Search

In the present study, the boiling mechanism in porous metals is investigated by Gong-Cheng phase-change lattice Boltzmann method. The complex geometry of porous metals is implemented in this study, which is different from the previous upscaling studies of boiling in porous metals, such as the method of representative elementary volume (REV). The detailed ebullition process at pore scale level is revealed, and the effects of wettability, porosity and porous structures on bubble behaviors and heat transfer are further analyzed. We found that bubble coalescence helps bubbles to be squeezed out of pores and bubbles accumulate more closely at the solid surface with increasing static contact angle. A thin liquid film forms between the bubble and solid surface with the static contact angle of 46.2°. Bubbles at the bottom of the porous metals are hard to escape, and bubbles coalesce frequently at the bottom of porous metals with lower porosity. Bubbles escape more easily in gradient porous metals than uniform porous metals in flow boiling simulation.

Gradient metal foams (GMFs) are novel porous structures and have great potential in heat transfer performance. In the present study, fully developed forced convective heat transfer in tubes sintered with partially-filled GMFs is numerically investigated. The inner surface of the tubes is subjected to constant heat flux. In the GMF region, the Brinkman extended Darcy flow model and the local thermal non-equilibrium (LTNE) model are used to predict fluid and thermal transport. At the layer-layer interface in GMF, fluid temperature and solid energy are assumed to be continuous. At the GMF-fluid interface, no-slip coupling conditions are used to couple flow and heat transfer of the foam and free regions. Velocity distribution, temperature profile, the friction factor and Nusselt number in partially-filled GMF tubes are calculated. The results show that the heat transfer performance and flow resistance of GMFs are heavily dependent on porosity, pore density and GMF thickness gradients.

Pyrene excimer usually serves as a chromogenic unit for developing ratiometric fluorescent sensors. But this study used excimer as a large hydrophobic group to regulate the molecular hydrophobicity, and obtained a new fluorescent sensor, N, N-bi[4(1-pyrene)-butyroyl]ornithine (1), for detection and removal of Fe3 + and Pb2 + from aqueous solutions. The coordination of 1 and Fe3 + in the aqueous solution or even pure water forms removable flocculent precipitates, accompanied by obvious fluorescent quenching of emission spectra. In aqueous solutions containing 40% (v/v) acetonitrile, the special responses exhibit a high selectivity and sensitivity to Fe3 + over other common metal ions. However, in aqueous solutions containing 40% (v/v) dimethylsulfoxide, the probe exhibits the analogous fluorescent quenching responses and the removable flocculent precipitates in the presence Fe3 + and Pb2 +. These results indicate that the extremely hydrophobic 1-Fe3 +/Pb2 + complexes are not only a supplement to the fluorescent sensing of Fe3 + and Pb2 +, but also a requirement to the removal of Fe3 + and Pb2 + from aqueous solutions.

DNA is the carrier of genetic information. DNA modifications play a central role in essential physiological processes. Phosphorothioation (PT) modification involves the replacement of an oxygen atom on the DNA backbone with a sulfur atom. PT modification can cause genomic instability in Salmonella enterica under hypochlorous acid stress. This modification restores hydrogen peroxide (H(2)O(2)) resistance in the catalase-deficient Escherichia coli Hpx(−) strain. Here, we report biochemical characterization results for a purified PT modification protein complex (DndCDE) from S. enterica. We observed multiplex oligomeric states of DndCDE by using native PAGE. This protein complex bound avidly to PT-modified DNA. DndCDE with an intact iron-sulfur cluster (DndCDE-FeS) possessed H(2)O(2) decomposition activity, with a V(max) of 10.58 ± 0.90 mM min(−1) and a half-saturation constant, K(0.5S), of 31.03 mM. The Hill coefficient was 2.419 ± 0.59 for this activity. The protein’s activity toward H(2)O(2) was observed to be dependent on the intact DndCDE and on the formation of an iron-sulfur (Fe-S) cluster on the DndC subunit. In addition to cysteine residues that mediate the formation of this Fe-S cluster, other cysteine residues play a catalytic role. Finally, catalase activity was also detected in DndCDE from Pseudomonas fluorescens Pf0-1. The data and conclusions presented suggest that DndCDE-FeS is a short-lived catalase. Our experiments also indicate that the complex binds to PT sites, shielding PT DNA from H(2)O(2) damage. This catalase shield might be able to extend from PT sites to the entire bacterial genome. IMPORTANCE DNA phosphorothioation has been reported in many bacteria. These PT-hosting bacteria live in very different environments, such as the human body, soil, or hot springs. The physiological function of DNA PT modification is still elusive. A remarkable property of PT modification is that purified genomic PT DNA is susceptible to oxidative cleavage. Among the oxidants, hypochlorous acid and H(2)O(2) are of physiological relevance for human pathogens since they are generated during the human inflammation response to bacterial infection. However, expression of PT genes in the catalase-deficient E. coli Hpx(−) strain restores H(2)O(2) resistance. Here, we seek to solve this obvious paradox. We demonstrate that DndCDE-FeS is a short-lived catalase that binds tightly to PT DNA. It is thus possible that by docking to PT sites the catalase activity protects the bacterial genome against H(2)O(2) damage.

