[Show abstract][Hide abstract] ABSTRACT: A wide variety of prokaryotes possess DNA modifications consisting of sequence-specific phosphorothioates (PT) inserted by members of a five-gene cluster. Recent genome mapping studies revealed two unusual features of PT modifications: short consensus sequences and partial modification of a specific genomic site in a population of bacteria. To better understand the mechanism of target selection of PT modifications that underlies these features, we characterized the substrate recognition of the PT-modifying enzymes termed DptC, D and E in a cell extract system from Salmonella. The results revealed that double-stranded oligodeoxynucleotides underwent de novo PT modification in vitro, with the same modification pattern as in vivo, i. e., GpsAAC/GpsTTC motif. Unexpectedly, in these in vitro analyses we observed no significant effect on PT modification by sequences flanking GAAC/GTTC motif, while PT also occurred in the GAAC/GTTC motif that could not be modified in vivo. Hemi-PT DNA also served as substrate of the PT-modifying enzymes, but not single-stranded DNA. The PT-modifying enzymes were then found to function as a large protein complex, with all of three subunits in tetrameric conformations. This study provided the first demonstration of in vitro DNA PT modification by PT-modifying enzymes that function as a large protein complex.
[Show abstract][Hide abstract] ABSTRACT: DNA phosphorothioate (PT) modifications, with one non-bridging phosphate oxygen replaced with sulfur, are widely but sporadically distributed in prokaryotic genomes. Short consensus sequences surround the modified linkage in each strain, although each site is only partially modified. The mechanism that maintains this low-frequency modification status is still unknown. In Salmonella enterica serovar Cerro 87, PT modification is mediated by a four-gene cluster called dptBCDE. Here, we found that deletion of dptB led to a significant increase of intracellular PT modification level. In this deletion, transcription of downstream genes was elevated during rapid cell growth. Restoration of dptB on a plasmid restored wild type levels of expression of downstream genes and PT modification. In vitro, DptB directly protected two separate sequences within the dpt promoter region from DNase I cleavage. Each protected sequence contained a direct repeat (DR). Mutagenesis assays of the DRs demonstrated that each DR was essential for DptB binding. The observation of two shifted species by gel-shift analysis suggests dimer conformation of DptB protein. These DRs are conserved among the promoter regions of dptB homologs, suggesting that this regulatory mechanism is widespread. These findings demonstrate that PT modification is regulated at least in part at the transcriptional level.
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[Show abstract][Hide abstract] ABSTRACT: Prokaryotes protect their genomes from foreign DNA with a diversity of defense mechanisms, including a widespread restriction-modification (R-M) system involving phosphorothioate (PT) modification of the DNA backbone. Unlike classical R-M systems, highly partial PT-modification of consensus motifs in bacterial genomes suggests an unusual mechanism of PT-dependent restriction. In Salmonella enterica, PT modification is mediated by four genes dptB-E, while restriction involves additional three genes dptF-H. Here, we performed a series of studies to characterize the PT-dependent restriction, and found that it presented several features distinct with traditional R-M systems. The presence of restriction genes in a PT-deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling revealed the expression of >600 genes was affected by restriction enzymes in cells lacking PT, including induction of bacteriophage, SOS response and DNA repair-related genes. These transcriptional responses are consistent with the observation that restriction enzymes caused extensive DNA cleavage in the absence of PT modifications in vivo. However, over-expression of restriction genes was lethal to the host in spite of the presence PT modifications. These results point to an unusual mechanism of PT-dependent DNA cleavage by restriction enzymes in the face of partial PT modification.
