Biology subjects

Kepplinger, B.

Publications and source records attributed to Kepplinger, B..

4 recordsLinked to original sources

bioRxiv2024

Roles of PknB and CslA in cell wall morphogenesis of Streptomyces

The bacterial cell wall is essential for maintaining cellular integrity and defining the mode of growth, with different species adopting distinct strategies for cell wall synthesis and remodelling. Streptomyces are filamentous bacteria predominantly found in soil and renowned for producing specialised metabolites, including antibiotics. They grow through tip extension and branching hyphal filaments, forming a multicellular mycelium. New branches are established by forming a new growth zone on the lateral cell wall. Proteins involved in this process are organised into complexes called polarisomes, with DivIVA being the most well-characterised component. To investigate the tip growth requirements in Streptomyces albus we developed a genetic screen utilising toxic DivIVA overproduction and searched for suppressors of its lethality, reasoning that such suppressors would likely encode components functionally linked to DivIVA or the tip growth machinery. Among the identified genes was pknB, encoding a serine/threonine protein kinase implicated in the regulation of cell growth and morphogenesis. We confirmed that deletion of pknB restored the growth phenotype of S. albus following DivIVA overproduction. The phosphoproteome analysis revealed that the absence of PknB alters the phosphorylation state of CslA, a cellulose synthase-like protein. We demonstrate that a phosphoablative mutant of CslA impairs {beta}-glucan synthesis and causes hypersensitivity to lysozyme. Overproduction of CslA restored colony growth defects arising from DivIVA-induced hyperbranching, without however suppressing the hyperbranching phenotype. These findings collectively identify PknB-dependent phosphorylation of CslA as a central regulatory point in Streptomyces cell envelope construction, revealing how modulation of {beta}-glucan synthesis can mitigate the cellular consequences of DivIVA dysregulation.

microbiology↗

bioRxiv2023

The interplay between the polar growth determinant DivIVA, the segregation protein ParA and their novel interaction partner PapM controls the Mycobacterium smegmatis cell cycle by modulation of DivIVA subcellular distribution

Bacterial chromosome segregation is facilitated by the ParABS system. The ParB protein binds centromere-like parS sequences and forms nucleoprotein complexes. These nucleoprotein complexes are segregated by the dynamic ATPase ParA. In mycobacteria, ParA also interacts with the polar growth determinant DivIVA (Wag31). This interaction was earlier shown to facilitate the segregation of ParB complexes but also to affect cell extension. Here, we identify an additional partner of ParA in Mycobacterium smegmatis, named PapM. Using E. coli based analysis, we show that PapM likewise interacts with DivIVA and that the tripartite interaction of ParA-PapM-DivIVA is phosphorylation-dependent: ParA binding to DivIVA is diminished, while PapM binding is promoted upon phosphorylation of DivIVA. The presence of PapM enhances the dissociation of ParA from the DivIVA complex upon its phosphorylation. Studies of M. smegmatis mutant strains reveal that altered PapM levels influence chromosome segregation and cell length. The elimination of PapM affects ParA dynamics. Further, ParA and, to a lesser extent, PapM modulates the subcellular distribution of DivIVA. Altogether our studies show that the tripartite interplay between ParA-DivIVA and PapM controls the switch between cell division and cell elongation and in this way affects the mycobacterial cell cycle. ImportanceThe genus of Mycobacteria includes important clinical pathogens (M. tuberculosis). Bacteria of this genus share the unusual features of their cell cycle such as asymmetric polar cell elongation and long generation time. Markedly, control of the mycobacterial cell cycle still remains not fully understood. The main cell growth determinant in Mycobacteria is an essential protein DivIVA, which is also involved in cell division. DivIVA activity is controlled by phosphorylation but the mechanism and significance of this process is unknown. Here, we show how the previously established protein interaction partner of DivIVA in Mycobacteria, the segregation protein ParA, affects the DivIVA subcellular distribution. We also demonstrate the role of a newly identified M. smegmatis DivIVA and ParA interaction partner, a protein named PapM, and we establish how their interactions are modulated by phosphorylation. Demonstrating that the tripartite interplay affects the mycobacterial cell cycle contributes to general understanding of mycobacterial growth regulation.

microbiology↗

bioRxiv2022

Mircubactin B rescues the lethal effect of cell wall biosynthesis mutations in Bacillus subtilis.

