scholarly journals Characterization of rrdA, a TetR Family Protein Gene Involved in the Regulation of Secondary Metabolism in Streptomyces coelicolor

2009 ◽  
Vol 75 (7) ◽  
pp. 2158-2165 ◽  
Author(s):  
Xijun Ou ◽  
Bo Zhang ◽  
Lin Zhang ◽  
Guoping Zhao ◽  
Xiaoming Ding
Keyword(s):  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Transcript Level ◽  
Biosynthetic Gene Cluster ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Family Protein ◽  
Severe Reduction ◽  
High Level

ABSTRACT Streptomyces not only exhibits complex morphological differentiation but also produces a plethora of secondary metabolites, particularly antibiotics. To improve our general understanding of the complex network of undecylprodigiosin (Red) biosynthesis regulation, we used an in vivo transposition system to identify novel regulators that influence Red production in Streptomyces coelicolor M145. Using this screening system, we obtained 25 Red-deficient mutants. Twenty-four of these mutants had a transposon inserted in the previously described Red biosynthetic gene cluster and produced different amounts of another secondary metabolite, actinorhodin (Act). One mutant was shown to have an insertion in a different region of the chromosome upstream of the previously uncharacterized gene rrdA (regulator of redD, sco1104), which encodes a putative TetR family transcription factor. Compared with wild-type strain M145, the rrdA null mutant exhibited increased Red production and decreased Act production. A high level of rrdA expression resulted in a severe reduction in Red production and Act overproduction. Reverse transcription-PCR analysis showed that RrdA negatively regulated Red production by controlling redD mRNA abundance, while no change was observed at the transcript level of the Act-specific activator gene, actII-orf4. The effects on Act biosynthesis might arise from competition for precursors that are common to both pathways.

scholarly journals Identification of a gene from Streptomyces rimosus M527 negatively affecting antibiotic biosynthesis and morphological differentiation

2020 ◽  
Author(s):  
Zheng Ma ◽  
Zhangqing Song ◽  
Jie Xu ◽  
Andreas Bechthold ◽  
Xiaoping Yu
Keyword(s):  
Plant Pathogens ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Negative Regulator ◽  
Antibiotic Biosynthesis ◽  
Pcr Analysis ◽  
Streptomyces Rimosus ◽  
Over Expression ◽  
Reverse Transcription Pcr ◽  
Fungal Plant Pathogens

Abstract Background: The polyene macrolide rimocidin, produced by Streptomyces rimosus M527, was found to be highly effective against a broad range of fungal plant pathogens. Current understanding of the regulatory mechanism of rimocidin biosynthesis and morphological differentiation in S. rimosus M527 is limited. NsdA is considered as a negative regulator involved in morphological differentiation and biosynthesis of secondary metabolites in some Streptomyces.Results: In this study, nsdAsr was cloned from S. rimosus M527. The role of nsdAsr in rimocidin biosynthesis and morphological differentiation was investigated by gene deletion, complementation, and over-expression. A ΔnsdAsr mutant was obtained using CRISPR/Cas9. The mutant produced more rimocidin (46%) and generated more spores than the wild-type strain. Over-expression of nsdAsr led to a decrease in rimocidin production and impairment of sporulation. Quantitative reverse transcription-PCR (qRT-PCR) analysis revealed that transcription of rim genes responsible for rimocidin biosynthesis was up-regulated in the ΔnsdAsr mutant but down-regulated in the nsdAsr over-expression strain. Similar effects have been described for Streptomyces coelicolor M145 and the industrial toyocamycin-producing strain Streptomyces diastatochromogenes 1628.Conclusion: NsdAsr is identified as a negative regulator of sporluation and antibiotic biosynthesis as well as the transcription of biosynthetic genes both in its host S. rimosus M527 and in model strain S. coelicolor and industrial producer strain S. diastatochromogenes 1628. This work will provide further information for understanding regulatory mechanisms controlling rimocidin biosynthesis in S. rimosus M527.

scholarly journals MicroRNA-182-5p relieves murine allergic rhinitis via TLR4/NF-κB pathway

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 1202-1212
Author(s):  
Aichun Zhang ◽  
Yangzi Jin
Keyword(s):  
Allergic Rhinitis ◽  
Inflammatory Cells ◽  
Lavage Fluid ◽  
Specific Ige ◽  
Inflammatory Factors ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Pathway Activation ◽  
Nasal Lavage Fluid

