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

2021 ◽  
Vol 9 (2) ◽  
pp. 284 ◽  
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.

scholarly journals Role of Phosphopantetheinyl Transferase Genes in Antibiotic Production by Streptomyces coelicolor

2008 ◽  
Vol 190 (20) ◽  
pp. 6903-6908 ◽  
Author(s):  
Ya-Wen Lu ◽  
Adrianna K. San Roman ◽  
Amy M. Gehring
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Morphological Development ◽  
Phosphopantetheinyl Transferase ◽  
Calcium Dependent ◽  
Actinorhodin Production

ABSTRACT The phosphopantetheinyl transferase genes SCO5883 (redU) and SCO6673 were disrupted in Streptomyces coelicolor. The redU mutants did not synthesize undecylprodigiosin, while SCO6673 mutants failed to produce calcium-dependent antibiotic. Neither gene was essential for actinorhodin production or morphological development in S. coelicolor, although their mutation could influence these processes.

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

2020 ◽  
Author(s):  
Julian Haist ◽  
Sara Alina Neumann ◽  
Mahmoud M Al-Bassam ◽  
Sandra Lindenberg ◽  
Marie A Elliot ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Second Messenger ◽  
Natural Product Synthesis ◽  
Regulatory Role ◽  
Adverse Conditions ◽  
Promising Tool ◽  
Diguanylate Cyclases ◽  
Genome Wide ◽  
Protein Variants

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.

scholarly journals Functional Characterization of a New Member of the Cdk9 Family in Aspergillus nidulans

2010 ◽  
Vol 9 (12) ◽  
pp. 1901-1912 ◽  
Author(s):  
Friederike Bathe ◽  
Claudia Kempf ◽  
Stephen A. Osmani ◽  
Aysha H. Osmani ◽  
Sabrina Hettinger ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Transcription Elongation ◽  
Functional Characterization ◽  
Asexual Development ◽  
Full Spectrum ◽  
Content Type ◽  
Essential Components

ABSTRACT Cdk9-like kinases in complex with T-type cyclins are essential components of the eukaryotic transcription elongation machinery. The full spectrum of Cdk9/cyclin T targets, as well as the specific consequences of phosphorylations, is still largely undefined. We identify and characterize here a Cdk9 kinase (PtkA) in the filamentous ascomycete Aspergillus nidulans. Deletion of ptkA had a lethal effect in later stages of vegetative growth and completely impeded asexual development. Overexpression of ptkA affected directionality of polarized growth and the initiation of new branching sites. A green fluorescent protein-tagged PtkA version localized inside the nucleus during interphase, supporting a role of PtkA in transcription elongation, as observed in other organisms. We also identified a putative cyclin T homolog, PchA, in the A. nidulans genome and confirmed its interaction with PtkA in vivo. Surprisingly, the Pcl-like cyclin PclA, previously described to be involved in asexual development, was also found to interact with PtkA, indicating a possible role of PtkA in linking transcriptional activity with development and/or morphogenesis in A. nidulans. This is the first report of a Cdk9 kinase interacting with a Pcl-like cyclin, revealing interesting new aspects about the involvement of this Cdk-subfamily in differential gene expression.

scholarly journals The Heat Shock Repressor HspR Directly Controls Avermectin Production, Morphological Development, and H 2 O 2 Stress Response in Streptomyces avermitilis

2021 ◽  
Author(s):  
Xiaorui Lu ◽  
Qian Wang ◽  
Mengyao Yang ◽  
Zhi Chen ◽  
Jilun Li ◽  
...  
Keyword(s):  
Heat Shock ◽  
Stress Responses ◽  
Cellular Response ◽  
Antibiotic Production ◽  
Streptomyces Avermitilis ◽  
Morphological Development ◽  
Biological Processes ◽  
Transcriptional Repressors ◽  
Important Species

