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

AbstractLevels 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 σWhiG. 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 5’pGpG. 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 ΔcdgB, ΔcdgC, ΔrmdA and ΔrmdB 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.

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Diguanylate cyclase and phosphodiesterase interact to maintain the specificity of c-di-GMP signaling in the regulation of antibiotic synthesis in Lysobacter enzymogenes

Applied and Environmental Microbiology ◽

10.1128/aem.01895-21 ◽

2021 ◽

Author(s):

Gaoge Xu ◽

Lichuan Zhou ◽

Guoliang Qian ◽

Fengquan Liu

Keyword(s):

Direct Evidence ◽

Antibiotic Production ◽

Indirect Evidence ◽

Second Messenger ◽

Antibiotic Biosynthesis ◽

Diguanylate Cyclase ◽

Signaling Specificity ◽

Lysobacter Enzymogenes ◽

Subsequent Investigation ◽

Diguanylate Cyclases

Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. The large number of c-di-GMP-related diguanylate cyclases (DGCs), phosphodiesterases (PDEs) and effectors are responsible for the complexity and dynamics of c-di-GMP signaling. Some of these components deploy various methods to avoid undesired crosstalk to maintain signaling specificity. Synthesis of the antibiotic HSAF ( H eat S table A ntifungal F actor) in Lysobacter enzymogenes is regulated by a specific c-di-GMP signaling pathway that includes a PDE LchP and a c-di-GMP effector Clp (also a transcriptional regulator). In the present study, from among 19 DGCs, we identified a diguanylate cyclase, LchD, which participates in this pathway. Subsequent investigation indicates that LchD and LchP physically interact and that the catalytic center of LchD is required for both the formation of the LchD-LchP complex and HSAF production. All the detected phenotypes support that LchD and LchP dispaly local c-di-GMP signaling to regulate HSAF biosynthesis. Although direct evidence is lacking, our investigation, which shows that the interaction between a DGC and a PDE maintains the specificity of c-di-GMP signaling, suggests the possibility of the existence of local c-di-GMP pools in bacteria. Importance Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. Signaling of c-di-GMP is complex and dynamic, and it is mediated by a large number of components, including c-di-GMP synthases (diguanylate cyclases. DGCs), c-di-GMP degrading enzymes (phosphodiesterases, PDEs), and c-di-GMP effectors. These components deploy various methods to avoid undesired crosstalk to maintain signaling specificity. In the present study, we identified a DGC that interacted with a PDE to specifically regulate antibiotic biosynthesis in L. enzymogenes . We provide direct evidence to show that the DGC and PDE form a complex, and also indirect evidence to argue that they may balance a local c-di-GMP pool to control the antibiotic production. The results represent an important finding regarding the mechanism of a pair of DGC and PDE to control the expression of specific c-di-GMP signaling pathways.

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The Bacterial Second Messenger cdiGMP Exhibits Promising Activity as a Mucosal Adjuvant

Clinical and Vaccine Immunology ◽

10.1128/cvi.00119-07 ◽

2007 ◽

Vol 14 (8) ◽

pp. 952-958 ◽

Cited By ~ 48

Author(s):

Thomas Ebensen ◽

Kai Schulze ◽

Peggy Riese ◽

Michael Morr ◽

Carlos A. Guzmán

Keyword(s):

Cytotoxic T Lymphocyte ◽

Second Messenger ◽

Secretory Iga ◽

Specific Lysis ◽

Mucosal Adjuvant ◽

Promising Tool ◽

Iga Production ◽

Cellular Immune ◽

Lymphocyte Responses

ABSTRACT The development of mucosal adjuvants is still a critical need in vaccinology. In the present work, we show that bis(3′,5′)-cyclic dimeric GMP (cdiGMP), a second messenger that modulates cell surface properties of several microorganisms, exerts potent activity as a mucosal adjuvant. BALB/c mice were immunized intranasally with the model antigen β-galactosidase (β-Gal) coadministered with cdiGMP. Animals receiving cdiGMP as an adjuvant showed significantly higher anti-β-Gal immunoglobulin G (IgG) titers in sera than controls (i.e., 512-fold [P < 0.05]). Coadministration of cdiGMP also stimulated efficient β-Gal-specific secretory IgA production in the lung (P < 0.016) and vagina (P < 0.036). Cellular immune responses were observed in response to both the β-Gal protein and a peptide encompassing its major histocompatibility complex class I-restricted epitope. The IgG1-to-IgG2a ratio of anti-β-Gal antibodies and the observed profiles of secreted cytokines suggest that a dominant Th1 response pattern is promoted by mucosal coadministration of cdiGMP. Finally, the use of cdiGMP as a mucosal adjuvant also led to the stimulation of in vivo cytotoxic T-lymphocyte responses in C57BL/6 mice intranasally immunized with ovalbumin and cdiGMP (up to 30% of specific lysis). The results obtained indicate that cdiGMP is a promising tool for the development of mucosal vaccines.

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Identification and Characterization of Four c-di-GMP-Metabolizing Enzymes from Streptomyces ghanaensis ATCC14672 Involved in the Regulation of Morphogenesis and Moenomycin A Biosynthesis

Microorganisms ◽

10.3390/microorganisms9020284 ◽

2021 ◽

Vol 9 (2) ◽

pp. 284 ◽

Cited By ~ 1

Author(s):

Desirèe Nuzzo ◽

Roman Makitrynskyy ◽

Olga Tsypik ◽

Andreas Bechthold

Keyword(s):

Antibiotic Production ◽

Functional Characterization ◽

Morphological Development ◽

Metabolizing Enzymes ◽

Regulatory Cascade ◽

Diguanylate Cyclases ◽

Streptomyces Ghanaensis ◽

Moenomycin A

Diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) are essential enzymes deputed to maintain the intracellular homeostasis of the second messenger cyclic dimeric (3′→5′) GMP (c-di-GMP). Recently, c-di-GMP has emerged as a crucial molecule for the streptomycetes life cycle, governing both morphogenesis and secondary metabolite production. Indeed, in Streptomyces ghanaensis ATCC14672 c-di-GMP was shown to be involved in the regulatory cascade of the peptidoglycan glycosytransferases inhibitor moenomycin A (MmA) biosynthesis. Here, we report the role of four c-di-GMP-metabolizing enzymes on MmA biosynthesis as well as morphological progression in S. ghanaensis. Functional characterization revealed that RmdAgh and CdgAgh are two active PDEs, while CdgEgh is a DGC. In vivo, overexpression of rmdAgh and cdgAgh led to precocious sporulation, whereas overexpression of cdgEgh and cdgDgh (encoding a predicted DGC) caused an arrest of morphological development. Furthermore, we demonstrated that individual deletion of rmdAgh, cdgAgh, and cdgDgh enhances MmA accumulation, whereas deletion of cdgEgh has no impact on antibiotic production. Conversely, an individual deletion of each studied gene does not affect morphogenesis. Altogether, our results show that manipulation of c-di-GMP-metabolizing enzymes represent a useful approach to improving MmA production titers in S. ghanaensis.

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