Background How the assist parameters affect synchronization and inspiratory workload in proportional assist ventilation (PAV) remains unknown. Purpose This bench study aimed to optimize the PAV parameters by evaluating their effects on patient-ventilator synchrony and work of breathing (WOB) in a chronic obstructive pulmonary disease (COPD) model during noninvasive ventilation, compared with the pressure support ventilation (PSV) mode. Methods The Respironics V60 ventilator was connected to an ASL5000 lung simulator, which simulates lung mechanics in COPD (compliance, 50 mL/cmH2O; expiratory resistance, 20 cmH2O/L/s; respiratory rate, 15 breaths/min; inspiratory time, 1.6 s). PAV was applied with different assistance levels, including flow assist (FA, 40–90% respiratory resistance) and volume assist (VA, 50–90% elastance). PSV was assessed using the same model. Measurements were obtained at a leak flow rate of 25–28 L/min. Performance characteristics, simulator-ventilator synchrony, and WOB were assessed. Results Runaway was prone to occur, and severe premature cycling was observed with VA75+FA level > 65%. Compared with PSV, lower tidal volume (≤400 mL) was observed during PAV with VA75+FA40-50 and FA50+VA40-80; similar and improved cycling synchrony was observed for FA50+VA80 and FA50+VA90 (cycling delay: −117.60 ± 6.13 and −61.50 ± 8.03 vs. −101.20 ± 7.32 ms). The reduced triggering workload was observed for VA75+FA60-65 and FA50+VA80-90. Total and patient WOB was improved with all tested assist level combinations, except for FA50+VA90. Conclusions PAV reduces WOB but can induce asynchrony if improper settings are set, but the most optimal settings still need more clinical observations.

In the flavin-reductase-catalyzed reducing condition, a mild and efficient α-hydroxylation and decarboxylation procedure using natural flavins as a catalyst and atmospheric oxygen as an external oxidizing agent has been successfully developed and applied to the synthesis of streptonigrone from streptonigrin. This reaction was achieved not only with streptonigrin analogues but also with structurally diverse electron-rich picolinic acid derivatives. The hydroxylation and decarboxylation may take place in a concerted manner.

Calcimycin, N-demethyl calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis . A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between calcimycin and cezomycin. In this study, we observed that when a Cal − ( calB1 mutant) derivative of a calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, calcimycin production was restored. This suggested that calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal − S. chartreusis mutants lacking the ability to convert cezomycin to calcimycin allowed the identification of a gene, provisionally named calC , which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein's apparent K m and k cat for cezomycin were observed to be 190 μM and 3.98 min −1 , respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form. IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both calcimycin's biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues', divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for calcimycin's production.

