scholarly journals The Phosin PptA Plays a Negative Role in the Regulation of Antibiotic Production in Streptomyces lividans

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 325
Author(s):  
Noriyasu Shikura ◽  
Emmanuelle Darbon ◽  
Catherine Esnault ◽  
Ariane Deniset-Besseau ◽  
Delin Xu ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Lividans ◽  
Positive Control ◽  
Phosphate Limitation ◽  
Antibiotic Biosynthesis ◽  
Nudix Hydrolase ◽  
Energetic Stress ◽  
Accessory Factor ◽  
Negative Role ◽  
Weak Expression

In Streptomyces, antibiotic biosynthesis is triggered in phosphate limitation that is usually correlated with energetic stress. Polyphosphates constitute an important reservoir of phosphate and energy and a better understanding of their role in the regulation of antibiotic biosynthesis is of crucial importance. We previously characterized a gene, SLI_4384/ppk, encoding a polyphosphate kinase, whose disruption greatly enhanced the weak antibiotic production of Streptomyces lividans. In the condition of energetic stress, Ppk utilizes polyP as phosphate and energy donor, to generate ATP from ADP. In this paper, we established that ppk is co-transcribed with its two downstream genes, SLI_4383, encoding a phosin called PptA possessing a CHAD domain constituting a polyphosphate binding module and SLI_4382 encoding a nudix hydrolase. The expression of the ppk/pptA/SLI_4382 operon was shown to be under the positive control of the two-component system PhoR/PhoP and thus mainly expressed in condition of phosphate limitation. However, pptA and SLI_4382 can also be transcribed alone from their own promoter. The deletion of pptA resulted into earlier and stronger actinorhodin production and lower lipid content than the disruption of ppk, whereas the deletion of SLI_4382 had no obvious phenotypical consequences. The disruption of ppk was shown to have a polar effect on the expression of pptA, suggesting that the phenotype of the ppk mutant might be linked, at least in part, to the weak expression of pptA in this strain. Interestingly, the expression of phoR/phoP and that of the genes of the pho regulon involved in phosphate supply or saving were strongly up-regulated in pptA and ppk mutants, revealing that both mutants suffer from phosphate stress. Considering the presence of a polyphosphate binding module in PptA, but absence of similarities between PptA and known exo-polyphosphatases, we proposed that PptA constitutes an accessory factor for exopolyphosphatases or general phosphatases involved in the degradation of polyphosphates into phosphate.

scholarly journals Engineering of Primary Carbon Metabolism for Improved Antibiotic Production in Streptomyces lividans

2002 ◽  
Vol 68 (10) ◽  
pp. 4731-4739 ◽  
Author(s):  
Michael J. Butler ◽  
Per Bruheim ◽  
Srdjan Jovetic ◽  
Flavia Marinelli ◽  
Pieter W. Postma ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Lividans ◽  
Pentose Phosphate ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild ◽  
Commercially Important ◽  
Biochemical Product ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Wild Type Control

ABSTRACT Deletions were made in Streptomyces lividans in either of two genes (zwf1 and zwf2) encoding isozymes of glucose-6-phosphate dehydrogenase, the first enzyme in the oxidative pentose phosphate pathway (PPP). Each mutation reduced the level of Zwf activity to approximately one-half that observed in the wild-type strain. When the mutants were transformed with multicopy plasmids carrying the pathway-specific transcriptional activator genes for either the actinorhodin (ACT) or undecylprodigiosin (RED) biosynthetic pathway, they produced higher levels of antibiotic than the corresponding wild-type control strains. The presumed lower flux of carbon through the PPP in each of the Δzwf mutants may allow more efficient glucose utilization via glycolysis, resulting in higher levels of antibiotic production. This appears to occur without lowering the concentration of NADPH (the major biochemical product of the oxidative PPP activity) to a level that would limit antibiotic biosynthesis. Consistent with this hypothesis, deletion of the gene (devB) encoding the enzyme that catalyzes the next step in the oxidative PPP (6-phosphogluconolactonase) also resulted in increased antibiotic production. However, deletion of both zwf genes from the devB mutant resulted in reduced levels of ACT and RED production, suggesting that some of the NADPH made by the PPP is utilized, directly or indirectly, for antibiotic biosynthesis. Although applied here to the model antibiotics ACT and RED, such mutations may prove to be useful for improving the yield of commercially important secondary metabolites.

