Browsing by Person "Commichau, Fabian M."

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Adaptation of model organisms and environmental bacilli to glyphosate gives insight to species-specific peculiarities of the shikimate pathway
(2024) Schwedt, Inge; Commichau, Fabian M.
Glyphosate (GS), the active ingredient of the popular herbicide Roundup, inhibits the 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase of the shikimate pathway, which is present in archaea, bacteria, Apicomplexa, algae, fungi, and plants. In these organisms, the shikimate pathway is essential for de novo synthesis of aromatic amino acids, folates, quinones and other metabolites. Therefore, the GS-dependent inhibition of the EPSP synthase results in cell death. Previously, it has been observed that isolates of the soil bacteria Burkholderia anthina and Burkholderia cenocepacia are resistant to high amounts of GS. In the framework of this PhD thesis, it could be demonstrated that B. anthina isolates are not intrinsically resistant to GS. However, B. anthina rapidly adapts to the herbicide at the genome level and the characterization of GS-resistant suppressor mutants led to the discovery of a novel GS resistance mechanism. In B. anthina, the acquisition of loss-of-function mutations in the ppsR gene increases GS resistance. The ppsR gene encodes a regulator of the phosphoenolpyruvate (PEP) synthetase PpsA. In the absence of a functional PpsR protein, the bacteria synthesize more PEP, which competes with GS for binding in the active site of the EPSP synthase, increasing GS resistance. The EPSP synthase in B. anthina probably does not allow changes in the amino acid sequence as it is the case in other organisms. Indeed, the Gram-negative model organism Escherichia coli evolves GS resistance by the acquisition of mutations that either reduce the sensitivity of the EPSP synthase or increase the cellular concentration of the enzyme. Unlike E. coli, the EPSP synthase is also critical for the viability of Gram-positive model bacterium Bacillus subtilis. This observation is surprising because the enzyme belongs to the class of GS-insensitive EPSP synthases. In fact, the EPSP synthase is essential for growth of B. subtilis. The determination of the nutritional requirements allowing the growth of B. subtilis and E. coli mutants lacking EPSP synthase activity revealed that the demand for shikimate pathway intermediates is higher in the former organism. This finding explains why laboratory as well as environmental Bacilli exclusively adapt to GS by the mutational inactivation of glutamate transporter genes. Here, it was also shown that a B. subtilis mutant lacking EPSP synthase activity grows in minimal medium only when additional mutations accumulate in genes involved in the regulation of aerobic/anaerobic metabolism and central carbon metabolism. The characterization of these additional mutants will help to elucidate the peculiarities of the shikimate pathway in B. subtilis. Moreover, the mutants could be useful to identify the aromatic amino acid transporters that still await their discovery.
The Bacillus phage SPβ : a model system to study the lysis-lysogeny regulatory network and antiphage defense systems
(2024) Kohm, Katharina; Commichau, Fabian M.