Type II thioesterases typically function as editing enzymes, removing acyl groups that have been misconjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic, and we have identified the presence of a putative type II thioesterase-like sequence, calG , within the biosynthetic gene cluster involved in the antibiotic's synthesis. However, targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in calcimycin production but rather reduced the strain's production of its biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin coenzyme A (cezomycin-CoA), as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC; the mimic of the polyketide chain-releasing precursor). Further investigation of the enzyme's activity showed that it possessed a cezomycin-CoA hydrolysis K m of 0.67 mM and a k cat of 17.77 min −1 and was significantly inhibited by the presence of Mn 2+ and Fe 2+ divalent cations. Interestingly, when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO 2 , it was observed that the production of calcimycin rather than cezomycin was promoted. Also, supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca 2+ , Mg 2+ , Mn 2+ , and Fe 2+ had a similar effect. Taken together, these observations suggest that CalG is not responsible for megasynthase polyketide precursor chain release during the synthesis of calcimycin or for retaining the catalytic efficiency of the megasynthase enzyme complex as is supposed to be the function for type II thioesterases. Rather, our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of type II thioesterases. IMPORTANCE Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production, thereby maintaining the efficiency of the megasynthase involved in this class of secondary metabolites' biosynthesis. Specifically, this class of enzyme is believed to be involved in editing misprimed precursors, controlling initial units, providing key intermediates, and releasing final synthetic products in the biosynthesis of this class of secondary metabolites. Our results indicate that the putative TEII CalG present in the calcimycin (A23187)-producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or for the release of the product compound from, the megasynthase involved in calcimycin biosynthesis. Rather, the enzyme is involved in regulating/controlling the pool size of the calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolites.

Analysis of the oligomeric state of a protein may provide insights into its physiological functions. Because membrane proteins are considered to be the workhorses of energy generation and polypeptide and nutrient transportation, in this study we characterized the membrane-associated proteome of Streptomyces coelicolor by two-dimensional (2D) blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high-resolution clear native/native PAGE, and native/SDS-PAGE. A total of 77 proteins were identified, and 20 proteins belonging to 15 complexes were characterized. Moreover, the resolution of high-resolution clear native/SDS-PAGE is much higher than that of blue native/SDS-PAGE. OBP (SCO5477) and BldKB (SCO5113) were identified as the main protein spots from the membrane fractions of S. coelicolor M145, suggesting that these two proteins are involved in extracellular peptide transportation. These two transporters exhibited multiple oligomeric states in the native PAGE system, which may suggest their multiple physiological functions in the development of S. coelicolor.

Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 μM, kcat 1.26 min(-1) and kcat/Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.

DNA phosphorothioation, conferred by dnd genes, was originally discovered in the soil-dwelling bacterium Streptomyces lividans, and thereafter found to exist in various bacterial genera. However, the physiological significance of this sulfur modification of the DNA backbone remains unknown in S. lividans. Our studies indicate that DNA phosphorothioation has a major role in resistance to oxidative stress in the strain. Although Streptomyces species express multiple catalase/peroxidase and organic hydroperoxide resistance genes to protect them against peroxide damage, a wild type strain of S. lividans exhibited two-fold to 10-fold higher survival, compared to a dnd (-) mutant, following treatment with peroxides. RNA-seq experiments revealed that, catalase and organic hydroperoxide resistance gene expression were not up-regulated in the wild type strain, suggesting that the resistance to oxidative stress was not due to the up-regulation of these genes by DNA phosphorothioation. Quantitative RT-PCR analysis was conducted to trace the expression of the catalase and the organic hydroperoxide resistance genes after peroxides treatments. A bunch of these genes were activated in the dnd (-) mutant rather than the wild type strain in response to peroxides. Moreover, the organic hydroperoxide peracetic acid was scavenged more rapidly in the presence than in the absence of phosphorothioate modification, both in vivo and in vitro. The dnd gene cluster can be up-regulated by the disulfide stressor diamide. Overall, our observations suggest that DNA phosphorothioate modification functions as a peroxide resistance system in S. lividans.

A highly selective and sensitive ratiometric and "turn-on" fluorescent probe for Fe(3+), 2-(1-pyrenyl) benzimidazole (L), was synthesized by a one-step process. In emission spectra, the relative intensity ratio of excimer to monomer fluorescence (IE450/IM387) of L increased 510-fold upon the addition of 30 equiv. of Fe(3+) with a detection limit of 0.2 μM (11.2 ppb) in aqueous solution. Meanwhile, the fluorescence excitation spectra of L showed a fluorescent "turn-on" probe for Fe(3+) with 30-fold enhancement in excitation band intensity of excimer.