Heat shock response (HSR) is a universal cellular response that promotes survival following temperature increase. In filamentous Streptomyces , which account for ∼70% commercial antibiotic production, HSR is regulated by transcriptional repressors; in particular, the widespread MerR-family regulator HspR has been identified as a key repressor. However, functions of HspR in other biological processes are unknown. The present study demonstrates that HspR pleiotropically controls avermectin production, morphological development, and heat shock and H 2 O 2 stress responses in industrially important species S. avermitilis . HspR directly activated ave structural genes ( aveA1 , aveA2 ) and H 2 O 2 stress-related genes ( katA1 , catR , katA3 , oxyR , ahpC , ahpD ), whereas it directly repressed heat shock genes (HSGs) ( dnaK1-grpE1-dnaJ1-hspR operon, clpB1p , clpB2p , lonAp ) and developmental genes ( wblB , ssgY , ftsH ). HspR interacted with PhoP (response regulator of the widespread PhoPR two-component system) at dnaK1p to co-repress the important dnaK1-grpE1-dnaJ1-hspR operon. PhoP exclusively repressed target HSGs ( htpG , hsp18_1 , hsp18_2 ) different from those of HspR ( clpB1p , clpB2p , lonAp ). A consensus HspR-binding site, 5′-TTGANBBNNHNNNDSTSHN-3′, was identified within HspR target promoter regions, allowing prediction of the HspR regulon involved in broad cellular functions. Taken together, our findings demonstrate a key role of HspR in coordination of a variety of important biological processes in Streptomyces species. IMPORTANCE Our findings are significant to clarify the molecular mechanisms underlying HspR function in Streptomyces antibiotic production, development, and H 2 O 2 stress responses through direct control of its target genes associated with these biological processes. HspR homologs described to date function as transcriptional repressors, but not as activators. Results of the present study demonstrate that HspR acts as a dual repressor/activator. PhoP was shown to crosstalk with HspR at dnaK1p to co-regulate HSR and have its exclusive target HSGs. The novel role of PhoP in HSR further demonstrates the importance of this regulator in Streptomyces . Overexpression of hspR strongly enhanced avermectin production in S. avermitilis wild-type and industrial strains. These findings provide new insights into the regulatory roles and mechanisms of HspR and PhoP, and facilitate methods for antibiotic overproduction in Streptomyces species.

scholarly journals Functional Characterization of Membrane Sialyltransferase Activity of Human Platelets

1977 ◽  
Author(s):  
K. K. Wu ◽  
C. Ku ◽  
C. Smith
Keyword(s):  
Enzyme Activity ◽  
Functional Characterization ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Transferase Activity ◽  
Platelet Surface

To evaluate the role of membrane sialyltransferase in the initiation of platelet aggregation, we studied the stimulatory effect of epinephrine and adenosine diphosphate and the inhibitory effect of aspirin on the platelet surface sialyl transferase activity. The enzyme activity was assayed under optimal conditions as determined previously. The assay mixture consisted of intact washed human platelets, CMP-14C-sialic acid, desialated fetuin, Mn2+ and buffer to a final volume of 1 ml. The enzyme activity was enhanced to 172% of control by 1μH, 152% by 5μM and 146% by 10μM epinephrine. Adenosine diphosphate enhanced the enzyme activity to a lesser extent:103% at 1μM and 113% at 5μM. In contrast, aspirin inhibited the enzyme activity to 46% of control when 10μg/ml of aspirin was used. Higher concentrations of aspirin failed to cause further inhibition. In the in-vivo experiment, 600 mg aspirin was given to normal subjects and the surface enzyme activity was determined 12 hours later. The enzyme activity reduced to 43% following aspirin administration. Furthermore, we studied the enzyme activity in a patient with “aspirin-like” release disorder. While the mean surface enzyme activity of 10 normal subjects was 1.56 + 0.21 (S. D.) pmole-hr-1 per 108 platelets, the enzyme activity of the patient was only 0.91 pmole.hr-l. The results strongly suggest that the membrane sialyltransferase plays an important part in the initiation of platelet release reaction.

scholarly journals Role of GntR Family Regulatory Gene SCO1678 in Gluconate Metabolism in Streptomyces coelicolor M145

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Olga Tsypik ◽  
Roman Makitrynskyy ◽  
Agnieszka Bera ◽  
Lijiang Song ◽  
Wolfgang Wohlleben ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Functional Characterization ◽  
Bioinformatic Analysis ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Putative Operon

Here we report functional characterization of the Streptomyces coelicolor M145 gene SCO1678, which encodes a GntR-like regulator of the FadR subfamily. Bioinformatic analysis suggested that SCO1678 is part of putative operon (gnt) involved in gluconate metabolism. Combining the results of SCO1678 knockout, transcriptional analysis of gnt operon, and Sco1678 protein-DNA electromobility shift assays, we established that Sco1678 protein controls the gluconate operon. It does so via repression of its transcription from a single promoter located between genes SCO1678 and SCO1679. The knockout also influenced, in a medium-dependent manner, the production of secondary metabolites by S. coelicolor. In comparison to the wild type, on gluconate-containing minimal medium, the SCO1678 mutant produced much less actinorhodin and accumulated a yellow-colored pigment, likely to be the cryptic polyketide coelimycin. Possible links between gluconate metabolism and antibiotic production are discussed.

scholarly journals An Inducible and Secreted Eukaryote-Like Serine/Threonine Kinase of Salmonella enterica Serovar Typhi Promotes Intracellular Survival and Pathogenesis