scholarly journals Impact of Phosphate Availability on Membrane Lipid Content of the Model Strains, Streptomyces lividans and Streptomyces coelicolor

2021 ◽  
Vol 12 ◽  
Author(s):  
Clara Lejeune ◽  
Sonia Abreu ◽  
Pierre Chaminade ◽  
Thierry Dulermo ◽  
Michelle David ◽  
...  
Keyword(s):  
Streptomyces Coelicolor ◽  
Streptomyces Lividans ◽  
Positive Control ◽  
Membrane Lipid ◽  
Phosphate Limitation ◽  
Phospholipid Content ◽  
Acyltransferase Activity ◽  
Over Expression ◽  
Phosphate Availability ◽  
Total Disappearance

In this issue we demonstrated that the phospholipid content of Streptomyces lividans varies greatly with Pi availability being was much lower in Pi limitation than in Pi proficiency whereas that of Streptomyces coelicolor varied little with Pi availability. In contrast the content in phosphate free ornithine lipids was enhanced in both strains in condition of phosphate limitation. Ornithine lipids biosynthesis starts with the N-acylation of ornithine to form lyso-ornithine that is then O-acylated to yield ornithine lipid. The operon sco1222-23 was proposed to be involved in the conversion of specific amino acids into ornithine in condition of phosphate limitation whereas the sco0921-20 operon encoding N- and O-acyltransferase, respectively, was shown to be involved in the biosynthesis of these lipids. The expression of these two operons was shown to be under the positive control of the two components system PhoR/PhoP and thus induced in phosphate limitation. The expression of phoR/phoP being weak in S. coelicolor, the poor expression of these operons resulted into a fivefold lower ornithine lipids content in this strain compared to S. lividans. In the deletion mutant of the sco0921-20 operon of S. lividans, lyso-ornithine and ornithine lipids were barely detectable and TAG content was enhanced. The complementation of this mutant by the sco0921-20 operon or by sco0920 alone restored ornithine lipids and TAG content to wild type level and was correlated with a twofold increase in the cardiolipin content. This suggested that SCO0920 bears, besides its broad O-acyltransferase activity, an N-acyltransferase activity and this was confirmed by the detection of lyso-ornithine in this strain. In contrast, the complementation of the mutant by sco0921 alone had no impact on ornithine lipids, TAG nor cardiolipin content but was correlated with a high lyso-ornithine content. This confirmed that SCO0921 is a strict N-acyltransferase. However, interestingly, the over-expression of the sco0921-20 operon or of sco0921 alone in S. coelicolor, led to an almost total disappearance of phosphatidylinositol that was correlated with an enhanced DAG and TAG content. This suggested that SCO0921 also acts as a phospholipase C, degrading phosphatidylinositol to indirectly supply of phosphate in condition of phosphate limitation.

Fresh approaches to antibiotic production

1980 ◽  
Vol 290 (1040) ◽  
pp. 313-328 ◽  
Keyword(s):  
Polyethylene Glycol ◽  
Plant Pathogens ◽  
Growth Promotion ◽  
Antibiotic Production ◽  
Strain Improvement ◽  
Antibiotic Biosynthesis ◽  
Yield Improvement ◽  
Acquired Drug Resistance ◽  
Intensive Farming ◽  
New Antibiotics