Pyrene excimer usually serves as a chromogenic unit for developing ratiometric fluorescent sensors. But this study used excimer as a large hydrophobic group to regulate the molecular hydrophobicity, and obtained a new fluorescent sensor, N, N-bi[4(1-pyrene)-butyroyl]ornithine (1), for detection and removal of Fe

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.

During the fermentation of Streptomyces hygroscopicus TL01 to produce validamycin A (18 g/L), a considerable amount of an intermediate validoxylamine A (4.0 g/L) is accumulated. Chemical or enzymatic hydrolysis of validamycin A was not observed during the fermentation process. Over-expression of glucosyltransferase ValG in TL01 did not increase the efficiency of glycosylation. However, increased validamycin A and decreased validoxylamine A production were observed in both the cell-free extract and fermentation broth of TL01 supplemented with a high concentration of UDP-glucose. The enzymatic activity of UDP-glucose pyrophosphorylase (Ugp) in TL01, which catalyzes UDP-glucose formation, was found to be much lower than the activities of other enzymes involved in the biosynthesis of UDP-glucose and the glucosyltransferase ValG. An ugp gene was cloned from S. hygroscopicus 5008 and verified to code for Ugp. In TL01 with an extra copy of ugp, the transcription of ugp was increased for 1.5 times, and Ugp activity was increased by 100%. Moreover, 22 g/L validamycin A and 2.5 g/L validoxylamine A were produced, and the validamycin A/validoxylamine A ratio was increased from 3.15 in TL01 to 5.75. These data prove that validamycin A biosynthesis is limited by the supply of UDP-glucose, which can be relieved by Ugp over-expression.

Avermectins are 16-membered macrocyclic polyketides with potent antiparasitic activities, produced by Streptomyces avermitilis. Upstream of the avermectin biosynthetic gene cluster, there is the avtAB operon encoding the ABC transporter AvtAB, which is highly homologous to the mammalian multidrug efflux pump P-glycoprotein (Pgp). Inactivation of avtAB had no effect, but increasing the concentration of avtAB mRNA 30-500-fold, using a multi-copy plasmid in S. avermitilis, enhanced avermectin production about two-fold both in the wild-type and in a high-yield producer strain on agar plates. In liquid industrial fermentation medium, the overall productivity of avermectin B1a in the engineered high-yield producer was improved for about 50%, from 3.3 to 4.8 g/l. In liquid YMG medium, moreover, the ratio of intracellular to extracellular accumulation of avermectin B1a was dropped from 6:1 to 4.5:1 in response to multiple copies of avtAB. Additionally, the overexpression of avtAB did not cause any increased expression of the avermectin biosynthetic genes through RT-PCR analysis. We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production inside the cell. This strategy may be useful for enhancing the production of other antibiotics.

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 remaining within bacterial protein samples from Streptomyces lividans and Escherichia coli were found to interfere significantly with blue native polyacrylamide gel electrophoresis (BN-PAGE), a technique used frequently for analyzing bacterial protein complexes in proteomics studies. We have used ultracentrifugation and/or precipitation of cell lysates with streptomycin sulfate to eliminate nucleic acids from total and/or membrane protein samples. Nucleic acid-binding proteins were first enriched by precipitation with streptomycin sulfate, and contaminating nucleic acids were then eliminated by precipitation by adding polyethyleneimine. The performance of BN-PAGE was found to be dramatically improved by these sample preparation steps.

Cultivation shift from 30° to 37° significantly enhanced validamycin (VAL) production. Analyzed by reverse-transcription PCR, the transcription of three val genes, valA, valK and valG, representing the three operons of the cluster was simultaneously increased at elevated temperature. Furthermore, the transcription of valP and valQ, a pair of two-component regulators in validamycin biosynthetic gene cluster, was also increased at 37°. Inactivation of valP and valQ reduced validamycin production at 37° to the yield level of wild type strain at 30°, and the val genes showed reduced expression in the mutant LL-8 at 37°. These results revealed that the two-component regulator valP and valQ contribute to the elevated validamycin production.