AbstractAllergic rhinitis (AR) is one of the most common chronic diseases. This study examined whether microRNA (miR)-182-5p plays a role in AR by regulating toll-like receptor 4 (TLR4). First, data demonstrated that TLR4 was a target of miR-182-5p. Subsequently, AR mouse model was established to explore the role of miR-182-5p and TLR4 in AR in vivo. Initially, quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that miR-182-5p was downregulated, while TLR4 expression was upregulated in AR mice. Then we found that miR-182-5p mimic reduced the frequency of sneezing and nose rubbing of the AR mice. In addition, miR-182-5p mimic significantly increased ovalbumin (OVA)-specific IgE and leukotriene C4 expression levels in nasal lavage fluid (NLF) and serum of AR mice. miR-182-5p mimic decreased the number of inflammatory cells in NLF of AR mice. It also reduced the levels of inflammatory factors in the serum of AR mice, such as interleukin (IL)-4, IL-5, IL-13, IL-17 and tumor necrosis factor (TNF)-α, while increasing the release of IFN-γ and IL-2. Finally, miR-182-5p mimic inhibited NF-κB signaling pathway activation in AR mice. However, all effects of miR-182-5p mimic on AR mice were reversed by TLR4-plasmid. In conclusion, miR-182-5p/TLR4 axis may represent a novel therapeutic target for AR.

scholarly journals Organelle relationships in cultured 3T3-L1 preadipocytes.

1980 ◽  
Vol 87 (1) ◽  
pp. 180-196 ◽  
Author(s):  
A B Novikoff ◽  
P M Novikoff ◽  
O M Rosen ◽  
C S Rubin
Keyword(s):  
Spatial Relationship ◽  
Morphological Differentiation ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Exogenous Lipid ◽  
Acetate Esterase ◽  
Alpha Naphthyl Acetate Esterase ◽  
High Level ◽  
Relationship Of

In differentiating 3T3-L1 cells, lipid spheres, the endoplasmic reticulum (ER), microperoxisomes, and mitochondria form "constellations" that may reflect the interplay of lipid metabolizing enzymes in these organelles. ER cisternae are also situated very close to "rosettes,"plasmalemmal specializations found in mature adipocytes in vivo. As in hepatocytes and absorptive cells of the intestine, this spatial relationship of ER and plasmalemma suggests a role for rosettes in the uptake of exogenous lipid precursors. The morphological differentiation of 3T3-L1 preadipocytes includes the loss of "stress fibers" and the appearance of microfilament like structures that encase, in a complex manner, the cytosolic lipid spheres that appear during differentiation. Other features described for the first time in 3T3-L1 preadipocytes include: (a) the presence of an extensive acid phosphatase (AcPase) positive GERL from which coated vesicles apparently arise (these coated vesicles display AcPase activity and are much smaller and far more numerous than the coated vesicles that seem to arise from the plasmalemmal coated pits); (b) the abundance of AcPase-positive autophagic vacuoles; and (c) a high level of alpha-naphthyl-acetate-esterase activity which, by light microscopy cytochemistry, appears to be localized in the cytosol.

scholarly journals Effects ofibeADeletion on Virulence and Biofilm Formation of Avian PathogenicEscherichia coli

2010 ◽  
Vol 79 (1) ◽  
pp. 279-287 ◽  
Author(s):  
Shaohui Wang ◽  
Chunling Niu ◽  
Zhenyu Shi ◽  
Yongjie Xia ◽  
Muhammad Yaqoob ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Wild Type Strain ◽  
Chicken Embryo Fibroblast ◽  
Neonatal Meningitis ◽  
Pcr Analysis ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Animal System ◽  
The Brain

ABSTRACTTheibeAgene is located on a genomic island, GimA, which is involved in the pathogenesis of neonatal meningitisEscherichia coli(NMEC) and avian pathogenicE. coli(APEC). The prevalence ofibeAin the APEC collection in China was investigated, and 20 of 467 strains (4.3%) were positive. In addition, analysis of the association of theE. colireference (ECOR) groups with positive strains revealed thatibeAwas linked to group B2. TheibeAgene in DE205B was analyzed and compared to those of APEC and NMEC, which indicated that the specificity ofibeAwas not consistent along pathotypes. The invasion of chicken embryo fibroblast DF-1 cells by APEC DE205B and RS218 was observed, which suggested that DF-1 cells could be a model to study the mechanism of APEC invasion. The inactivation ofibeAin APEC DE205B led to the reduced capacity to invade DF-1 cells, defective virulencein vivo, and decreased biofilm formation compared to the wild-type strain. In addition, strain AAEC189 expressingibeAexhibited enhanced invasion capacity and biofilm formation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) analysis and animal system infection experiments indicated that the loss ofibeAdecreased the colonization and proliferation capacities of APEC in the brain during system infection.

scholarly journals The Level of AdpA Directly Affects Expression of Developmental Genes in Streptomyces coelicolor

2011 ◽  
Vol 193 (22) ◽  
pp. 6358-6365 ◽  
Author(s):  
Marcin Wolański ◽  
Rafał Donczew ◽  
Agnieszka Kois-Ostrowska ◽  
Paweł Masiewicz ◽  
Dagmara Jakimowicz ◽  
...  
Keyword(s):  
Early Stage ◽  
Response Regulator ◽  
Streptomyces Coelicolor ◽  
Streptomyces Griseus ◽  
Morphological Differentiation ◽  
Aerial Mycelium ◽  
Content Type ◽  
Protein Functions

AdpA is a key regulator of morphological differentiation inStreptomyces. In contrast toStreptomyces griseus, relatively little is known about AdpA protein functions inStreptomyces coelicolor. Here, we report for the first time the translation accumulation profile of theS. coelicoloradpA(adpASc) gene; the level ofS. coelicolorAdpA (AdpASc) increased, reaching a maximum in the early stage of aerial mycelium formation (after 36 h), and remained relatively stable for the next several hours (48 to 60 h), and then the signal intensity decreased considerably. AdpAScspecifically binds theadpAScpromoter regionin vitroandin vivo, suggesting that its expression is autoregulated; surprisingly, in contrast toS. griseus, the protein presumably acts as a transcriptional activator. We also demonstrate a direct influence of AdpAScon the expression of several genes whose products play key roles in the differentiation ofS. coelicolor: STI, a protease inhibitor; RamR, an atypical response regulator that itself activates expression of the genes for a small modified peptide that is required for aerial growth; and ClpP1, an ATP-dependent protease. The diverse influence of AdpAScprotein on the expression of the analyzed genes presumably results mainly from different affinities of AdpAScprotein to individual promoters.

scholarly journals Unique Posttranslational Modifications of Chitin-Binding Lectins of Entamoeba invadens Cyst Walls

2006 ◽  
Vol 5 (5) ◽  
pp. 836-848 ◽  
Author(s):  
Katrina L. Van Dellen ◽  
Anirban Chatterjee ◽  
Daniel M. Ratner ◽  
Paula E. Magnelli ◽  
John F. Cipollo ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Cyst Wall ◽  
Low Complexity ◽  
Pcr Analysis ◽  
Major Protein ◽  
Chitin Binding Domain ◽  
Binding Domains ◽  
Reverse Transcription Pcr ◽  
Entamoeba Invadens

ABSTRACT Entamoeba histolytica, which causes amebic dysentery and liver abscesses, is spread via chitin-walled cysts. The most abundant protein in the cyst wall of Entamoeba invadens, a model for amebic encystation, is a lectin called EiJacob1. EiJacob1 has five tandemly arrayed, six-Cys chitin-binding domains separated by low-complexity Ser- and Thr-rich spacers. E. histolytica also has numerous predicted Jessie lectins and chitinases, which contain a single, N-terminal eight-Cys chitin-binding domain. We hypothesized that E. invadens cyst walls are composed entirely of proteins with six-Cys or eight-Cys chitin-binding domains and that some of these proteins contain sugars. E. invadens genomic sequences predicted seven Jacob lectins, five Jessie lectins, and three chitinases. Reverse transcription-PCR analysis showed that mRNAs encoding Jacobs, Jessies, and chitinases are increased during E. invadens encystation, while mass spectrometry showed that the cyst wall is composed of an ∼30:70 mix of Jacob lectins (cross-linking proteins) and Jessie and chitinase lectins (possible enzymes). Three Jacob lectins were cleaved prior to Lys at conserved sites (e.g., TPSVDK) in the Ser- and Thr-rich spacers between chitin-binding domains. A model peptide was cleaved at the same site by papain and E. invadens Cys proteases, suggesting that the latter cleave Jacob lectins in vivo. Some Jacob lectins had O-phosphodiester-linked carbohydrates, which were one to seven hexoses long and had deoxysugars at reducing ends. We concluded that the major protein components of the E. invadens cyst wall all contain chitin-binding domains (chitinases, Jessie lectins, and Jacob lectins) and that the Jacob lectins are differentially modified by site-specific Cys proteases and O-phosphodiester-linked glycans.

scholarly journals Involvement of the C-Terminal Extension of the α Polypeptide and of the PucC Protein in LH2 Complex Biosynthesis in Rubrivivax gelatinosus

2004 ◽  
Vol 186 (10) ◽  
pp. 3143-3152 ◽  
Author(s):  
Anne-Soisig Steunou ◽  
Soufian Ouchane ◽  
Françoise Reiss-Husson ◽  
Chantal Astier
Keyword(s):  
Purple Bacteria ◽  
Growth Conditions ◽  
Initiation Site ◽  
Northern Blotting ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Rubrivivax Gelatinosus

ABSTRACT The facultative phototrophic nonsulfur bacterium Rubrivivax gelatinosus exhibits several differences from other species of purple bacteria in the organization of its photosynthetic genes. In particular, the puc operon contains only the pucB and pucA genes encoding the β and α polypeptides of the light-harvesting 2 (LH2) complex. Downstream of the pucBA operon is the pucC gene in the opposite transcriptional orientation. The transcription of pucBA and pucC has been studied. No pucC transcript was detected either by Northern blotting or by reverse transcription-PCR analysis. The initiation site of pucBA transcription was determined by primer extension, and Northern blot analysis revealed the presence of two transcripts of 0.8 and 0.65 kb. The half-lives of both transcripts are longer in cells grown semiaerobically than in photosynthetically grown cells, and the small transcript is the less stable. It was reported that the α polypeptide, encoded by the pucA gene, presents a C-terminal extension which is not essential for LH2 function in vitro. The biological role of this alanine- and proline-rich C-terminal extension in vivo has been investigated. Two mutants with C-terminal deletions of 13 and 18 residues have been constructed. Both present the two pucBA transcripts, while their phenotypes are, respectively, LH2+ and LH2−, suggesting that a minimal length of the C-terminal extension is required for LH2 biogenesis. Another important factor involved in the LH2 biogenesis is the PucC protein. To gain insight into the function of this protein in R. gelatinosus, we constructed and characterized a PucC mutant. The mutant is devoid of LH2 complex under semiaerobiosis but still produces a small amount of these antennae under photosynthetic growth conditions. This conditional phenotype suggests the involvement of another factor in LH2 biogenesis.

scholarly journals In Vivo Selection of Reduced Susceptibility to Carbapenems in Acinetobacter baumannii Related to ISAba1-Mediated Overexpression of the Natural blaOXA-66 Oxacillinase Gene

2009 ◽  
Vol 53 (6) ◽  
pp. 2657-2659 ◽  
Author(s):  
Samy Figueiredo ◽  
Laurent Poirel ◽  
Jacques Croize ◽  
Christine Recule ◽  
Patrice Nordmann
Keyword(s):  
Acinetobacter Baumannii ◽  
Reverse Transcription ◽  
Resistance Pattern ◽  
Pcr Analysis ◽  
In Vivo Selection ◽  
Reverse Transcription Pcr ◽  
Naturally Occurring ◽  
Carbapenemase Gene ◽  
Selection Of

ABSTRACT Two clonally related Acinetobacter baumannii isolates, A1 and A2, were obtained from the same patient. Isolate A2, selected after an imipenem-containing treatment, showed reduced susceptibility to carbapenems. This resistance pattern was related to insertion of the ISAba1 element upstream of the naturally occurring bla OXA-66 carbapenemase gene as demonstrated by sequencing, reverse transcription-PCR analysis, and inactivation of the bla OXA-66 gene.

scholarly journals Genome-wide analysis of the role of the antibiotic biosynthesis regulator AbsA2 inStreptomyces coelicolorA3(2)

2018 ◽  
Author(s):  
Richard A. Lewis ◽  
Abdul Wahab ◽  
Giselda Bucca ◽  
Emma E. Laing ◽  
Carla Möller-Levet ◽  
...  
Keyword(s):  
Large Scale ◽  
Time Course ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Negative Influence ◽  
Antibiotic Biosynthesis ◽  
Biosynthetic Gene

AbstractThe AbsA1-AbsA2 two component signalling system ofStreptomyces coelicolorhas long been known to exert a powerful negative influence on the production of the antibiotics actinorhodin, undecylprodiginine and the Calcium-Dependent Antibiotic (CDA). Here we report the analysis of aΔabsA2deletion strain, which exhibits the classic precocious antibiotic hyper-production phenotype, and its complementation by an N-terminal triple-FLAG-tagged version of AbsA2. The complemented and non-complementedΔabsA2mutant strains were used in large-scale microarray-based time-course experiments to investigate the effect of deletingabsA2on gene expression and to identify thein vivoAbsA2 DNA-binding target sites using ChIP-on chip. We show that in addition to binding to the promoter regions ofredZandactII-orfIVAbsA2 binds to several previously unidentified sites within thecdabiosynthetic gene cluster within and/or upstream ofSCO3215-SCO3216,SCO3217,SCO3229-SCO3230, andSCO3226, and we relate the pattern of AbsA2 binding to the results of the transcriptomic study and antibiotic phenotypic assays. Interestingly, dual ‘biphasic’ ChIP peaks were observed with AbsA2 binding across the regulatory genesactII-orfIVandredZand theabsA2gene itself, while more conventional single promoter-proximal peaks were seen at the CDA biosynthetic genes suggesting a different mechanism of regulation of the former loci. Taken together the results shed light on the complex mechanism of regulation of antibiotic biosynthesis inStreptomyces coelicolorand the important role of AbsA2 in controlling the expression of three antibiotic biosynthetic gene clusters.

scholarly journals Reverse Transcription-PCR Analysis of the Regulation of the Manganese Peroxidase Gene Family

1998 ◽  
Vol 64 (2) ◽  
pp. 569-574 ◽  
Author(s):  
Jessica M. Gettemy ◽  
Biao Ma ◽  
Margaret Alic ◽  
Michael H. Gold
Keyword(s):  
Gene Expression ◽  
Reverse Transcription ◽  
Manganese Peroxidase ◽  
Transcript Level ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Total Rna ◽  
Nitrogen Levels ◽  
Reverse Transcription Pcr ◽  
Low Levels

ABSTRACT Manganese peroxidase (MnP) gene expression in the lignin-degrading fungus Phanerochaete chrysosporium is regulated by nutrient nitrogen levels and by Mn(II), the substrate for the enzyme, as well as by heat shock and other factors. Reverse transcription-PCR (RT-PCR) of total RNA can distinguish the mRNAs of each of the three sequencedP. chrysosporium mnp genes, i.e., mnp1,mnp2, and mnp3. Quantitative RT-PCR demonstrates that each of the three transcripts is present at a similar low basal level in nitrogen-sufficient cultures, with or without Mn, and in nitrogen-limited cultures lacking Mn. However, in 5-day-old, nitrogen-limited, stationary cultures supplemented with 180 μM Mn, the levels of the mnp1 and mnp2 transcripts increased approximately 100- and 1,700-fold, respectively, over basal levels. In contrast, under these conditions, the level of themnp3 transcript did not increase significantly over the basal level. Quantitative RT-PCR of total RNA extracted from nitrogen-deficient, Mn-supplemented cultures on days 2 through 7 demonstrates that whereas the mnp1 transcript was present at relatively low levels on days 3 through 7, the mnp2transcript level peaked on day 5 and the mnp3 transcript level peaked on day 3. Comparison of total RNA extracted on day 5 from nitrogen-deficient, Mn-supplemented stationary and agitated cultures indicates that in stationary cultures, mnp2 was the major expressed mnp gene, whereas in large agitated cultures,mnp1 was the major expressed mnp gene.

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