Growth of most rod-shaped bacteria is accompanied by the insertion of new peptidoglycan into the cylindrical cell wall. This insertion, which helps maintain and determine the shape of the cell, is guided by a complex protein machinery called the rod complex or elongasome. Although most of the proteins in this complex are essential under normal growth conditions, cell viability can be rescued, for reasons that are not understood, by the presence of a high (mM) Mg2+ concentration. We screened for natural product compounds that could rescue the growth of mutants affected in rod-complex function. By screening >2,000 extracts from a diverse collection of actinobacteria, we identified a new compound, mirubactin B, related to the known iron siderophore mirubactin A, which rescued growth in the low micromolar range, and this activity was confirmed by synthesising mirubactin B. The compound also displayed toxicity at higher concentrations, and this effect appears related to iron homeostasis. However, several lines of evidence suggest that the mirubactin B rescuing activity is not due simply to iron sequestration. The results demonstrate a novel antibacterial compound and add to growing evidence that bacterial siderophores have a range of activities beyond simple iron sequestration.

microbiology↗

bioRxiv2022

Systematic whole-genome sequencing reveals an unexpected diversity among actinomycetoma pathogens and provides insights into their antibacterial susceptibilities

Mycetoma is a neglected tropical chronic granulomatous inflammatory disease of the skin and subcutaneous tissues. More than 70 species with a broad taxonomic diversity have been implicated as agents of mycetoma. Understanding the full range of causative organisms and their antibiotic sensitivity profiles are essential for the appropriate treatment of infections. The present study focuses on the analysis of full genome sequences and antibiotic resistance profiles of actinomycetoma strains from patients seen at the Mycetoma Research Centre in Sudan with a view to developing rapid diagnostic tests. Seventeen pathogenic isolates obtained by surgical biopsies were sequenced using MinION and Illumina methods, and their antibiotic resistance profiles determined. The results highlight an unexpected diversity of actinomycetoma causing pathogens, including three Streptomyces isolates assigned to species not previously associated with human actinomycetoma and one new Streptomyces species. Thus, current approaches for clinical and histopathological classification of mycetoma may need to be updated. The standard treatment for actinomycetoma is a combination of sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Most tested isolates were not susceptible to sulfamethoxazole/trimethoprim or to amoxicillin alone. However, the addition of the {beta}-lactamase inhibitor clavulanic acid to amoxicillin increased susceptibility, particularly for Streptomyces somaliensis and Streptomyces sudanensis. Actinomadura madurae isolates appear to be particularly resistant under laboratory conditions, suggesting that alternative agents, such as amikacin, should be considered for more effective treatment. The results obtained will inform future diagnostic methods for the identification of actinomycetoma and treatment. Author SummaryMycetoma is a common health and medical problem that is endemic in many tropical and subtropical countries and has devastating effects on patients. The destructive nature of late-stage infection means that treatment often requires long term use of antibiotic therapy, massive surgical excisions and amputation. Several different bacterial species have been described as causing this disease but our understanding of the true diversity of mycetoma causing bacteria has been limited by a lack of molecular sequence data. We have now sequenced the genomes of 17 samples isolated from patients at the Mycetoma Research Centre in Sudan, revealing a diverse range of species associated with infection including one new Streptomyces species, and three species with no previous association with human mycetoma. Crucially, all isolates had a high level of resistance against the current first-line antibiotics used to treat actinomycetoma under laboratory conditions. This resistance was strongest in Actinomadura madurae, which was also the most frequently observed species isolated from patients in our study. We hope that these results will aid in the development of future rapid diagnostic tools and the improvement of treatment outcomes.

genomics↗