Biology subjects

Tschowri, N.

Publications and source records attributed to Tschowri, N..

4 recordsLinked to original sources

bioRxiv2025

Spatiotemporal Dynamics of the Dihydrolipoyl Dehydrogenase LpdA Determine the Intrinsic Autofluorescence in Filamentous Actinobacteria

The hyphal nature of filamentous streptomycetes poses unique challenges to their multicellular lifestyle, since it requires organizing cellular functions at the scale of hundreds of micrometers length. Streptomycetes exhibit a strong and patchy autofluorescence of so far unknown origin in their hyphae that - as we demonstrate - is a natural property of filamentous actinobacteria. The foci are dynamic and evenly distributed throughout the hyphae, including the hyphal tips, where they are cell membrane-associated. Here, we resolved the high spatiotemporal dynamics of these foci during spore germination and vegetative growth in Streptomyces venezuelae. We isolated a fluorescent protein band and identified the responsible protein as dihydrolipoyl dehydrogenase LpdA. An lpdA deletion mutant lacked these fluorescent foci and showed minor deficiencies in growth and development. Heterologous LpdA production in E. coli and characterization of the enzyme verified that a flavin cofactor is responsible for the green autofluorescence. LpdA is highly conserved in actinomycetes as a part of multienzyme complexes involved in central metabolism. Such delocalized metabolic centers provide a potential solution to mycelial multicellular lifestyle, where diffusion becomes a major challenge.

microbiology↗

bioRxiv2021

BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP

The widespread bacterial second messenger bis-(3-5)-cyclic diguanosine monophosphate (c-di-GMP) is an important regulator of biofilm formation, virulence and cell differentiation. C-di-GMP-specific biosensors that allow detection and visualization of c-di-GMP levels in living cells are key to our understanding of how c-di-GMP fluctuations drive cellular responses. Here, we describe a novel c-di-GMP biosensor, CensYBL, that is based on c-di-GMP-induced dimerization of the effector protein BldD from Streptomyces resulting in bimolecular fluorescence complementation of split-YPet fusion proteins. As a proof-of-principle, we demonstrate that CensYBL is functional in detecting fluctuations in intracellular c-di-GMP levels in the Gram-negative model bacteria Escherichia coli and Salmonella enterica serovar Typhimurium. Using deletion mutants of c-di-GMP diguanylate cyclases and phosphodiesterases, we show that c-di-GMP dependent dimerization of CBldD-YPet results in fluorescence complementation reflecting intracellular c-di-GMP levels. Overall, we demonstrate that the CensYBL biosensor is a user-friendly and versatile tool that allows to investigate c-di-GMP variations using single-cell and population-wide experimental set-ups. ImportanceThe second messenger c-di-GMP controls various bacterial functions including development of resistant biofilm communities and transition into dormant spores. In vivo detection of c-di-GMP levels is therefore crucial for a better understanding of how intracellular c-di-GMP levels induce changes of bacterial physiology. Here, we describe the design of a novel c-di-GMP biosensor and demonstrate its effective application in investigating fluctuations in intracellular c-di-GMP levels in Escherichia coli and Salmonella enterica serovar Typhimurium on a population-based and single-cell level.

microbiology↗

bioRxiv2020

Specialized and shared functions of diguanylate cyclases and phosphodiesterases in Streptomyces development.

Levels of the second messenger bis-3-5-cyclic di-guanosinemonophosphate (c-di-GMP) determine when Streptomyces initiate sporulation to survive under adverse conditions. c-di-GMP signals are integrated into the genetic differentiation network by the regulator BldD and the sigma factor {sigma}WhiG. However, functions of the development-specific c-di-GMP diguanylate cyclases (DGCs) CdgB and CdgC, and the phosphodiesterases (PDEs) RmdA and RmdB, are poorly understood. Here, we provide biochemical evidence that the GGDEF-EAL domain protein RmdB from S. venezuelae is a monofunctional PDE that hydrolyzes c-di-GMP to 5pGpG. Despite having an equivalent GGDEF-EAL domain arrangement, RmdA cleaves c-di-GMP to GMP and exhibits residual DGC activity. We show that an intact EAL motif is crucial for the in vivo function of both enzymes since strains expressing protein variants with an AAA motif instead of EAL are delayed in development, similar to null mutants. Global transcriptome analysis of {Delta}cdgB, {Delta}cdgC, {Delta}rmdA and {Delta}rmdB strains revealed that the c-di-GMP specified by these enzymes has a global regulatory role, with about 20 % of all S. venezuelae genes being differentially expressed in the cdgC mutant. Our data suggest that the major c-di-GMP-controlled targets determining the timing and mode of sporulation are genes involved cell division and the production of the hydrophobic sheath that covers Streptomyces aerial hyphae and spores. Altogether, this study provides a global view of the c-di-GMP-dependent genes that contribute to the hyphae-to-spores transition and sheds light on the shared and specific functions of the key enzymes involved in c-di-GMP metabolism in S. venezuelae. ImportanceStreptomyces are important producers of clinical antibiotics. The ability to synthesize these natural products is connected to their developmental biology, which includes a transition from filamentous cells to spores. The widespread bacterial second messenger c-di-GMP controls this complex switch and is a promising tool to improve antibiotic production. Here, we analyzed the enzymes that make and break c-di-GMP in S. venezuelae by studying the genome-wide transcriptional effects of the DGCs CdgB and CdgC and the PDEs RmdA and RmdB. We found that the c-di-GMP specified by these enzymes has a global regulatory role. However, despite shared enzymatic activities, the four c-di-GMP enzymes have specialized inputs into differentiation. Altogether, we demonstrate that altering c-di-GMP levels through the action of selected enzymes yields characteristically distinct transcriptional profiles; this can be an important consideration when modulating c-di-GMP for the purposes of natural product synthesis in Streptomyces.

microbiology↗

bioRxiv2019

c-di-AMP hydrolysis by a novel type of phosphodiesterase promotes differentiation of multicellular bacteria

Antibiotic-producing Streptomyces use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP but the mechanism for terminating c-di-AMP signaling and the proteins that bind the molecule to effect signal transduction are unknown. Here, we identify the AtaC protein as a new type of c-di-AMP-specific phosphodiesterase that is also conserved in pathogens such as Streptococcus pneumoniae and Mycobacterium tuberculosis. AtaC is monomeric in solution and binds Mn2+ to specifically hydrolyze c-di-AMP to AMP via the intermediate 5-pApA. As an effector of c-di-AMP signaling, we characterize the RCK-domain protein CpeA as the first c-di-AMP-binding protein to be identified in Streptomyces. CpeA interacts with the predicted cation / proton antiporter, CpeB, linking c-di-AMP signaling to ion homeostasis in actinobacteria. Hydrolysis of c-di-AMP is critical for normal growth and differentiation in Streptomyces, connecting osmotic stress to development. Thus, we present the discovery of two novel components of c-di-AMP signaling in bacteria and show that precise control of this second messenger is essential for osmoregulation and coordinated development in Streptomyces.

microbiology↗