@misc{MichalikRederRichtsetal., author = {Michalik, Stephan and Reder, Alexander and Richts, Bj{\"o}rn and Faßhauer, Patrick and M{\"a}der, Ulrike and Pedreira, Tiago and Poehlein, Anja and Heel, Auke van and Tilburg, Amanda van and Altenbuchner, Josef and Klewing, Anika and Reuß, Daniel and Daniel, Rolf and Commichau, Fabian M. and Kuipers, Oscar and Hamoen, Leendert and V{\"o}lker, Uwe and St{\"u}lke, J{\"o}rg}, title = {The Bacillus subtilis Minimal Genome Compendium}, series = {ACS Synthetic Biology}, volume = {10}, journal = {ACS Synthetic Biology}, number = {10}, issn = {2161-5063}, doi = {10.1021/acssynbio.1c00339}, pages = {2767 -- 2771}, abstract = {To better understand cellular life, it is essential to decipher the contribution of individual components and their interactions. Minimal genomes are an important tool to investigate these interactions. Here, we provide a database of 105 fully annotated genomes of a series of strains with sequential deletion steps of the industrially relevant model bacterium Bacillus subtilis starting with the laboratory wild type strain B. subtilis 168 and ending with B. subtilis PG38, which lacks approximately 40\% of the original genome. The annotation is supported by sequencing of key intermediate strains as well as integration of literature knowledge for the annotation of the deletion scars and their potential effects. The strain compendium presented here represents a comprehensive genome library of the entire MiniBacillus project. This resource will facilitate the more effective application of the different strains in basic science as well as in biotechnology.}, language = {en} } @misc{KohmFloccariLutzetal., author = {Kohm, Katharina and Floccari, Valentina and Lutz, Veronika and Nordmann, Birthe and Mittelst{\"a}dt, Carolin and Poehlein, Anja and Dragos, Anna and Commichau, Fabian M. and Hertel, Robert}, title = {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}, series = {bioRxiv beta}, journal = {bioRxiv beta}, doi = {10.1101/2021.11.22.469490}, pages = {44}, abstract = {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.}, language = {en} } @misc{HertelGibhardtMartienssenetal., author = {Hertel, Robert and Gibhardt, Johannes and Martienssen, Marion and Kuhn, Ramona and Commichau, Fabian M.}, title = {Molecular mechanisms underlying glyphosate resistance in bacteria}, series = {Environmental Microbiology}, volume = {23}, journal = {Environmental Microbiology}, number = {6}, issn = {1462-2920}, doi = {10.1111/1462-2920.15534}, pages = {2891 -- 2905}, abstract = {Glyphosate is a nonselective herbicide that kills weeds and other plants competing with crops. Glyphosate specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, thereby depleting the cell of EPSP serving as a precursor for biosynthesis of aromatic amino acids. Glyphosate is considered to be toxicologically safe for animals and humans. Therefore, it became the most-important herbicide in agriculture. However, its intensive application in agriculture is a serious environmental issue because it may negatively affect the biodiversity. A few years after the discovery of the mode of action of glyphosate, it has been observed that bacteria evolve glyphosate resistance by acquiring mutations in the EPSP synthase gene, rendering the encoded enzyme less sensitive to the herbicide. The identification of glyphosate-resistant EPSP synthase variants paved the way for engineering crops tolerating increased amounts of the herbicide. This review intends to summarize the molecular mechanisms underlying glyphosate resistance in bacteria. Bacteria can evolve glyphosate resistance by (i) reducing glyphosate sensitivity or elevating production of the EPSP synthase, by (ii) degrading or (iii) detoxifying glyphosate and by (iv) decreasing the uptake or increasing the export of the herbicide. The variety of glyphosate resistance mechanisms illustrates the adaptability of bacteria to anthropogenic substances due to genomic alterations.}, language = {en} } @misc{HertelSchoeneMittelstaedtetal., author = {Hertel, Robert and Sch{\"o}ne, Kerstin and Mittelst{\"a}dt, Carolin and Meißner, Janek and Zschoche, Nick and Collignon, Madeline and Kohler, Christian and Friedrich, Ines and Schneider, Dominik and Hoppert, Michael and Kuhn, Ramona and Schwedt, Inge and Scholz, Patricia and Poehlein, Anja and Martienssen, Marion and Ischebeck, Till and Daniel, Rolf and Commichau, Fabian M.}, title = {Characterization of glyphosate-resistant Burkholderia anthina and Burkholderia cenocepacia isolates from a commercial Roundup® solution}, series = {Environmental Microbiology Reports}, volume = {14}, journal = {Environmental Microbiology Reports}, number = {1}, issn = {1758-2229}, doi = {10.1111/1758-2229.13022}, pages = {70 -- 84}, abstract = {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.}, language = {en} } @misc{WangWampGibhardtetal., author = {Wang, Mengyi and Wamp, Sabrina and Gibhardt, Johannes and Holland, Gudrun and Schwedt, Inge and Schmidtke, Kai-Uwe and Scheibner, Katrin and Halbedel, Sven and Commichau, Fabian M.