The complete DNA sequence of plasmid pHZ1358, a widely used vector for targeted gene disruption and replacement experiments in many Streptomyces hosts, has been determined. This has allowed a detailed analysis of the basis of its structural and segregational instability, compared to the high copy number plasmid pIJ101 of Streptomyces lividans 1326 from which it was derived. A 574-bp DNA region containing sti (strong incompatibility locus) was found to be a determinant for segregational instability in its original S. lividans 1326 host, while the structural instability was found to be related to the facile deletion of the entire Escherichia coli-derived part of pHZ1358, mediated by recombination between 36-bp direct repeats. A point mutation removing the BamHI site inside the rep gene encoding a replication protein (rep*) and/or a spontaneous deletion of the 694-bp region located between rep and sti including the uncharacterized ORF85 (orf85(-)) produced little or no effect on stability. A pHZ1358 derivative (pJTU412, sti(-), rep*, orf85(-)) was then constructed which additionally lacked one of the 36-bp direct repeats. pJTU412 was demonstrated to be structurally stable but segregationally unstable and, in contrast to sti(+) pHZ1358, allowed efficient targeted gene replacement in S. lividans 1326.

Gene fscTE , encoding a putative type II thioesterase (TEII), was associated with the FR-008/candicidin gene cluster. Deletion of fscTE reduced approximately 90% of the FR-008/candicidin production, while the production level was well restored when fscTE was added back to the mutant in trans . FscTE was unable to compensate for the release of the maturely elongated polyketide as site-directed inactivation of the type I thioesterase (TEI) totally abolished FR-008/candicidin production. Direct biochemical analysis of FscTE in parallel with its homologue TylO from the tylosin biosynthetic pathway demonstrated their remarkable preferences for acyl-thioesters (i.e., propionyl- S - N -acetylcysteamine [SNAC] over methylmalonyl-SNAC and acetyl-SNAC over malonyl-SNAC) and thus concluded that TEII could maintain effective polyketide biosynthesis by selectively removing the nonelongatable residues bound to acyl carrier proteins. Overexpression of FscTE under the strong constitutive ermE * p promoter in the wild-type strain did not suppress FR-008/candicidin formation, which confirmed its substrate specificity in vivo. Furthermore, successful complementation of the fscTE mutant was obtained with fscTE and tylO , whereas no complementation was detected with nonribosomal peptide synthetase (NRPS) TEII tycF and srfAD , reflecting substrate specificities of TEIIs distinctive from those of either polyketide synthases or NRPSs.

SummaryThe polyketide backbone of the polyether ionophore antibiotic nanchangmycin (1) is assembled by a modular polyketide synthase in Streptomyces nanchangensis NS3226. The ACP-bound polyketide is thought to undergo a cascade of oxidative cyclizations to generate the characteristic polyether. Deletion of the glycosyl transferase gene nanG5 resulted in accumulation of the corresponding nanchangmycin aglycone (6). The discrete thioesterase NanE exhibited a nearly 17-fold preference for hydrolysis of 4, the N-acetylcysteamine (SNAC) thioester of nanchangmycin, over 7, the corresponding SNAC derivative of the aglycone, consistent with NanE-catalyzed hydrolysis of ACP-bound nanchangmycin being the final step in the biosynthetic pathway. Site-directed mutagenesis established that Ser96, His261, and Asp120, the proposed components of the NanE catalytic triad, were all essential for thioesterase activity, while Trp97 was shown to influence the preference for polyether over polyketide substrates.

Sequence analysis of oxazolomycin (OZM) biosynthetic gene cluster from Streptomyces albus JA3453 revealed a gene, ozmH, encoding a hybrid polyketide and non-ribosomal peptide enzyme. Tandem ketosynthase (KS) domains (KS10-1 and KS10-2) were characterized and they show significant homology with known KSs. Using an alternative method that involves RecA-mediated homologous recombination, the negative selection marker sacB gene, and temperature-sensitive replications, site-directed mutagenesis of the catalytic triad amino acid cysteines were carried out in each of the tandem KS domains to test the function they play in OZM biosynthesis. HPLC-mass spectrometry analysis of the resulting mutant strains showed that KS10-2 is essential for OZM biosynthesis but KS10-1 is not indispensable and might serve as a “redundant” domain. These results confirmed the existence of an “extra domain” in complex polyketide synthase.