[Show abstract][Hide abstract] ABSTRACT: Alteration of sugar moieties of natural products often leads to novel antibiotics with different chemical and physical properties. fscMI is a putative glycosyltransferase (GT) in a gene cluster for the production of candicidin, a polyene macrolide antibiotic, produced by Streptomyces sp. FR-008. In this report, we established an in vivo biochemical detection system by inactivating fscMI and the DH11 domain of polyketide synthase (PKS) through double homologous recombination to unveil the interaction between polyene GTs and their substrates. We found that homologous GT genes including amphDI, nysDI and pimK can catalyze the conversion of candicidin aglycone into candicidin/FR-008-III in fscMI mutant, suggesting that homologous polyene GTs show some tolerance toward aglycones and that it is possible to create new polyene analogues with altered aglycones through genetic engineering. Inactivation of the DH11 domain of PKS led to novel polyene derivatives with mycosamine added to the altered polyketide backbones, further confirming the loose substrate specificity of polyene GTs. Furthermore, mutation of Ser346, Ser361, His362 or Cys387 of FscMI by site-directed mutagenesis significantly reduced its catalytic activity. Further analysis suggested that Ser361 and Cys387 are likely the critical donor interacting residues that could affect the activity of GT FscMI. To our knowledge, this is the first report of the critical residues in a polyene GT.
[Show abstract][Hide abstract] ABSTRACT: A polyene macrolide antibiotic tetramycin biosynthetic gene cluster was identified by genome mining and isolated from Streptomyces hygrospinosus var. beijingensis. Genetic and in silico analyses gave insights into the mechanism of biosynthesis of tetramycin, and a model of the tetramycin biosynthetic pathway is proposed. Inactivation of a cytochrome P450 monooxygenase gene, tetrK, resulted in the production of a tetramycin B precursor: tetramycin A, which lacks a hydroxy group in its polyol region. TetrK was subsequently overexpressed heterologously in E. coli with a His(6) tag, and purified TetrK efficiently hydroxylated tetramycin A to afford tetramycin B. Kinetic studies revealed no inhibition of TetrK by substrate or product. Surprisingly, sequence-alignment analysis showed that TetrK, as a hydroxylase, has much higher homology with epoxidase PimD than with hydroxylases NysL and AmphL. The 3D structure of TetrK was then constructed by homology modeling with PimD as reference. Although TetrK and PimD catalyzed different chemical reactions, homology modeling indicated that they might share the same catalytic sites, despite also possessing some different sites correlated with substrate binding and substrate specificity. These findings offer good prospects for the production of improved antifungal polyene analogues.
[Show abstract][Hide abstract] ABSTRACT: Diverse bacteria contain DNA with sulfur incorporated stereo-specifically into their DNA backbone at specific sequences (phosphorothioation). We found that in vitro oxidation of phosphorothioate (PT) DNA by hydrogen peroxide (H(2)O(2)) or peracetic acid has two possible outcomes: DNA backbone cleavage or sulfur removal resulting in restoration of normal DNA backbone. The physiological relevance of this redox reaction was investigated by challenging PT DNA hosting Salmonella enterica cells using H(2)O(2). DNA phosphorothioation was found to correlate with increasing resistance to the growth inhibition by H(2)O(2). Resistance to H(2)O(2) was abolished when each of the three dnd genes, required for phosphorothioation, was inactivated. In vivo, PT DNA is more resistant to the double-strand break damage caused by H(2)O(2) than PT-free DNA. Furthermore, sulfur on the modified DNA was consumed and the DNA was converted to PT-free state when the bacteria were incubated with H(2)O(2). These findings are consistent with a hypothesis that phosphorothioation modification endows DNA with reducing chemical property, which protects the hosting bacteria against peroxide, explaining why this modification is maintained by diverse bacteria.
Nucleic Acids Research 07/2012; 40(18):9115-24. DOI:10.1093/nar/gks650 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Piericidins are a class of α-pyridone antibiotics that inhibit mitochondrial respiratory chain and exhibit antimicrobial, antifungal, and antitumor activities. Sequential analysis of Streptomyces piomogeues var. Hangzhouwanensis genome revealed six modular polyketide synthases, an amidotransferase, two methyltransferases, and a monooxygenase for piericidin A1 production. Gene functional analysis and deletion results provide overview of the biosynthesis pathway. Furthermore, in vitro characterization of the terminal polyketide synthase module with the thioesterase domain using β-ketoacyl substrates was performed. That revealed a pathway where the α-pyridone ring formation is dependent on hydrolysis of the product β, δ-diketo carboxylic acid by the C-terminal thioesterase followed by amidation and cyclization. These findings set the stage to investigate unusual enzymatic mechanisms in α-pyridone antibiotics biosynthesis, provide a foundation for genome mining of α-pyridone antibiotics, and produce analogs by molecular engineering.
