylpropanoid pathway, which is regulated by transcription factors of the MYB family, play crucial roles in this early phase of fruit development. The MYB46 transcription factor is a key regulator of secondary cell wall structure and lignin and flavonoid biosynthesis in many plants, but little is known about its activity in flowers and berries in F. vesca. For functional analysis of FvMYB46, we designed a CRISPR-Cas9 construct with an endogenous F. vesca specific U6-promoter for efficient and specific expression of two gRNAs targeting the first exon of FvMYB46. This generated mutants with an in frame 81-bp deletion of the first conserved MYB-domain or an out of frame 82-bp deletion potentially knocking out the gene function. In both types of mutant plants, pollen germination and the frequency of flowers developing to mature berries was significantly reduced compared to wild type. Transcriptomic analysis of flowers demonstrated that FvMYB46 is positively regulating the expression of genes involved in pollen germination, homeostasis of reactive oxygen species (ROS) and the phenylpropanoid pathway, including secondary cell wall biosynthesis and flavonoid biosynthesis, while has a negative impact on carbohydrate metabolism. In FvMYB46-mutant flowers, the flavonols and flavan-3-olscontent, especially epicatechin, quercetin-glucoside and kaempferol-3-coumaroylhexoside were reduced, and we observed a local reduction of lignin content in anthers. Together these results suggest that MYB46 control fertility and efficient fruit set by regulating cell wall structure, flavonoid biosynthesis, carbohydrate metabolism and ROS-signaling in flowers and early fruit development in F. vesca.

plant biology↗

bioRxiv2019

Induced and primed defence responses of Fragaria vesca to Botrytis cinerea infection

1.Strawberry is a high-value crop that suffers huge losses from diseases such as grey mould caused by the necrotrophic fungal pathogen Botrytis cinerea. Pesticides are heavily used to protect the strawberry crop, which raises environmental and human health concerns and promotes the evolution of pesticide resistant strains. Upregulating or priming the plants defences may be a more environmentally sustainable way of increasing disease resistance. Using Fragaria vesca as a model for the commercially grown octaploid strawberry Fragaria x ananassa, we investigated the transcriptional reprogramming of strawberry upon B. cinerea infection and the effectiveness of four priming chemicals in protecting strawberry against grey mould. First, we found that the transcriptional reprogramming of strawberry upon B. cinerea infection overlapped substantially with the transcriptome responses induced by Phytophthora cactorum (Toljamo et al., 2016), including the genes involved in jasmonic acid (JA), salicylic acid (SA), ethylene (ET) and terpenoid pathways. Furthermore, we investigated the effectiveness of previously identified priming chemicals in protecting strawberry against B. cinerea. The level of upregulated or primed resistance depended on the priming chemical itself ({beta}-aminobutyric acid (BABA), methyl jasmonate (MeJ), (R)-{beta}-homoserine (RBH), prohexadione-calcium (ProCa)) and the application method used (foliar spray, soil drench, seed treatment). Overall, RBH effectively primed strawberry defences against B. cinerea, whereas BABA and ProCa were not effective and MeJ showed mixed effects. Our results not only identify ways to effectively upregulate or prime strawberry defences against B. cinerea, but also provide novel insights about strawberry defences that may be applied in future crop protection schemes.

plant biology↗

bioRxiv2018

Over 2000-fold increased production of the leaderless bacteriocin garvicin KS by genetic engineering and optimization of culture conditions

The leaderless bacteriocin Garvicin KS (GarKS) is a potent antimicrobial, being active against a wide range of important pathogens. GarKS production by the native producer Lactococcus garvieae KS1546 was however relatively low (80 BU/ml) under standard laboratory growth conditions (batch culture in GM17 at 30{degrees}C). To improve the production of GarKS, we systematically evaluated the impact of different media and media components on bacteriocin production. Based on the outcomes a new medium formulation was made to greatly improve bacteriocin production. The new medium composed of pasteurized milk and tryptone (PM-T), increased GarKS production about 60-fold compared to that achieved in GM17. GarKS production was increased further 4-fold (i.e., to 20,000 BU/ml) by increasing gene dose of the bacteriocin gene cluster (gak) in the native producer. Finally, a combination of the newly composed medium (PM-T), an increased gene dose and a cultivation at a constant pH 6 and a 50-60% dissolved oxygen level in growth medium, gave rise to a GarKS production of 164,000 BU/ml. This high production, which is about 2000-fold higher compared to that initially achieved in GM17, corresponds to a GarKS production of 1.2 g/L. To our knowledge, this is one of the highest bacteriocin production reported hitherto.\n\nImportanceLow bacteriocin production is a well-known bottle-neck in developing bacteriocins into large-scaled and useful applications. The present study shows different approaches that significantly improve bacteriocin production. This is an important research field to better exploit the antimicrobial potential of bacteriocins, especially with regard to the decreasing effect of antibiotics in infection treatments due to the global emergence of antibiotic resistance.

microbiology↗