The complete sequence (92 770 bp) of a genomic island (GI) named SLG from Streptomyces lividans 66, encoding a novel DNA S-modification system (dnd), was determined. Its overall G+C content was 67.8%, lower than those of three sequenced Streptomyces genomes. Among 85 predicted open reading frames (ORFs) in SLG, 22 ORFs showed little homology with previously known proteins. SLG displays a mosaic structure composed of four modules, indicative of multiple recombination events in its formation. Spontaneous excision and circularization of SLG was observed, and the excision rate appeared to be induced at least fivefold by MNNG exposure. Using constructed mini-islands of SLG, we demonstrated that Slg01, a P4-like integrase, was sufficient to promote SLG integration, excision and circularization. Eleven counterpart dnd clusters, which also mapped to GIs in 10 chromosomes and a plasmid, were found in taxonomically unrelated bacterial species from various geographic niches. Additionally, c. 10% of actinomycetes were found to possess a dnd cluster in a survey involving 74 strains. Comparison of dnd clusters in the 12 bacteria strongly suggests that these dnd-bearing elements might have evolved from a common ancestor similar to plasmid-originated chromosome II of Pseudoalteromonas haloplanktis TAC125.

Streptomyces coelicolor A3(2) does not have a canonical cell division cycle during most of its complex life cycle, yet it contains a gene ( ftsK SC ) encoding a protein similar to FtsK, which couples the completion of cell division and chromosome segregation in unicellular bacteria such as Escherichia coli . Here, we show that various constructed ftsK SC mutants all grew apparently normally and sporulated but upon restreaking gave rise to many aberrant colonies and to high frequencies of chloramphenicol-sensitive mutants, a phenotype previously associated with large terminal deletions from the linear chromosome. Indeed, most of the aberrant colonies had lost large fragments near one or both chromosomal termini, as if chromosome ends had failed to reach their prespore destination before the closure of sporulation septa. A constructed FtsK SC -enhanced green fluorescent protein fusion protein was particularly abundant in aerial hyphae, forming distinctive complexes before localizing to each sporulation septum, suggesting a role for FtsK SC in chromosome segregation during sporulation. Use of a fluorescent reporter showed that when ftsK SC was deleted, several spore compartments in most spore chains failed to express the late-sporulation-specific sigma factor gene sigF , even though they contained chromosomal DNA. This suggested that sigF expression is autonomously activated in each spore compartment in response to completion of chromosome transfer, which would be a previously unknown checkpoint for late-sporulation-specific gene expression. These results provide new insight into the genetic instability prevalent among streptomycetes, including those used in the industrial production of antibiotics.

SC7A1 is a cosmid with an insert of chromosomal DNA from Streptomyces coelicolor A3(2). Its insertion into the chromosome of S. coelicolor strains caused a duplication of a segment of ca. 40 kb and delayed actinorhodin antibiotic production and sporulation, implying that SC7A1 carried a gene negatively affecting these processes. The subcloning of SC7A1 insert DNA resulted in the identification of the open reading frame SCO5582 as nsdA , a gene n egatively affecting S treptomyces d ifferentiation. The disruption of chromosomal nsdA caused the overproduction of spores and of three of four known S. coelicolor antibiotics of quite different chemical types. In at least one case (that of actinorhodin), this was correlated with premature expression of a pathway-specific regulatory gene ( actII-orf4 ), implying that nsdA in the wild-type strain indirectly repressed the expression of the actinorhodin biosynthesis cluster. nsdA expression was up-regulated upon aerial mycelium initiation and was strongest in the aerial mycelium. NsdA has DUF921, a Streptomyces protein domain of unknown function and a conserved SXR site. A site-directed mutation (S458A) in this site in NsdA abolished its function. Blast searching showed that NsdA homologues are present in some Streptomyces genomes. Outside of streptomycetes, NsdA-like proteins have been found in several actinomycetes. The disruption of the nsdA -like gene SCO4114 had no obvious phenotypic effects on S. coelicolor . The nsdA orthologue SAV2652 in S. avermitilis could complement the S. coelicolor nsdA -null mutant phenotype.

