Revealing second messenger functions in bacterial stress response, cell differentiation and natural product biosynthesis

Project Information

SecMessFunctions

Grant agreement ID: 101039556

DOI

10.3030/101039556

EC signature date 7 February 2022

Start date 1 July 2022

End date 30 June 2027

Funded under

• European Research Council (ERC)

Total cost

€ 1 478 373,00

EU contribution

€ 1 478 373,00

Coordinated by

GOTTFRIED WILHELM LEIBNIZ UNIVERSITAET HANNOVER
Germany

Periodic Reporting for period 1 - SecMessFunctions (Revealing second messenger functions in bacterial stress response, cell differentiation and natural product biosynthesis)

Reporting period: 2022-07-01 to 2024-12-31

Summary of the context and overall objectives of the project

The key to bacterial life in almost every single niche on our planet is their versatile signalling systems. The current knowledge about the nucleotide-based signalling pathways and functions in bacteria is mainly based on studies on bacteria belonging to Proteobacteria and Firmicutes. Recent dynamics in the signalling research suggest that this is only a tip of an iceberg of the diversity, complexity and beauty of nucleotide-based signalling networks that bacteria use to sense and respond to their environment. The ubiquitous Actinobacteria are metabolically, physiologically and morphologically the most diverse group of bacteria in nature. The phylum includes deadly pathogens, such as Mycobacteria and the gifted Streptomyces that provide us with the majority of antibiotics used in clinic today.
The nucleotide second messenger cyclic dimeric adenosine monophosphate (c-di-AMP) is a key regulator of osmotic stress responses and plays an important role in adaption of environmental bacteria to rainfall and drought. How bacteria remodel their cell wall in order to survive stress signalled by c-di-AMP and how the dinucleotide controls cell differentiation remains poorly understood. The main goal of the project SecMessFunctions is to uncover new principles and pathways in second messenger-based signal transduction by exploiting a powerful model system with a unique developmental biology and a rich secondary metabolism. We use a combination of genetic, molecular and microscopy-based approaches to understand the following questions:
1. How are cell wall remodelling processes linked to c-di-AMP-mediated stress adaptation?
2. Which direct c-di-AMP effectors are involved in Streptomyces’ signalling cascades?
3. Can we establish c-di-AMP as a tool for activation of natural product biosynthesis in Streptomyces

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the first reporting period, we have discovered that decoration of the cell wall with glycopolymers and c-di-AMP signalling are physiologically linked. We have shown that deletion of the glycopolymer ligase CglA characterised by an LCP-LytR_C domain architecture suppresses the osmosensitive phenotype of the disA mutant that contains low levels of c-di-AMP. Our quantitative analysis of the cell wall glycopolymer content revealed that CglA acts as a glycopolymer ligase and deletion of the gene results in reduced amount of glycopolymers in the cell wall fraction. In addition, we found that deletion of cglA has a dramatic effect on Streptomyces development. The cglA mutant forms widened hyphae leading to failures in FtsZ-ring formation and positioning which in turn causes defects in cell septa placement and results in deformed spores with reduced vitality. We started to address the role of putative c-di-AMP-dependent riboswitches controlling expression of cell wall hydrolases in linking c-di-AMP signalling and cell wall stability and performed a screen for c-di-AMP effectors using a capture compound. Finally, while we could not detect a strong effect of c-di-AMP modulation on antibiotic production, we found that c-di-GMP is a highly potent tool for activation of antibiotic biosynthesis and leads to massive increase of chloramphenicol titre in S. venezuelae.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Despite the immense value of Streptomyces in biotechnology and medicine as antibiotic-producers, we know very little about their cell wall biogenesis, composition and functions. In SecMessFunctions, we have identified and characterized CglA as a novel component of cell wall biogenesis in Streptomyces, which is required for cell shape maintenance and cellular vitality in filamentous, multicellular bacteria. Our study on the function of CglA clearly advanced our knowledge about the role of cell wall glycopolymers in filamentous bacteria beyond the state-of-the art and set the stage for many exciting follow-up questions. We demonstrate for the first time, that the cell wall glycopolymer profile is important for the maintenance of healthy cell shape, for both, vegetative filaments and spores in Streptomycetes. We also provide mechanistic insights into the question, why cell shape is affected when the glycopolymer content is disturbed due to cglA deletion and show that division-competent FtsZ-rings cannot assemble and localize properly, due to increased width of hyphae in the cglA mutant. We also demonstrate for the first time, that the cell wall glycopolymer ligase CglA localizes explicitly in zones of growth, i.e. at tips and branching points in Streptomyces and thus is important for coordination of cell growth.