In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex

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.National Natural Science Foundation (China) (Grant 31470183)National Natural Science Foundation (China) (Grant 31400029)National Natural Science Foundation (China) (Grant 31170085)National Natural Science Foundation (China) (Grant 30570400)National Natural Science Foundation (China) (Grant 31070058)China. Ministry of Science and Technology (Grant 2012CB721004)China. Ministry of Science and Technology (Grant 2009ZX09501-008)Shanghai Municipal Council of Science and Technology (Shanghai Pujiang Program Grant 12PJD021)China Scholarship CouncilNational Science Foundation (U.S.) (Grant CHE-1019990)National Institute of Environmental Health Sciences (Grant ES002109)Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology

Pathological phenotypes and in vivo DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella

Prokaryotes protect their genomes from foreign DNA with a diversity of defence 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, overexpression 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.National Natural Science Foundation (China) (Grant 31170085)National Natural Science Foundation (China) (Grant 31070058)Ministry of Science and Technology of the People's Republic of China (973 and 863 Programs)China Scholarship CouncilNational Science Foundation (U.S.) (Grant CHE-1019990)Shanghai Municipal Council of Science and Technology. Shanghai Pujiang Program (Grant 12PJD021

Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences

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. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of G[subscript ps]AAC/G[subscript ps]TTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at C[subscript ps]CA, 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.National Natural Science Foundation (China)Ministry of Science and Technology of the People's Republic of China (973 and 863 Programs)Shanghai Municipal Council of Science and Technology. Shanghai Pujiang ProgramNational Science Foundation (U.S.) (CHE-1019990)National Institute of Environmental Health Sciences (ES002109)Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology

Characterization of the Replication, Transfer, and Plasmid/Lytic Phage Cycle of the Streptomyces Plasmid-Phage pZL12▿ †

We report here the isolation and recombinational cloning of a large plasmid, pZL12, from endophytic Streptomyces sp. 9R-2. pZL12 comprises 90,435 bp, encoding 112 genes, 30 of which are organized in a large operon resembling bacteriophage genes. A replication locus (repA) and a conjugal transfer locus (traA-traC) were identified in pZL12. Surprisingly, the supernatant of a 9R-2 liquid culture containing partially purified phage particles infected 9R-2 cured of pZL12 (9R-2X) to form plaques, and a phage particle (φZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of φZL12 virions were encoded by pZL12 genes. Like bacteriophage P1, linear φZL12 DNA contained ends from a largely random pZL12 sequence. There was also a hot end sequence in linear φZL12. φZL12 virions efficiently infected only one host, 9R-2X, but failed to infect and form plaques in 18 other Streptomyces strains. Some 9R-2X spores rescued from lysis by infection of φZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lytic phage φZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces

Thermochemical processing of fuels involving the use of molecular oxygen

In a high-temperature (e.g., 1000 degrees C) atmosphere containing combustibles, it is naturally thought that air/O-2 should be kept away for fear of combustion and explosion. However, during thermochemical processing (TCP) of carbonaceous fuels (CFs) it&#39;s actually not that simple. TCP, the process for recovering CFs or converting problematic CFs into preferred ones, is a key technology for efficiently and relatively cleanly utilizing various CFs that supply the majority of the worldwide energy demand. However, TCP is often subject to some obstacles (e.g., high energy input and thermodynamic constraint). This review on extensive literature demonstrates that not only does introducing a controlled amount of O-2 (i.e., molecular oxygen) into TCP overcome its many obstacles, but also this O-2-assisted strategy is applicable to all of the main CFs currently known. Despite such a wide applicability to CFs, there lacks a comprehensive consideration of various aerobic TCP processes for understanding the O-2-assisted strategy as a kind of universal TCP technology for CFs. In this review, we for the first time summarize and discuss the use of O-2 in various TCP processes for all of the main CFs. We hope that this review can inspire more consideration of the various aerobic TCP processes so that these extremely complex processes can be communicated or consulted from each other&#39;s experience/research findings in order to enlighten, guide or promote their respective development.</p

Factors controlling soil organic carbon stability along a temperate forest altitudinal gradient.