In the arsenic resistance gene cluster from the large linear plasmid pHZ227, two novel genes, arsO (for a putative flavin-binding monooxygenase) and arsT (for a putative thioredoxin reductase), were coactivated and cotranscribed with arsR1-arsB and arsC , respectively. Deletion of the ars gene cluster on pHZ227 in Streptomyces sp. strain FR-008 resulted in sensitivity to arsenic, and heterologous expression of the ars gene cluster in the arsenic-sensitive Streptomyces strains conferred resistance on the new hosts. The pHZ227 ArsB protein showed homology to the yeast arsenite transporter Acr3p. The pHZ227 ArsC appears to be a bacterial thioredoxin-dependent ArsC-type arsenate reductase with four conserved cysteine thioredoxin-requiring motifs.

SummaryA 45 kb DNA sequencing analysis from Streptomyces hygroscopicus 5008 involved in validamycin A (VAL-A) biosynthesis revealed 16 structural genes, 2 regulatory genes, 5 genes related transport, transposition/integration or tellurium resistance; another 4 genes had no obvious identity. The VAL-A biosynthetic pathway was proposed, with assignment of the required genetic functions confined to the sequenced region. A cluster of eight reassembled genes was found to support VAL-A synthesis in a heterologous host, S. lividans 1326. In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for VAL production and resulted in accumulation of the VAL-A precursor, validoxylamine, while the normal production of VAL-A could be restored by complementation with valG. The role of valG in the glycosylation of validoxylamine to VAL-A was demonstrated in vitro by enzymatic assay.

Streptomyces lividans has a novel DNA modification, which sensitises its DNA to degradation during electrophoresis (the Dnd phenotype). The entire gene cluster (dnd) involved in this modification was localized on an 8 kb DNA fragment and was expressed in a S. lividans deletion mutant (dnd) and in several heterologous hosts. Disruption of the dnd locus abolishes the Dnd phenotype, and gain of the dnd locus conferred the Dnd phenotype respectively. Extensive analysis of the dnd gene cluster revealed five open reading frames, whose hypothetic functions suggested an incorporation of sulphur or a sulphur-containing substance into S. lividans genome, yet in an unknown manner. The Dnd phenotype was also discovered to exist in DNA of widespread bacterial species of variable origin and diverse habitat. Similarly organized gene clusters were found in several bacterial genomes representing different genera and in eDNA of marine organisms, suggesting such modification as a widespread phenomenon. A coincidence between the Dnd phenotype and DNA modification by sulphur was demonstrated to occur in several representative bacterial genomes by the in vivo(35)S-labelling experiments.

Dark brown haloes of melanin around colonies are an easily visualized phenotype displayed by many Streptomyces strains harboring plasmid pIJ702 carrying the melC operon of Streptomyces antibioticus IMRU3270. Spontaneous melanin-negative mutants of pIJ702 occur with a frequency of ca. 1%, and often mutation occurs in the melC operon, which removes the BglII site as part of an inverted repeat. Other melanin-negative mutations seem to occur spontaneously in Streptomyces lividans , resulting in white colonies from which intact, melanin-producing pIJ702 can be isolated by introduction into a new host. S. lividans ZX66 was found to be such a mutant and to have a secondary mutation influencing expression of the melC operon on the chromosome. A 3.3-kb DNA fragment was isolated from its progenitor strain, JT46, and a gene able to restore melC operon expression was found to encode a member of an AraC family of transcriptional regulators, which was equivalent to AdpA c in Streptomyces coelicolor and therefore was designated AdpA l . Lack of melC operon expression was correlated with a single A-to-C transversion, which altered a single key amino acid residue from Thr to Pro. The transcription of the melC operon was found to be greatly reduced in the adpA mutant background. The counterpart gene ( adpA a ) in the S. antibioticus strain in which the melC operon carried on pIJ702 originated was also isolated and was found to have an identical regulatory role. Thus, we concluded that the melC operon is under general direct positive control by AdpA family proteins, perhaps at the transcriptional level and certainly at the translational level via bldA , in Streptomyces .