New antibiotics are needed, ( a ) to control diseases that are refractory to existing ones either because of intrinsic or acquired drug resistance of the pathogen or because inhibition of the disease is difficult, at present, without damaging the host (fungal and viral diseases, and tumours), ( b ) for the control of plant pathogens and of invertebrates such as helminths, insects, etc., and ( c ) for growth promotion in intensive farming. Numerous new antibiotics are still being obtained from wild microbes, especially actinomycetes. Chemical modification of existing compounds has also had notable success. Here we explore the uses, actual and potential, of genetics to generate new antibiotics and to satisfy the ever-present need to increase yield. Yield improvement has depended in the past on mutation and selection, combined with optimization of fermentation conditions. Progress would be greatly accelerated by screening random recombinants between divergent high-yielding strains. Strain improvement may also be possible by the introduction of extra copies of genes of which the products are rate-limiting, or of genes conferring beneficial growth characteristics. Although new antibiotics can be generated by mutation, either through disturbing known biosyntheses or by activating ‘silent’ genes, we see more promise in interspecific recombination between strains producing different secondary metabolites, generating producers of ‘hybrid’ antibiotics. As with proposals for yield improvement, there are two major strategies for obtaining interesting recombinants of this kind: random recombination between appropriate strains, or the deliberate movement of particular biosynthetic abilities between strains. The development of protoplast technology in actinomycetes, fungi and bacilli has been instrumental in bringing these idealized strategies to the horizon. Protoplasts of the same or different species can be induced to fuse by polyethylene glycol. At least in intraspecific fusion of streptomycetes, random and high frequency recombination follows. Protoplasts can also be used as recipients for isolated DNA, again in the presence of polyethylene glycol, so that the deliberate introduction of particular genes into production strains can be realistically envisaged. Various kinds of DNA cloning vectors are being developed to this end. Gene cloning techniques also offer rich possibilities for the analysis of the genetic control of antibiotic biosynthesis, knowledge of which is, at present, minimal. The information that should soon accrue can be expected to have profound effects on the application of genetics to industrial microbiology.

scholarly journals Genetic and physiological characterization of rpoB mutations that activate antibiotic production in Streptomyces lividans

Microbiology ◽  
2002 ◽  
Vol 148 (11) ◽  
pp. 3365-3373 ◽  
Author(s):  
Caixia Lai ◽  
Jun Xu ◽  
Yuzuru Tozawa ◽  
Yoshiko Okamoto-Hosoya ◽  
Xingsheng Yao ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Lividans ◽  
Physiological Characterization

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

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.

185 THE PORCINE EPIBLAST AND NOT THE INNER CELL MASS HAS DEVELOPED CONVENTIONAL PATHWAYS FOR REGULATION OF PLURIPOTENCY

2009 ◽  
Vol 21 (1) ◽  
pp. 191
Author(s):  
V. J. Hall ◽  
J. Christensen ◽  
P. Maddox-Hyttel
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Positive Control ◽  
Total Rna ◽  
Weak Expression ◽  
Whole Transcriptome