[Show abstract][Hide abstract] ABSTRACT: A novel, site-specific, DNA backbone S-modification (phosphorothioation) has been discovered, but its in vivo function(s) have remained obscure. Here, we report that the enteropathogenic Salmonella enterica serovar Cerro 87, which possesses S-modified DNA, restricts DNA isolated from Escherichia coli, while protecting its own DNA by site-specific phosphorothioation. A cloned 15-kb gene cluster from S. enterica conferred both host-specific restriction and DNA S-modification on E. coli. Mutational analysis of the gene cluster proved unambiguously that the S-modification prevented host-specific restriction specified by the same gene cluster. Restriction activity required three genes in addition to at least four contiguous genes necessary for DNA S-modification. This functional overlap ensures that restriction of heterologous DNA occurs only when the host DNA is protected by phosphorothioation. Meanwhile, this novel type of host-specific restriction and modification system was identified in many diverse bacteria. As in the case of methylation-specific restriction systems, targeted inactivation of this gene cluster should facilitate genetic manipulation of these bacteria, as we demonstrate in Salmonella.
Nucleic Acids Research 11/2010; 38(20):7133-41. DOI:10.1093/nar/gkq610 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA phosphorothioation is widespread in many bacterial species. By homology analysis of the dnd gene cluster in Pseudomonas fluorescens Pf0-1, a spfBCDE gene cluster involved in DNA phosphorothioation was localized. Disruption of the spfD gene, a dndD homolog, caused the loss of the Dnd phenotype and demonstrated the involvement of spfD in DNA phosphorothioation in P. fluorescens Pf0-1. The ATPase activity of SpfD suggests that SpfD could hydrolyze ATP to provide the energy required in the DNA phosphorothioate modification process.
[Show abstract][Hide abstract] ABSTRACT: Finding the intermediate: ThiI is an enzyme that plays a role in the biosynthesis of both thiamine and 4-thiouridine at position 8 in bacterial tRNA. Using kinetic assays and mass spectrometry, we have identified ThiI as an ATP pyrophosphatase that catalyzes the hydrolysis of ATP to AMP and pyrophosphate. Our results provide direct evidence for the formation of an adenylation intermediate in the tRNA modification process.
[Show abstract][Hide abstract] ABSTRACT: A novel DNA modification system by sulfur (S) in Streptomyces lividans 66 was reported to be encoded by a cluster of five genes designated dndA-E [Zhou, X., He, X., Liang, J., Li, A., Xu, T., Kieser, T., Helmann, J. D., and Deng, Z. (2005) Mol. Microbiol. 57, 1428-1438]. The dndA gene was cloned and the protein product expressed in Escherichia coli, purified to homogeneity, and characterized as a homodimeric protein of ca. 91 kDa. Purified DndA has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate-containing enzyme and was proven to be a cysteine desulfurase able to catalyze removal of elemental S atoms from l-cysteine to produce l-alanine with substrate specificity similar to that of E. coli IscS. DndC was also purified to homogeneity and found to contain a 4Fe-4S cluster by spectral analysis and have obvious ATP pyrophosphatase activity. DndA could catalyze iron-sulfur cluster assembly by activation of apo-Fe DndC protein prepared by removal of its iron-sulfur cluster using alpha,alpha'-dipyridyl. A mutated DndA, with serine substituted for cysteine at position 327, which was confirmed to have lost its corresponding cysteine desulfurase activity, also lost its ability to reactivate the apo-Fe DndC. The likely involvement of an interaction between DndA and DndC in the biochemical pathway for the unusual site-specific DNA modification in S. lividans 66 is discussed.