Changes in soil organic carbon (SOC) stability may alter carbon release from the soil and, consequently, atmospheric CO2 concentration. The mean annual temperature (MAT) can change the soil physico-chemical characteristics and alter the quality and quantity of litter input into the soil that regulate SOC stability. However, the relationship between climate and SOC stability remains unclear. A 500-day incubation experiment was carried out on soils from an 11 °C-gradient mountainous system on Changbai Mountain in northeast China. Soil respiration during the incubation fitted well to a three-pool (labile, intermediate and stable) SOC decomposition model. A correlation analysis revealed that the MAT only influenced the labile carbon pool size and not the SOC stability. The intermediate carbon pool contributed dominantly to cumulative carbon release. The size of the intermediate pool was strongly related to the percentage of sand particle. The decomposition rate of the intermediate pool was negatively related to soil nitrogen availability. Because both soil texture and nitrogen availability are temperature independent, the stability of SOC was not associated with the MAT, but was heavily influenced by the intrinsic processes of SOC formation and the nutrient status

Factors controlling soil organic carbon stability along a temperate forest altitudinal gradient.

Changes in soil organic carbon (SOC) stability may alter carbon release from the soil and, consequently, atmospheric CO2 concentration. The mean annual temperature (MAT) can change the soil physico-chemical characteristics and alter the quality and quantity of litter input into the soil that regulate SOC stability. However, the relationship between climate and SOC stability remains unclear. A 500-day incubation experiment was carried out on soils from an 11 °C-gradient mountainous system on Changbai Mountain in northeast China. Soil respiration during the incubation fitted well to a three-pool (labile, intermediate and stable) SOC decomposition model. A correlation analysis revealed that the MAT only influenced the labile carbon pool size and not the SOC stability. The intermediate carbon pool contributed dominantly to cumulative carbon release. The size of the intermediate pool was strongly related to the percentage of sand particle. The decomposition rate of the intermediate pool was negatively related to soil nitrogen availability. Because both soil texture and nitrogen availability are temperature independent, the stability of SOC was not associated with the MAT, but was heavily influenced by the intrinsic processes of SOC formation and the nutrient status

Soil microbial community structure, metabolic potentials and influencing factors in a subtropical mountain forest ecosystem of China

This study investigated the variances in soil microbial community (SMC) structure and metabolic potentials in subtropical mountain forest ecosystem. Soil samples (0-10, 10-30 and 30-60 cm) were collected within 27 plots (20x20 m(2)) that were randomly assigned. Changes in SMC structure were mainly regulated by soil physicochemical property and tree diversity in 0-10 cm depth, by total nitrogen (TN) and clay in 10-30 cm depth, and by soil organic carbon (SOC) and pH in 30-60 cm depth. Metabolic potential variance was mainly driven by soil chemical property in 0-10 cm depth, by SOC and TN in 10-30 cm depth, and by TN in 30-60 cm depth. Collectively, soil physicochemical properties were the primary factors influencing SMC structure and its metabolic potentials along the soil profile, but the expected effects of tree diversity and topography were not evident

Lactic acid bacteria secrete toll like receptor 2 stimulating and macrophage immunomodulating bioactive factors

Lactic acid bacteria (LAB) can support host health and crosstalks between ligands on bacterial cells and Toll-like receptors (TLR) are known as key inducers of these health effects. LAB-secreted bioactive factors also possess health-promoting properties but the underlying mechanisms remain to be identified. Here, we report that secreted factors of different LAB strains induce cytokine (IL-10 and TNF-α) production by THP1-differentiated human macrophages in a species- and strain-dependent manner. LAB-secreted products also drive immune-stimulating and anti-pathogenic M1-macrophage polarization. Moreover, bacterial supernatants induce species- and strain-dependent TLR2 activation. Several bacterial strains such as L. casei CCFM9 and L. brevis CCFM498 cannot activate TLR signaling, but their secreted products strongly activate TLR2. This indicates that LAB-secreted components may further boost the functional effects of bacterial strains. Our results confirm direct interactions of LAB-secreted components with TLRs, and provide novel insights in how LABs may provide immune-modulation