Pluripotency in mice and human embryonic stem cells is regulated by a number of transcription factors, notably including Oct-4, Sox-2, and Nanog. However, in the pig, previous research indicates that Oct-4 protein and mRNA is not specifically localized to the inner cell mass (ICM) of the zona-intact (ZI) blastocyst. Levels of expression of Nanog mRNA, on the other hand, appear to be low in the ZI blastocyst, and protein has not been detected. Similarly, Sox-2 expression in the ZI blastocyst is relatively low and not specific to the ICM. In this study, we investigated the mRNA expression of Oct-4, Sox-2, and Nanog in D6/D7-derived ZI porcine in vivo-derived blastocysts compared with epiblasts mechanically isolated from hatched D10/D11 in vivo-derived blastocysts. We then investigated components involved in pathways important for regulating pluripotency, including JAK/STAT (i.e. gp130, LIFr), FGF (i.e. bFGF, FGFr1, FGFr2), and BMP (bmp4, smad4) signaling pathways and their downstream targets, stat3, c-myc, c-fos, by using RT-PCR. Sows were artificially inseminated, and embryos were flushed from uteri following slaughter. Single D6/D7 blastocysts (n = 3), single mechanically isolated D10/D11 epiblasts (n = 3), endometrium, and oviduct total RNA was isolated using the RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Total RNA from the blastocysts and epiblasts was then amplified to form cDNA using the QuantiTect Whole Transcriptome kit (Qiagen). Positive control tissues (oviduct and endometrium) were reverse transcribed using the RevertAid First Strand cDNA synthesis kit (Fermentas, Burlington, Ontario, Canada). Primers were designed to span introns in highly homologous sequences to human mRNA. Primers were tested in both oviduct and endometrium tissue, and products were sequenced to confirm specificity. PCR was performed at 55°C for 35 cycles. Results indicate that D6/D7 blastocysts only expressed Oct-4 and not Nanog and Sox-2. In contrast, all 3 transcripts were expressed in D10/D11 epiblasts. The D10/D11 epiblasts also expressed LIFr, bFGF, FGFr1, FGFr2, bmp4, smad4, stat3, c-myc, and c-fos. The cytokine receptor gp130 was only weakly expressed in a single epiblast. In contrast, the earlier stage D6/D7 blastocysts failed to express these messengers with the exception of weak expression of gp130 in all 3 blastocysts, and only a single blastocyst expressed LIFr, smad4, c-myc, and c-fos. In conclusion, this study indicates that the ICM of the porcine D6/D7 ZI blastocyst has not developed pluripotency signaling as observed in mice and humans at this developmental stage. Furthermore, without expression of gp130, the JAK/STAT pathway is unlikely to play a role in regulating pluripotency in the epiblast. It is likely that the later stage epiblast may be more amenable for the derivation of porcine embryonic stem cells.

scholarly journals Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727

2015 ◽  
Vol 197 (15) ◽  
pp. 2536-2544 ◽  
Author(s):  
Letizia Lo Grasso ◽  
Sonia Maffioli ◽  
Margherita Sosio ◽  
Mervyn Bibb ◽  
Anna Maria Puglia ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Antibiotic Production ◽  
Regulatory Protein ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Mechanisms ◽  
Antibiotic Biosynthesis ◽  
Strain Atcc ◽  
Protein Coding ◽  
Content Type

ABSTRACTThe actinomyceteNonomuraeasp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by thedbvgene cluster, which contains 37 protein-coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation ofdbv6had no effect. In addition, overexpression ofdbv3led to higher levels of A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons,dbv14-dbv8anddbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4,dbv29,dbv36, anddbv37) and of six operons (dbv2-dbv1,dbv14-dbv8,dbv17-dbv15,dbv21-dbv20,dbv24-dbv28, anddbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription ofdbv4and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export, and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 directly regulates biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation.IMPORTANCEThis report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomyceteNonomuraeasp. strain ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of skin infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control two other similar glycopeptides (balhimycin and teicoplanin) have been elucidated, and beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.

The Leeuwenhoek Lecture, 1987 - Towards an understanding of gene switching in Streptomyces , the basis of sporulation and antibiotic production

1988 ◽  
Vol 235 (1279) ◽  
pp. 121-138 ◽  
Keyword(s):  
Life Cycle ◽  
Secondary Metabolites ◽  
Vegetative Growth ◽  
Antibiotic Production ◽  
Morphological Differentiation ◽  
Aerial Mycelium ◽  
Secondary Phase ◽  
Antibiotic Biosynthesis ◽  
Regulatory Cascade ◽  
Pharmacologically Active

