Streptomyces sp. Tü 6176 produces the cytotoxic benzoxazole nataxazole. Bioinformatic analysis of the genome of this organism predicts the presence of 38 putative secondary-metabolite biosynthesis gene clusters, including those involved in the biosynthesis of AJI9561 and its derivative nataxazole, the antibiotic hygromycin B, and ionophores enterobactin and coelibactin. The nataxazole biosynthesis gene cluster was identified and characterized: it lacks the O-methyltransferase gene required to convert AJI9561 into nataxazole. This O-methyltransferase activity might act as a resistance mechanism, as AJI9561 shows antibiotic activity whereas nataxazole is inactive. Moreover, heterologous expression of the nataxazole biosynthesis gene cluster in S. lividans JT46 resulted in the production of AJI9561. Nataxazole biosynthesis requires the shikimate pathway to generate 3-hydroxyanthranilate and an iterative type I PKS to generate 6-methylsalicylate. Production of nataxazole was improved up to fourfold by disrupting one regulatory gene in the cluster. An additional benzoxazole, 5-hydroxynataxazole is produced by Streptomyces sp. Tü 6176. 5-Hydroxynataxazole derives from nataxazole by the activity of an as yet unidentified oxygenase; this implies cross-talk between the nataxazole biosynthesis pathway and an unknown pathway.
The QDR2 gene of Saccharomyces cerevisiae encodes a putative plasma membrane drug:H ؉ antiporter that confers resistance against quinidine, barban, bleomycin, and cisplatin. This work provides experimental evidence of defective K ؉ (Rb ؉ ) uptake in the absence of QDR2. The direct involvement of Qdr2p in K ؉ uptake is reinforced by the fact that increased K ؉ (Rb ؉ ) uptake due to QDR2 expression is independent of the Trk1p/Trk2p system. QDR2 expression confers a physiological advantage for the yeast cell during the onset of K ؉ limited growth, due either to a limiting level of K ؉ in the growth medium or to the presence of quinidine. This drug decreases the K ؉ uptake rate and K ؉ accumulation in the yeast cell, especially in the ⌬qdr2 mutant. Qdr2p also helps to sustain the decrease of intracellular pH in quinidine-stressed cells in growth medium at pH 5.5 by indirectly promoting H ؉ extrusion affected by the drug. The results are consistent with the hypothesis that Qdr2p may also couple K ؉ movement with substrate export, presumably with quinidine. Other clues to the biological role of QDR2 in the yeast cell come from two additional lines of experimental evidence. First, QDR2 transcription is activated under nitrogen (NH 4 ؉ ) limitation or when the auxotrophic strain examined enters stationary phase due to leucine limitation, this regulation being dependent on general amino acid control by Gcn4p. Second, the amino acid pool is higher in ⌬qdr2 cells than in wild-type cells, indicating that QDR2 expression is, directly or indirectly, involved in amino acid homeostasis.
Summary Streptomyces sp. NTK937, producer of benzoxazole antibiotic caboxamycin, produces in addition a methyl ester derivative, O‐methylcaboxamycin. Caboxamycin cluster, comprising one regulatory and nine structural genes, has been delimited, and each gene has been individually inactivated to demonstrate its role in the biosynthetic process. The O‐methyltransferase potentially responsible for O‐methylcaboxamycin synthesis would reside outside this cluster. Five of the genes, cbxR, cbxA, cbxB, cbxD and cbxE, encoding a SARP transcriptional regulator, salicylate synthase, 3‐oxoacyl‐ACP‐synthase, ACP and amidohydrolase, respectively, have been found to be essential for caboxamycin biosynthesis. The remaining five structural genes were found to have paralogues distributed throughout the genome, capable of partaking in the process when their cluster homologue is inactivated. Two of such paralogues, cbxC’ and cbxI’, coding an AMP‐dependent synthetase‐ligase and an anthranilate synthase, respectively, have been identified. However, the other three genes might simultaneously have more than one paralogue, given that cbxF (DAHP synthase), cbxG (2,3‐dihydro‐2,3‐dihydroxybenzoate dehydrogenase) and cbxH (isochorismatase) have three, three and five putative paralogue genes, respectively, of similar function within the genome. As a result of genetic manipulation, a novel benzoxazole (3′‐hydroxycaboxamycin) has been identified in the salicylate synthase‐deficient mutant strain ΔcbxA. 3′‐hydroxycaboxamycin derives from the cross‐talk between the caboxamycin and enterobactin pathways.
SummaryProtein phosphatases 2C are a family of conserved enzymes involved in many aspects of the cell biology. We reported that, in the yeast Saccharomyces cerevisiae, overexpression of the Ptc3p isoform resulted in increased lithium tolerance in the hypersensitive hal3 background. We have found that the tolerance induced by PTC3 overexpression is also observed in wild-type cells and that this is most probably the result of increased expression of the ENA1 Na + -ATPase mediated by the Hog1 MAP kinase pathway. This effect does not require a catalytically active protein. Surprisingly, deletion of PTC3 (similarly to that of PTC2, PTC4 or PTC5) does not confer a lithium-sensitive phenotype, but mutation of PTC1 does. Lack of PTC1 in an ena1-4 background did not result in additive lithium sensitivity and the ptc1 mutant showed a decreased expression of the ENA1 gene in cells stressed with LiCl. In agreement, under these conditions, the ptc1 mutant was less effective in extruding Li + and accumulated higher concentrations of this cation. Deletion of PTC1 in a hal3 background did not exacerbate the halosensitive phenotype of the hal3 strain. In addition, induction from the ENA1 promoter under LiCl stress decreased similarly (50%) in hal3, ptc1 and ptc1 hal3 mutants. Finally, mutation of PTC1 virtually abolishes the increased tolerance to toxic cations provided by overexpression of Hal3p. These results indicate that Ptc1p modulates the function of Ena1p by regulating the Hal3/Ppz1,2 pathway. In conclusion, overexpression of PTC3 and lack of PTC1 affect lithium tolerance in yeast, although through different mechanisms.
The AMP-activated protein kinase, AMPK, controls energy homeostasis in eukaryotic cells but little is known about the mechanisms governing the dynamics of its activation/deactivation. The yeast AMPK, SNF1, is activated in response to glucose depletion and mediates glucose de-repression by inactivating the transcriptional repressor Mig1. Here we show that overexpression of the Snf1-activating kinase Sak1 results, in the presence of glucose, in constitutive Snf1 activation without alleviating glucose repression. Co-overexpression of the regulatory subunit Reg1 of the Glc-Reg1 phosphatase complex partly restores glucose regulation of Snf1. We generated a set of 24 kinetic mathematical models based on dynamic data of Snf1 pathway activation and deactivation. The models that reproduced our experimental observations best featured (a) glucose regulation of both Snf1 phosphorylation and dephosphorylation, (b) determination of the Mig1 phosphorylation status in the absence of glucose by Snf1 activity only and (c) a regulatory step directing active Snf1 to Mig1 under glucose limitation. Hence it appears that glucose de-repression via Snf1-Mig1 is regulated by glucose via at least two independent steps: the control of activation of the Snf1 kinase and directing active Snf1 to inactivating its target Mig1.
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