TY - GEN A1 - Kohm, Katharina A1 - Floccari, Valentina A1 - Lutz, Veronika A1 - Nordmann, Birthe A1 - Mittelstädt, Carolin A1 - Poehlein, Anja A1 - Dragos, Anna A1 - Commichau, Fabian M. A1 - Hertel, Robert T1 - The Bacillus phage SPβ and its relatives: A temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components T2 - bioRxiv beta N2 - The Bacillus phage SPβ has been known for about 50 years, but only a few strains are avalible. We isolated four new wild type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SPβ-like phages. We could also reveal the SPβ-like phage genome replication strategy, the genome packaging mode, and the phage genome opening point. We extracted 55 SPβ-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resembled four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SPβ-like prophages consists of 38 proteins. The integration cassette proved to be not conserved even though present in all strains. It consists of distinct integrases. Analysis of SPβ transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system. Y1 - 2021 UR - https://www.biorxiv.org/content/10.1101/2021.11.22.469490v1 U6 - https://doi.org/10.1101/2021.11.22.469490 ER - TY - GEN A1 - Hertel, Robert A1 - Schöne, Kerstin A1 - Mittelstädt, Carolin A1 - Meißner, Janek A1 - Zschoche, Nick A1 - Collignon, Madeline A1 - Kohler, Christian A1 - Friedrich, Ines A1 - Schneider, Dominik A1 - Hoppert, Michael A1 - Kuhn, Ramona A1 - Schwedt, Inge A1 - Scholz, Patricia A1 - Poehlein, Anja A1 - Martienssen, Marion A1 - Ischebeck, Till A1 - Daniel, Rolf A1 - Commichau, Fabian M. T1 - Characterization of glyphosate-resistant Burkholderia anthina and Burkholderia cenocepacia isolates from a commercial Roundup® solution T2 - Environmental Microbiology Reports N2 - Roundup® is the brand name for herbicide solutions containing glyphosate, which specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase of the shikimate pathway. The inhibition of the EPSP synthase causes plant death because EPSP is required for biosynthesis of aromatic amino acids. Glyphosate also inhibits the growth of archaea, bacteria, Apicomplexa, algae and fungi possessing an EPSP synthase. Here, we have characterized two glyphosate-resistant bacteria from a Roundup solution. Taxonomic classification revealed that the isolates 1CH1 and 2CH1 are Burkholderia anthina and Burkholderia cenocepacia strains respectively. Both isolates cannot utilize glyphosate as a source of phosphorus and synthesize glyphosate-sensitive EPSP synthase variants. Burkholderia. anthina 1CH1 and B. cenocepacia 2CH1 tolerate high levels of glyphosate because the herbicide is not taken up by the bacteria. Previously, it has been observed that the exposure of soil bacteria to herbicides like glyphosate promotes the development of antibiotic resistances. Antibiotic sensitivity testing revealed that the only the B. cenocepacia 2CH1 isolate showed increased resistance to a variety of antibiotics. Thus, the adaptation of B. anthina 1CH1 and B. cenocepacia 2CH1 to glyphosate did not generally increase the antibiotic resistance of both bacteria. However, our study confirms the genomic adaptability of bacteria belonging to the genus Burkholderia. Y1 - 2022 U6 - https://doi.org/10.1111/1758-2229.13022 SN - 1758-2229 VL - 14 IS - 1 SP - 70 EP - 84 ER - TY - GEN A1 - Schwedt, Inge A1 - Collignon, Madeline A1 - Mittelstädt, Carolin A1 - Giudici, Florian A1 - Rapp, Johanna A1 - Meißner, Janek A1 - Link, Hannes A1 - Hertel, Robert A1 - Commichau, Fabian M. T1 - Genomic adaptation of Burkholderia anthina to glyphosate uncovers a novel herbicide resistance mechanism T2 - Environmental Microbiology Reports N2 - Glyphosate (GS) specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that converts phosphoenolpyruvate (PEP) and shikimate-3-phosphate to EPSP in the shikimate pathway of bacteria and other organisms. The inhibition of the EPSP synthase depletes the cell of the EPSP-derived aromatic amino acids as well as of folate and quinones. A variety of mechanisms (e.g., EPSP synthase modification) has been described that confer GS resistance to bacteria. Here, we show that the Burkholderia anthina strain DSM 16086 quickly evolves GS resistance by the acquisition of mutations in the ppsR gene. ppsR codes for the pyruvate/ortho-Pi dikinase PpsR that physically interacts and regulates the activity of the PEP synthetase PpsA. The mutational inactivation of ppsR causes an increase in the cellular PEP concentration, thereby abolishing the inhibition of the EPSP synthase by GS that competes with PEP for binding to the enzyme. Since the overexpression of the Escherichia coli ppsA gene in Bacillus subtilis and E. coli did not increase GS resistance in these organisms, the mutational inactivation of the ppsR gene resulting in PpsA overactivity is a GS resistance mechanism that is probably unique to B. anthina. Y1 - 2023 U6 - https://doi.org/10.1111/1758-2229.13184 SN - 1758-2229 VL - 15 IS - 6 SP - 727 EP - 739 ER - TY - GEN A1 - Schwedt, Inge A1 - Schöne, Kerstin A1 - Eckert, Maike A1 - Pizzinato, Manon A1 - Winkler, Laura A1 - Knotkova, Barbora A1 - Richts, Björn A1 - Hau, Jann‐Louis A1 - Steuber, Julia A1 - Mireles, Raul A1 - Noda‐Garcia, Lianet A1 - Fritz, Günter A1 - Mittelstädt, Carolin A1 - Hertel, Robert A1 - Commichau, Fabian M. T1 - The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates T2 - Environmental Microbiology N2 - Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that is required for aromatic amino acid, folate and quinone biosynthesis in Bacillus subtilis and Escherichia coli. The inhibition of the EPSP synthase by GS depletes the cell of these metabolites, resulting in cell death. Here, we show that like the laboratory B. subtilis strains also environmental and undomesticated isolates adapt to GS by reducing herbicide uptake. Although B. subtilis possesses a GS-insensitive EPSP synthase, the enzyme is strongly inhibited by GS in the native environment. Moreover, the B. subtilis EPSP synthase mutant was only viable in rich medium containing menaquinone, indicating that the bacteria require a catalytically efficient EPSP synthase under nutrient-poor conditions. The dependency of B. subtilis on the EPSP synthase probably limits its evolvability. In contrast, E. coli rapidly acquires GS resistance by target modification. However, the evolution of a GS-resistant EPSP synthase under non-selective growth conditions indicates that GS resistance causes fitness costs. Therefore, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability. This study also revealed that the uptake systems for folate precursors, phenylalanine and tyrosine need to be identified and characterized in B. subtilis. Y1 - 2023 U6 - https://doi.org/10.1111/1462-2920.16518 SN - 1462-2912 SN - 1462-2920 VL - 25 IS - 12 SP - 3604 EP - 3622 ER -