Streptomycetes are soil bacteria that differ from the genetically well-known Escherichia coli in two striking characteristics. (1) Instead of consisting of an alternation of growth and fission of morphologically simple, undifferentiated rods, the streptomycete life cycle involves the formation of a system of elongated, branching hyphae which, after a period of vegetative growth, respond to specific signals by producing specialized spore-bearing structures. (2) The streptomycetes produce an unrivalled range of chemically diverse ‘secondary metabolites’, which we recognize as antibiotics, herbicides and pharmacologically active molecules, and which presumably play an important role in the streptomycete life cycle in nature. This ‘physiological’ differentiation is often tem­porally associated with the morphological differentiation of sporulation and there are common elements in the regulation of the two sets of processes. In the model system provided by Streptomyces coelicolor A3(2), the isolation of several whole clusters of linked antibiotic biosynthetic pathway genes, and some key regulatory genes involved in sporulation, has made it possible to study the basis for the switching on and off of particular sets of genes during morphological and ‘physiological’ differen­tiation. Genetic analysis clearly reveals a regulatory cascade operating at several levels in a ‘physiological’ branch of the differentiation control system. At the lowest level, within individual clusters of antibiotic biosynthesis genes are genes with a role as activators of the structural genes for the pathway enzymes, and also resistance genes. It is attractive to speculate that the latter play a dual role: protecting the organism from self-destruction by its own potentially lethal product, and forming an essential component of a regulatory circuit that activates the biosyn­thetic genes, thus ensuring that resistance is established before any antibiotic is made. A next higher level of regulation is revealed by the isolation of mutations in a gene ( afsB ) required for expression (probably at the level of transcription) of all five known secondary metabolic pathways in the organism. At a higher level still, the bldA gene, whose product seems to be a tRNA essential to translate the rare (in high [G + C] Streptomyces DNA) TTA leucine codon, controls or influences the whole gamut of morphological and ‘physiological’ differentiation, because bldA mutants fail to produce either secondary metabolites or aerial mycelium and spores, while growing normally in the vegetative phase. Thus a decision to switch from vegetative growth to the secondary phase of colonial development may be taken at the level of translation. In the ‘morphological’ branch of the proposed regulatory cascade, a key gene is whiG whose product, essential for the earliest known step in the metamorphosis of aerial hyphae into spore chains, appears to be an RNA polymerase sigma factor which is not needed for transcription of vegetative genes, but seems to control, at the level of transcription, the decision to sporulate.

scholarly journals The Product of a Developmental Gene, crgA, That Coordinates Reproductive Growth in Streptomyces Belongs to a Novel Family of Small Actinomycete-Specific Proteins

2003 ◽  
Vol 185 (22) ◽  
pp. 6678-6685 ◽  
Author(s):  
Ricardo Del Sol ◽  
Andrew Pitman ◽  
Paul Herron ◽  
Paul Dyson
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Orthologous Gene ◽  
Streptomyces Avermitilis ◽  
Antibiotic Biosynthesis ◽  
Reproductive Growth ◽  
Solid Media ◽  
Aerial Hyphae ◽  
A Chain ◽  
Membrane Spanning

ABSTRACT On solid media, the reproductive growth of Streptomyces involves antibiotic biosynthesis coincident with the erection of filamentous aerial hyphae. Following cessation of growth of an aerial hypha, multiple septation occurs at the tip to form a chain of unigenomic spores. A gene, crgA, that coordinates several aspects of this reproductive growth is described. The gene product is representative of a well-conserved family of small actinomycete proteins with two C-terminal hydrophobic-potential membrane-spanning segments. In Streptomyces avermitilis, crgA is required for sporulation, and inactivation of the gene abolished most sporulation septation in aerial hyphae. Disruption of the orthologous gene in Streptomyces coelicolor indicates that whereas CrgA is not essential for sporulation in this species, during growth on glucose-containing media, it influences the timing of the onset of reproductive growth, with precocious erection of aerial hyphae and antibiotic production by the mutant. Moreover, CrgA subsequently acts to inhibit sporulation septation prior to growth arrest of aerial hyphae. Overexpression of CrgA in S. coelicolor, uncoupling any nutritional and growth phase-dependent regulation, results in growth of nonseptated aerial hyphae on all media tested, consistent with a role for the protein in inhibiting sporulation septation.

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