Although bacteriophages are considered the most abundant biological entities on our planet, they are less well-studied compared to their host. Being intracellular parasites, phages rely on the metabolic processes of their bacterial hosts for their replication. Phages that use the host exclusively to produce virions are called virulent phages and the reproduction cycle is called the lytic cycle. The lytic cycle is accompanied by lysis and, thus, the killing of the host cell. Temperate phages can choose between the virulent or lysogenic lifecycle. Lysogeny or the lysogenic cycle is a type of viral reproduction in which no virus particles are produced, instead, the genetic material of the phage is replicated and then passed on to the daughter cells. The viral genome can be present as part of the bacterial chromosome or as a circular or linear plasmid molecule and is referred to as a prophage. Since temperate phages can influence the mutual interactions with other bacteria, growth, metabolic pathways or pathogenicity of their host, it is important to understand how temperate phages control their lysogenic life cycle and which genes are involved. Repression usually occurs through the interaction between a repressor and specific binding sites, which are mostly located in the promoter regions of the lytic genes. SPβ is a temperate phage of the model bacterium Bacillus subtilis. In contrast to its host, many aspects of the life cycle of SPβ have been little studied and many genes have not been assigned a function. Not only are SPβ-like phages widespread within the genus Bacillus and of greater importance to their hosts than previously thought, but they also exhibit a novel lysogeny management system. With regard to the control and regulation of the lysis-lysogeny network, it is already partially known which gene products are involved in the decision, establishment and resolvement of lysogeny. The maintenance and resolvement of lysogeny of SPβ was investigated in more detail in this thesis. To gain more insight into the regulation and control of lysogeny, the SPβ c2 mutant was characterized in this work. This mutant is unable to maintain its lysogenic state when exposed to heat, suggesting the alteration of a key regulatory element. This work demonstrated that the SPβ c2 phenotype is due to a single nucleotide exchange in the mrpR (yopR) gene that renders the encoded MrpRG136E protein temperature-sensitive. Furthermore, it was shown that this protein acts as a repressor of lytic gene expression. This occurs through the binding of the repressor to several conserved elements in the genome of the SPβ prophage. Further biochemical analysis revealed that the G136E exchange makes MrpR less stable and reduces its affinity for DNA binding. Structural characterization of MrpR revealed that the protein is a DNA-binding protein with a similar protein fold to tyrosine recombinases. However, the repressor function is independent of functional recombinase activity. In addition, a mutagenesis approach was used to identify the region within the protein that is essential for the function of the repressor. This work also identified further players in the lysogeny management system, with the YosL protein being crucial for the induction of the lytic cycle. However, YosL cannot activate the lytic cycle of SPβ alone. In addition, the core genome of SPβ-like phages was defined and new integration loci were identified in this work. Apart from a better understanding of lysis-lysogeny regulation and phagehost relationships, the characterization of the SPβ c2 mutant also led to the identification of a previously unknown phage defense system. The defense system is encoded on a plasmid and leads to a decrease in phage titer and a change in plaque morphology. It could be shown that the spbB locus, which ensures the segregation stability of the plasmid and codes for two open reading frames, is also responsible for the resistance to SPβ c2 and related phages. Further studies have shown that the spbB gene and the downstream region, which presumably encodes an RNA element and a terminator, play a crucial role in mediating resistance. The second open reading frame of the spbB locus is irrelevant for the mediation of phage resistance. Overall, this work contributes to a better understanding of the phage-host relationship.
Genomic adaptation of Burkholderia anthina to glyphosate uncovers a novel herbicide resistance mechanism
(2023) Schwedt, Inge; Collignon, Madeline; Mittelstädt, Carolin; Giudici, Florian; Rapp, Johanna; Meißner, Janek; Link, Hannes; Hertel, Robert; Commichau, Fabian M.
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.
The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates
(2023) Schwedt, Inge; Schöne, Kerstin; Eckert, Maike; Pizzinato, Manon; Winkler, Laura; Knotkova, Barbora; Richts, Björn; Hau, Jann-Louis; Steuber, Julia; Mireles, Raul; Noda‐Garcia, Lianet; Fritz, Günter; Mittelstädt, Carolin; Hertel, Robert; Commichau, Fabian M.
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.
The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates
(2023) Schwedt, Inge; Schöne, Kerstin; Eckert, Maike; Pizzinato, Manon; Winkler, Laura; Knotkova, Barbora; Richts, Björn; Hau, Jann-Louis; Steuber, Julia; Mireles, Raul; Noda‐Garcia, Lianet; Fritz, Günter; Mittelstädt, Carolin; Hertel, Robert; Commichau, Fabian M.
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.
Metabolic rewiring enables ammonium assimilation via a non‐canonical fumarate‐based pathway
(2024) Mardoukhi, Mohammad Saba Yousef; Rapp, Johanna; Irisarri, Iker; Gunka, Katrin; Link, Hannes; Marienhagen, Jan; de Vries, Jan; Stülke, Jörg; Commichau, Fabian M.
Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, in the presence of arginine, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle.