scholarly journals Inducing Effect of Diamines on Transcription of the Cephamycin C Genes from the lat and pcbAB Promoters inNocardia lactamdurans

1999 ◽  
Vol 181 (8) ◽  
pp. 2379-2384 ◽  
Author(s):  
Ana Lucia Leitão ◽  
Francisco J. Enguita ◽  
Juan Luis De La Fuente ◽  
Paloma Liras ◽  
Juan F. Martin
Keyword(s):  
Cell Growth ◽  
Antibiotic Production ◽  
Protein A ◽  
Transcriptional Level ◽  
Intracellular Accumulation ◽  
Low Resolution ◽  
Cephamycin C ◽  
Nocardia Lactamdurans ◽  
Shake Flasks ◽  
Acid Precursor

ABSTRACT The diamines putrescine, cadaverine, and diaminopropane stimulate cephamycin biosynthesis in Nocardia lactamdurans, in shake flasks and fermentors, without altering cell growth. Intracellular levels of the P7 protein (a component of the methoxylation system involved in cephamycin biosynthesis) were increased by diaminopropane, as shown by immunoblotting studies. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase activities involved in biosynthesis of the α-aminoadipic acid precursor were also greatly stimulated. The diamine stimulatory effect is exerted at the transcriptional level, as shown by low-resolution S1 protection studies. The transcript corresponding to the pcbAB gene and to a lesser extent also the lat transcript were significantly increased in diaminopropane-supplemented cultures, whereas transcription from the cefD promoter was not affected. Coupling of the lat and pcbABpromoters to the reporter xylE gene showed that expression from the lat and pcbAB promoters was increased by addition of diaminopropane in Streptomyces lividans. Intracellular accumulation of diamines in Nocardia may be a signal to trigger antibiotic production.

scholarly journals Escherichia coli FadR Positively Regulates Transcription of the fabB Fatty Acid Biosynthetic Gene

2001 ◽  
Vol 183 (20) ◽  
pp. 5982-5990 ◽  
Author(s):  
John W. Campbell ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Unsaturated Fatty Acid ◽  
Protein A ◽  
Transcriptional Level ◽  
Biosynthetic Gene ◽  
Biochemical Tests ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

ABSTRACT In Escherichia coli expression of the genes of fatty acid degradation (fad) is negatively regulated at the transcriptional level by FadR protein. In contrast the unsaturated fatty acid biosynthetic gene, fabA, is positively regulated by FadR. We report that fabB, a second unsaturated fatty acid biosynthetic gene, is also positively regulated by FadR. Genomic array studies that compared global transcriptional differences between wild-type and fadR-null mutant strains, as well as in cultures of each strain grown in the presence of exogenous oleic acid, indicated that expression of fabBwas regulated in a manner very similar to that of fabAexpression. A series of genetic and biochemical tests confirmed these observations. Strains containing both fabB andfadR mutant alleles were constructed and shown to exhibit synthetic lethal phenotypes, similar to those observed infabA fadR mutants. A fadR strain was hypersensitive to cerulenin, an antibiotic that at low concentrations specifically targets the FabB protein. A transcriptional fusion of chloramphenicol acetyltransferase (CAT) to the fabBpromoter produces lower levels of CAT protein in a strain lacking functional FadR. The ability of a putative FadR binding site within thefabB promoter to form a complex with purified FadR protein was determined by a gel mobility shift assay. These experiments demonstrate that expression of fabB is positively regulated by FadR.

Abstract LB-272: KPNA7 nuclear import protein - a key regulator of cancer cell growth and nuclear morphology

2017 ◽  
Author(s):  
Elisa M. Vuorinen ◽  
Nina Rajala ◽  
Teemu Ihalainen ◽  
Hanna E. Rauhala ◽  
Anssi Nurminen ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cancer Cell ◽  
Nuclear Import ◽  
Protein A ◽  
Nuclear Morphology ◽  
Cancer Cell Growth

The Citrate Transport System of Lactococcus lactis subsp. lactis biovar diacetylactis Is Induced by Acid Stress

1998 ◽  
Vol 64 (3) ◽  
pp. 850-857 ◽  
Author(s):  
Nieves García-Quintáns ◽  
Christian Magni ◽  
Diego de Mendoza ◽  
Paloma López
Keyword(s):  
Stress Response ◽  
Cell Growth ◽  
Lactococcus Lactis ◽  
Growth Medium ◽  
Transport System ◽  
Acid Stress ◽  
Selective Advantage ◽  
Transcriptional Level ◽  
Transport Activity ◽  
Citrate Transport

ABSTRACT Citrate transport in Lactococcus lactis subsp.lactis biovar diacetylactis is catalyzed by citrate permease P (CitP), which is encoded by the plasmidic citPgene. We have shown previously that citP is included in thecitQRP operon, which is mainly transcribed from the P1 promoter in L. lactis subsp. lactis biovar diacetylactis. Furthermore, transcription of citQRP and citrate transport are not induced by the presence of citrate in the growth medium. In this work, we analyzed the influence of the extracellular pH on the expression of citP. The citrate transport system is induced by natural acidification of the medium during cell growth and by a shift to media buffered at acidic pHs. This inducible response to acid stress takes place at the transcriptional level and seems to be due to increased utilization of the P1 promoter. Increased transcription correlates with increased synthesis of CitP and results in higher citrate transport activity catalyzed by the cells. Finally, this acid stress response seems to provide L. lactis subsp. lactis biovar diacetylactis with a selective advantage resulting from cometabolism of glucose and citrate at low pHs.

scholarly journals mgr, a Novel Global Regulator in Staphylococcus aureus

2003 ◽  
Vol 185 (13) ◽  
pp. 3703-3710 ◽  
Author(s):  
Thanh T. Luong ◽  
Steven W. Newell ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Capsular Polysaccharide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Binding Motif ◽  
Transcriptional Level ◽  
Virulence Determinants ◽  
Alpha Toxin ◽  
Extracellular Protein Production

ABSTRACT The virulence determinants of Staphylococcus aureus are coordinately controlled by several unlinked chromosomal loci. Here, we report the identification of CYL5614, derived from strain Becker, with a mutation that affects the expression of type 8 capsular polysaccharide (CP8), nuclease, alpha-toxin, coagulase, protease, and protein A. This novel locus, named mgr, was linked by transposon Tn917 and mapped by three-factorial transduction crosses. The region containing the mgr locus was cloned and sequenced. Deletion mutagenesis and genetic complementation showed that the locus consisted of one gene, mgrA. Interestingly, mgrA-null mutants exhibited a phenotype opposite to that of CYL5614. This was due to a T-to-C mutation upstream of mgrA that resulted in a four- to eightfold increase in mgrA transcription in strain CYL5614. Thus, these results indicate that mgrA is an activator of CP8 and nuclease but a repressor of alpha-toxin, coagulase, protease, and protein A. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the mgr locus profoundly affected extracellular protein production, suggesting that the locus may regulate many other genes as well. The translated MgrA protein has a region of significant homology, which includes the helix-turn-helix DNA-binding motif, with the Escherichia coli MarR family of transcriptional regulators. Northern slot blot analyses suggested that mgr affected CP8, alpha-toxin, nuclease, and protein A at the transcriptional level.

scholarly journals A Novel AdpA Homologue Negatively Regulates Morphological Differentiation inStreptomyces xiamenensis318

2019 ◽  
Vol 85 (7) ◽  
Author(s):  
Xu-Liang Bu ◽  
Jing-Yi Weng ◽  
Bei-Bei He ◽  
Min-Juan Xu ◽  
Jun Xu
Keyword(s):  
Cell Growth ◽  
Secondary Metabolism ◽  
Gene Cluster ◽  
Morphological Differentiation ◽  
Gene Clusters ◽  
Transcriptional Level ◽  
Negative Effects ◽  
Promoter Regions ◽  
Content Type ◽  
Enhanced Production

ABSTRACTThe pleiotropic transcriptional regulator AdpA positively controls morphological differentiation and regulates secondary metabolism in mostStreptomycesspecies.Streptomyces xiamenensis318 has a linear chromosome 5.96 Mb in size. How AdpA affects secondary metabolism and morphological differentiation in such a naturally minimized genomic background is unknown. Here, we demonstrated that AdpASx, an AdpA orthologue inS. xiamenensis, negatively regulates cell growth and sporulation and bidirectionally regulates the biosynthesis of xiamenmycin and polycyclic tetramate macrolactams (PTMs) inS. xiamenensis318. Overexpression of theadpASxgene inS. xiamenensis318 had negative effects on morphological differentiation and resulted in reduced transcription of putativessgA,ftsZ,ftsH,amfC,whiB,wblA1,wblA2,wblE, and a gene encoding sporulation-associated protein (sxim_29740), whereas the transcription of putativebldDandbldAgenes was upregulated. Overexpression ofadpASxled to significantly enhanced production of xiamenmycin but had detrimental effects on the production of PTMs. As expected, the transcriptional level of theximgene cluster was upregulated, whereas the PTM gene cluster was downregulated. Moreover, AdpASxnegatively regulated the transcription of its own gene. Electrophoretic mobility shift assays revealed that AdpASxcan bind the promoter regions of structural genes of both theximand PTM gene clusters as well as to the promoter regions of genes potentially involved in the cell growth and differentiation ofS. xiamenensis318. We report that an AdpA homologue has negative effects on morphological differentiation inS. xiamenensis318, a finding confirmed when AdpASxwas introduced into the heterologous hostStreptomyces lividansTK24.IMPORTANCEAdpA is a key regulator of secondary metabolism and morphological differentiation inStreptomycesspecies. However, AdpA had not been reported to negatively regulate morphological differentiation. Here, we characterized the regulatory role of AdpASxinStreptomyces xiamenensis318, which has a naturally streamlined genome. In this strain, AdpASxnegatively regulated cell growth and morphological differentiation by directly controlling genes associated with these functions. AdpASxalso bidirectionally controlled the biosynthesis of xiamenmycin and PTMs by directly regulating their gene clusters rather than through other regulators. Our findings provide additional evidence for the versatility of AdpA in regulating morphological differentiation and secondary metabolism inStreptomyces.

Δ-1-Piperideine-6-carboxylate dehydrogenase, a new enzyme that forms α-aminoadipate in Streptomyces clavuligerus and other cephamycin C-producing actinomycetes

1997 ◽  
Vol 327 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Juan L. de la FUENTE ◽  
Angel RUMBERO ◽  
Juan F. MARTÍN ◽  
Paloma LIRAS
Keyword(s):  
Dehydrogenase Activity ◽  
Maximal Activity ◽  
Streptomyces Clavuligerus ◽  
Electron Acceptors ◽  
Native Protein ◽  
Cephamycin C ◽  
Second Stage ◽  
Streptomyces Cattleya ◽  
Nocardia Lactamdurans ◽  
Blue Sepharose

Δ-1-Piperideine-6-carboxylate (P6C) dehydrogenase activity, which catalyses the conversion of P6C into α-aminoadipic acid, has been studied in the cephamycin C producer Streptomyces clavuligerus by both spectrophotometric and radiometric assays. The enzyme has been purified 124-fold to electrophoretic homogeneity with a 26% yield. The native protein is a monomer of 56.2 kDa that efficiently uses P6C (apparent Km 14 μM) and NAD+ (apparent Km 115 μM), but not NADP+ or other electron acceptors, as substrates. The enzyme activity was inhibited (by 66%) by its end product NADH at 0.1 mM concentration. It did not show activity towards pyrroline-5-carboxylate and was separated by Blue-Sepharose chromatography from pyrroline-5-carboxylate dehydrogenase, an enzyme involved in the catabolism of proline. P6C dehydrogenase reached maximal activity later than other early enzymes of the cephamycin pathway. The P6C dehydrogenase activity was decreased in ammonium (40 mM)-supplemented cultures, as was that of lysine 6-aminotransferase. P6C dehydrogenase activity was also found in other cephamycin C producers (Streptomyces cattleya and Nocardia lactamdurans) but not in actinomycetes that do not produce β-lactams, suggesting that it is an enzyme specific for cephamycin biosynthesis, involved in the second stage of the two-step conversion of lysine to α-aminoadipic acid.

scholarly journals Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions

2013 ◽  
Vol 79 (12) ◽  
pp. 3802-3812 ◽  
Author(s):  
Juergen Mairhofer ◽  
Theresa Scharl ◽  
Karoline Marisch ◽  
Monika Cserjan-Puschmann ◽  
Gerald Striedner
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Gene Dosage ◽  
Expression System ◽  
Transcriptional Level ◽  
Strong Impact ◽  
Expression Systems ◽  
Free System ◽  
Recombinant Gene Expression

ABSTRACTPlasmid-basedEscherichia coliBL21(DE3) expression systems are extensively used for the production of recombinant proteins. However, the combination of a high gene dosage with strong promoters exerts extremely stressful conditions on producing cells, resulting in a multitude of protective reactions and malfunctions in the host cell with a strong impact on yield and quality of the product. Here, we provide in-depth characterization of plasmid-based perturbations in recombinant protein production. A plasmid-free T7 system with a single copy of the gene of interest (GOI) integrated into the genome was used as a reference. Transcriptomics in combination with a variety of process analytics were used to characterize and compare a plasmid-free T7-based expression system to a conventional pET-plasmid-based expression system, with both expressing human superoxide dismutase in fed-batch cultivations. The plasmid-free system showed a moderate stress response on the transcriptional level, with only minor effects on cell growth. In contrast to this finding, comprehensive changes on the transcriptome level were observed in the plasmid-based expression system and cell growth was heavily impaired by recombinant gene expression. Additionally, we found that the T7 terminator is not a sufficient termination signal. Overall, this work reveals that the major metabolic burden in plasmid-based systems is caused at the level of transcription as a result of overtranscription of the multicopy product gene and transcriptional read-through of T7 RNA polymerase. We therefore conclude that the presence of high levels of extrinsic mRNAs, competing for the limited number of ribosomes, leads to the significantly reduced translation of intrinsic mRNAs.

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