Biology subjects

Stegmann, E.

Publications and source records attributed to Stegmann, E..

4 recordsLinked to original sources

bioRxiv2026

Conserved cis and trans communication domains mediate module interaction in glycopeptide antibiotic NRPS assembly lines

Non-ribosomal peptide synthetases (NRPSs) assemble structurally complex and clinically important natural products, yet the mechanisms coordinating communication between multiple enzymes remain incompletely understood. Here, we dissected the NRPS system underlying biosynthesis of the glycopeptide antibiotic (GPA) balhimycin in Amycolatopsis balhimycina to elucidate principles of inter-enzyme communication. Genetic perturbation of short terminal structural elements markedly reduced balhimycin production, demonstrating their critical role in biosynthesis. AlphaFold3 predictions identified these elements as trans-COM domains mediating specific NRPS interactions, primarily through hydrophobic contacts. Quantitative binding studies using microscale thermophoresis confirmed the importance of the trans-COM domains for multiprotein interaction, extending current models of NRPS communication. Comparative structural analysis further uncovered a conserved class of cis-COM domains within condensation domains across GPA NRPS assembly lines. Together, our findings establish a unified model in which trans- and cis-interfaces cooperatively maintain assembly line fidelity, redefining NRPS architecture and enabling rational engineering strategies.

microbiology↗

bioRxiv2025

Identification of the negamycin split biosynthetic gene cluster in Kitasatospora purpeofusca ATCC21470

Negamycin is a ribosome-targeting antibiotic with activity against Gram-positive and Gram-negative bacteria including ESKAPE pathogens. Furthermore, it promotes premature stop codon readthrough. Its therapeutic potential is limited by low natural production and synthetic complexity. To enable scalable biosynthesis, we identified and characterized its genetic basis in Kitasatospora purpeofusca ATCC 21470. Two distant chromosomal regions, neg1 and neg2, were found to be essential. Deletion of neg1, involved in nitrite provision for N-N bond formation, reduced production to [~]10%, while deletion of neg2, which directs {beta}-lysine generation and scaffold assembly, abolished it completely. Isotope-labeling experiments confirmed nitrite incorporation. Transcriptomic and proteomic analyses further supported the involvement of both regions. The heterologous expression of neg1 along with the neg2 region in Streptomyces albidoflavus reconstituted negamycin biosynthesis, confirming the unusual involvement of two distant gene clusters in the biosynthesis, and provides a foundation for biotechnological production and further development of this promising antibiotic. SIGNIFICANCEThe rapid rise of antimicrobial resistance (AMR), particularly among Gram-negative ESKAPE pathogens, represents one of the most urgent global health threats. Despite this, the discovery and development of new antibiotics have stagnated. Addressing this challenge requires the exploration of natural products with novel mechanisms of action, alongside the development of scalable production strategies. Negamycin has emerged as a compelling candidate in this regard, characterized by an unusual mechanism of action and therapeutic potential extending beyond traditional antibacterial use. However, its development has been constrained by low production yields in the native producer. In this study, we identify and characterize the biosynthetic genes responsible for negamycin production, providing a foundation for pathway engineering, yield optimization, and the rational design of new analogs.

microbiology↗

bioRxiv2023

Phylogenetic distance and structural diversity directing a reclassification of glycopeptide antibiotics

Glycopeptide antibiotics (GPAs) are key agents against multidrug-resistant Gram-positive pathogens, yet both the term "glycopeptide" and the current GPA type I-V classification framework have become increasingly strained as structurally and mechanistically divergent members continue to be discovered. In particular, compounds historically grouped as "type V GPAs" differ from classical GPAs in features such as glycosylation, peptide length, and reported mode of action, raising the question of whether they belong to the same natural product class. Here, a curated dataset of GPA-associated biosynthetic gene clusters (BGCs) is analysed by combining fingerprint similarity of the products with phylogenetic analysis of the BGCs. Fingerprint-based structural similarity networks and BGC similarity comparisons reveal a pronounced separation between classical lipid II-binding GPAs (types I-IV) and type V GPAs. Multi-locus phylogenetic analyses of conserved biosynthetic components further support two deeply divergent evolutionary subclasses, consistent with subclass-specific biosynthetic signatures. Together, these results motivate a revised, unambiguous framework in which the broader class is termed xyclopeptides, comprising the subclasses dalabactins (legacy GPA types I-IV) and murobactins (legacy type V).

genetics↗

bioRxiv2020

Mechanism of BceAB-type transporter: Resistance by lipid II flipping

Treatment of bacterial infections are the great challenge of our era due to the evolved resistance mechanisms against antibiotics. The Achilles heel of bacteria is the cell wall especially during the needs of its synthesis and cell division. Here lipid II is an essential cell wall precursor component synthesized in the cytosol and flipped into the outer leaflet of the membrane prior to its incorporation into the cell wall. Compounds targeting the cell wall or its biosynthesis precursors have been around for decades and have been used as antibiotics against bacterial infections like meningitis, pneumonia and endocarditis. Antimicrobial peptides (AMPs) have proven to be a promising weapon against multiresistant bacteria. However, the Bacitracin efflux (BceAB)-type ATP binding cassette transporters expressed in the membrane of human pathogenic bacteria have been shown to confer resistance to these alternative antibiotics, thereby hampering their medical development. In Streptococcus agalactiae COH1 the BceAB-type transporter NsrFP (SaNsrFP) confers high-level resistance against the antimicrobial peptide nisin, a member of the lantibiotic subfamily. We showed that SaNsrFP provides a novel resistance mechanism by flipping lipid II back into the cytosol, thereby preventing the binding of nisin as well as other lipid II targeting compounds. This is intriguing since a relatively simple reaction mediates resistance to human pathogenic bacteria to lipid II targeting antibiotics, regardless of their structure. Significance StatementThe ABC-transporter NsrFP from Streptococcus agalactiae (SaNsrFP) belongs to the BceAB-type transporters. Several BceAB-type transporters are known to confer resistance against multiple antimicrobial peptides. In this study a new resistance mechanism was identified, which is based on the reduction of the number of cell wall precursor lipid II molecules on the cell surface mediated by SaNsrFP. SaNsrFP flips lipid II, which are considered to be the target for many antibiotics, back into the cytoplasm. With this newly gained knowledge about the resistance mechanism of BceAB-type transporters, novel strategies can be established to overcome or bypass this resistance in human pathogenic bacteria.

microbiology↗