2014 ◽  
Vol 83 (2) ◽  
pp. 522-533 ◽  
Author(s):  
Nagaraja Theeya ◽  
Atri Ta ◽  
Sayan Das ◽  
Rahul S. Mandal ◽  
Oishee Chakrabarti ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Functional Characterization ◽  
Systemic Infection ◽  
Serine Threonine Kinase ◽  
Content Type ◽  
Threonine Kinase ◽  
Universal Substrate

Eukaryote-like serine/threonine kinases (eSTKs) constitute an important family of bacterial virulence factors. Genome analysis had predicted putative eSTKs inSalmonella entericaserovar Typhi, although their functional characterization and the elucidation of their role in pathogenesis are still awaited. We show here that the primary sequence and secondary structure of thet4519locus ofSalmonellaTyphi Ty2 have all the signatures of eukaryotic superfamily kinases.t4519encodes a ∼39-kDa protein (T4519), which shows serine/threonine kinase activitiesin vitro. Recombinant T4519 (rT4519) is autophosphorylated and phosphorylates the universal substrate myelin basic protein. Infection of macrophages results in decreased viability of the mutant (Ty2Δt4519) strain, which is reversed by gene complementation. Moreover, reactive oxygen species produced by the macrophages signal to the bacteria to induce T4519, which is translocated to the host cell cytoplasm. That T4519 may target a host substrate(s) is further supported by the activation of host cellular signaling pathways and the induction of cytokines/chemokines. Finally, the role of T4519 in the pathogenesis ofSalmonellaTyphi is underscored by the significantly decreased mortality of mice infected with the Ty2Δt4519strain and the fact that the competitive index of this strain for causing systemic infection is 0.25% that of the wild-type strain. This study characterizes the first eSTK ofSalmonellaTyphi and demonstrates its role in promoting phagosomal survival of the bacteria within macrophages, which is a key determinant of pathogenesis. This, to the best of our knowledge, is the first study to describe the essential role of eSTKs in thein vivopathogenesis ofSalmonellaspp.

scholarly journals Identification and characterization of the Streptomyces globisporus 1912 regulatory gene lndYR that affects sporulation and antibiotic production

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1240-1249 ◽  
Author(s):  
Bohdan Ostash ◽  
Yuriy Rebets ◽  
Maksym Myronovskyy ◽  
Olga Tsypik ◽  
Iryna Ostash ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Functional Characterization ◽  
Signal Features ◽  
Abc Transporter Genes ◽  
Identification And Characterization

Here, we report the identification and functional characterization of the Streptomyces globisporus 1912 gene lndYR, which encodes a GntR-like regulator of the YtrA subfamily. Disruption of lndYR arrested sporulation and antibiotic production in S. globisporus. The results of in vivo and in vitro studies revealed that the ABC transporter genes lndW–lndW2 are targets of LndYR repressive action. In Streptomyces coelicolor M145, lndYR overexpression caused a significant increase in the amount of extracellular actinorhodin. We suggest that lndYR controls the transcription of transport system genes in response to an as-yet-unidentified signal. Features that distinguish lndYR-based regulation from other known regulators are discussed.

scholarly journals miR-1272 Exerts Tumor-Suppressive Functions in Prostate Cancer via HIP1 Suppression

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 435
Author(s):  
Federica Rotundo ◽  
Denis Cominetti ◽  
Rihan El Bezawy ◽  
Stefano Percio ◽  
Valentina Doldi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Functional Characterization ◽  
Developed Countries ◽  
Membrane Turnover ◽  
Normal Tissues ◽  
Treat Prostate Cancer

The development of novel therapies or the improvement of currently used approaches to treat prostate cancer (PCa), the most frequently diagnosed male tumor in developed countries, is an urgent need. In this regard, the functional characterization of microRNAs, molecules shown to regulate a number of cancer-related pathways, is instrumental to their possible clinical exploitation. Here, we demonstrate the tumor-suppressive role of the so far uncharacterized miR-1272, which we found to be significantly down-modulated in PCa clinical specimens compared to normal tissues. Through a gain-of-function approach using miRNA mimics, we showed that miR-1272 supplementation in two PCa cell models (DU145 and 22Rv1) reverted the mesenchymal phenotype by affecting migratory and invasive properties, and reduced cell growth in vitro and in vivo in SCID mice. Additionally, by targeting HIP1 encoding the endocytic protein HIP1, miR-1272 balanced EGFR membrane turnover, thus affecting the downstream AKT/ERK pathways, and, ultimately, increasing PCa cell response to ionizing radiation. Overall, our results show that miR-1272 reconstitution can affect several tumor traits, thus suggesting this approach as a potential novel therapeutic strategy to be pursued for PCa, with the multiple aim of reducing tumor growth, enhancing response to radiotherapy and limiting metastatic dissemination.

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