The PKS genes for biosynthesis of the polyether nanchangmycin are organized to encode two sets of proteins (six and seven ORFs, respectively), but are separated by independent ORFs that encode an epimerase, epoxidase, and epoxide hydrolase, and, notably, an independent ACP. One of the PKS modules lacks a corresponding ACP. We propose that the process of oxidative cyclization to form the polyether structure occurs when the polyketide chain is still anchored on the independent ACP before release. 4-O-methyl-L-rhodinose biosynthesis and its transglycosylation involve four putative genes, and regulation of nanchangmycin biosynthesis seems to involve activation as well as repression. In-frame deletion of a KR6 domain generated the nanchangmycin aglycone with loss of 4-O-methyl-L-rhodinose and antibacterial activity, in agreement with the assignments of the PKS domains catalyzing specific biosynthetic steps.

Streptomyces hygroscopicus 10-22 harbors a conjugative, autonomously replicating linear plasmid pHZ6 of ca. 70 kb, which shows no obvious homology with chromosomal DNA and is temperature-sensitive for replication, being stable in the host at 28 degrees C but easily lost at 37 degrees C. On a lawn of the wild-type S. hygroscopicus 10-22 cured of pHZ6, pHZ6 elicit pocks. Temperature sensitivity seemed to be a unique property for pHZ6 among six linear plasmids tested, including the well-known linear plasmids SLP2 in Streptomyces lividans 1326 and SCP1 in Streptomyces coelicolor A3(2). The distinct identity of pHZ6 from previously identified pHZ1-pHZ5 was demonstrated by the profile of relevant plasmids in six well-defined strains originated from S. hygroscopicus 10-22.

Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. To better understand PT biology, we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: real-time (SMRT) sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of GpsAAC/GpsTTC motifs, but with only 18% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at CpsCA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT modification proteins and rule out known R-M mechanisms.

DNA phosphorothioate modification means substituting a non-bridging oxygen with a sulfur in DNA. The modification endows DNA with such chemical property that protects the hosting bacteria against peroxide. The modification is controlled by a dnd gene cluster. Salmonella entericaserovar Cerro 87 is one of the bacteria that harbor the DNA phosphorothioate modification. The modification is carried out by dptB, C, DandE. Ourstudy is designed to clone and express dptC, to optimize the expressing condition, and then to purify the DptC.dptC DNA fragment was amplified by KOD PCR with the special primers and S. entericaserovar Cerro 87 genomic DNA template. A fusion expression vector pJTU3622 was constructed by inserting the dptC DNA fragment into pGEX-6P-1 inSmaI and XhoI sites. The positive clone was verified by antibiotics resistance gene screening and sequenced, and then transferred into host strain E. coli BL21 (DE3) pLysS to producean engineering bacterium Anxh103. After optimizing the expression condition for dptC, we purified DptC from Anxh103 by Aikta FPLC with a GST-Trap column.A fusion expression vector pJTU3622 and an engineering bacterium Anxh103 were produced. The optimizing expressing condition for dptC is as follows: induced at 18 degrees C for 8 - 18 h; 0.6 mmol/L IPTG, LB with 50 micromol/L Fe2+.The anchor redeemed for high throughput expression of dptC. The TEV site in pJTU3622 made the process of purifying DptC easier and simpler. This helps lay the ground work for future study on the function of DptC. Also, the light brown color of DptC and the medium with 50 micromol/L Fe2+ showed us DptC has the same character with DndC which belongs to an iron-sufur protein with 4Fe - 4S.

DNA phosphorothioate modification (DNA sulfur modification, a non-bridging oxygen swapped with a sulfur) exists in diverse bacteria. Salmonella enterica serovar Cerro 87 is one of the bacteria that harbor the DNA sulfur modification. The modification is carried out by the products of a four-membered gene cluster, dptBCDE. Transformation of Escherichia coli DH10B with the dptBCDE gene cluster endows the strain with DNA sulfur modification capability. Deletion of dptC abolished the modification. Here, we studied the function of dptC in DNA sulfur modification.Six cysteine residues in dptC were mutated individually within the dptBCDE gene cluster. Mutants were then tested for DNA sulfur modification.Among the 6 cysteine mutations (C39, C146, C262, C273, C280, and C283), 5 abolished DNA modification except for C39, suggesting that C146, C262, C273, C280, and C283 are essential for DNA sulfur modification. Sequence alignment shows that these five cysteine residues are conserved among different strains.Mutation at anyone of C146, C262, C273, C280 and C283 of dptC abolished DNA modification. Our results shed light on further study of DNA sulfur modification biochemical pathway.