}, title = {Adaptation of Listeria monocytogenes to perturbation of c-di-AMP metabolism underpins its role in osmoadaptation and identifies a fosfomycin uptake system}, series = {Environmental microbiology}, volume = {24}, journal = {Environmental microbiology}, number = {9}, issn = {1462-2920}, doi = {10.1111/1462-2920.16084}, pages = {4466 -- 4488}, abstract = {The human pathogen Listeria monocytogenes synthesizes and degrades c-di-AMP using the diadenylate cyclase CdaA and the phosphodiesterases PdeA and PgpH respectively. c-di-AMP is essential because it prevents the uncontrolled uptake of osmolytes. Here, we studied the phenotypes of cdaA, pdeA, pgpH and pdeA pgpH mutants with defects in c-di-AMP metabolism and characterized suppressor mutants restoring their growth defects. The characterization of the pdeA pgpH mutant revealed that the bacteria show growth defects in defined medium, a phenotype that is invariably suppressed by mutations in cdaA. The previously reported growth defect of the cdaA mutant in rich medium is suppressed by mutations that osmotically stabilize the c-di-AMP-free strain. We also found that the cdaA mutant has an increased sensitivity against isoleucine. The isoleucine-dependent growth inhibition of the cdaA mutant is suppressed by codY mutations that likely reduce the DNA-binding activity of encoded CodY variants. Moreover, the characterization of the cdaA suppressor mutants revealed that the Opp oligopeptide transport system is involved in the uptake of the antibiotic fosfomycin. In conclusion, the suppressor analysis corroborates a key function of c-di-AMP in controlling osmolyte homeostasis in L. monocytogenes.}, language = {en} } @misc{SchwedtCollignonMittelstaedtetal., author = {Schwedt, Inge and Collignon, Madeline and Mittelst{\"a}dt, Carolin and Giudici, Florian and Rapp, Johanna and Meißner, Janek and Link, Hannes and Hertel, Robert and Commichau, Fabian M.}, title = {Genomic adaptation of Burkholderia anthina to glyphosate uncovers a novel herbicide resistance mechanism}, series = {Environmental Microbiology Reports}, volume = {15}, journal = {Environmental Microbiology Reports}, number = {6}, issn = {1758-2229}, doi = {10.1111/1758-2229.13184}, pages = {727 -- 739}, abstract = {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.}, language = {en} } @misc{SchwedtSchoeneEckertetal., author = {Schwedt, Inge and Sch{\"o}ne, Kerstin and Eckert, Maike and Pizzinato, Manon and Winkler, Laura and Knotkova, Barbora and Richts, Bj{\"o}rn and Hau, Jann-Louis and Steuber, Julia and Mireles, Raul and Noda-Garcia, Lianet and Fritz, G{\"u}nter and Mittelst{\"a}dt, Carolin and Hertel, Robert and Commichau, Fabian M.}, title = {The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates}, series = {Environmental Microbiology}, volume = {25}, journal = {Environmental Microbiology}, number = {12}, issn = {1462-2912}, doi = {10.1111/1462-2920.16518}, pages = {3604 -- 3622}, abstract = {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.}, language = {en} } @misc{RiedelCommichauBenndorfetal., author = {Riedel, Ramona and Commichau, Fabian M. and Benndorf, Dirk and Martienssen, Marion}, title = {Biologischer Abbau von Aminophosphonaten}, series = {BIOspektrum : Das Magazin f{\"u}r Biowissenschaften}, volume = {30}, journal = {BIOspektrum : Das Magazin f{\"u}r Biowissenschaften}, issn = {1868-6249}, doi = {10.1007/s12268-024-2179-9}, pages = {348 -- 350}, abstract = {Application of commercial aminophosphonates such as glyphosate (GS) or chelating agents in detergent are daily used at large scale worldwide. Only little is known about the potential biodegradation of the latter. Here we describe the characterization of the new strain Ochrobactrum sp. BTU1 isolated from a LC/MS standard solution. The strain is capable to degrade all investigated aminophosphonates including GS.}, language = {de} } @misc{RiedelCommichauBenndorfetal., author = {Riedel, Ramona and Commichau, Fabian M. and Benndorf, Dirk and Hertel, Robert and Holzer, Katharina and Mardoukhi, Mohammad Saba Yousef and Noack, Laura and Martienssen, Marion}, title = {Biodegradation of selected aminophosphonates by the bacterial isolate Ochrobactrum sp. BTU1}, series = {Microbial Research}, volume = {280}, journal = {Microbial Research}, issn = {0944-5013}, doi = {10.1016/j.micres.2024.127600}, pages = {1 -- 12}, abstract = {Aminophosphonates, like glyphosate (GS) or metal chelators such as ethylenediaminetetra(methylenephosphonic acid) (EDTMP), are released on a large scale worldwide. Here, we have characterized a bacterial strain capable of degrading synthetic aminophosphonates. The strain was isolated from LC/MS standard solution. Genome sequencing indicated that the strain belongs to the genus Ochrobactrum. Whole-genome classification using pyANI software to compute a pairwise ANI and other metrics between Brucella assemblies and Ochrobactrum contigs revealed that the bacterial strain is designated as Ochrobactrum sp. BTU1. Degradation batch tests with Ochrobactrum sp. BTU1 and the selected aminophosphonates GS, EDTMP, aminomethylphosphonic acid (AMPA), iminodi(methylene-phosphonic) (IDMP) and ethylaminobis(methylenephosphonic) acid (EABMP) showed that the strain can use all phosphonates as sole phosphorus source during phosphorus starvation. The highest growth rate was achieved with AMPA, while EDTMP and GS were least supportive for growth. Proteome analysis revealed that GS degradation is promoted by C-P lyase via the sarcosine pathway, i.e., initial cleavage at the C-P bond. We also identified C-P lyase to be responsible for degradation of EDTMP, EABMP, IDMP and AMPA. However, the identification of the metabolite ethylenediaminetri(methylenephosphonic acid) via LC/MS analysis in the test medium during EDTMP degradation indicates a different initial cleavage step as compared to GS. For EDTMP, it is evident that the initial cleavage occurs at the C-N bond. The detection of different key enzymes at regulated levels, form the bacterial proteoms during EDTMP exposure, further supports this finding.}, language = {en} }