The Putative Methyltransferase TlLAE1 Is Involved in the Regulation of Peptaibols Production in the Biocontrol Fungus Trichoderma longibrachiatum SMF2.

The biocontrol fungus Trichoderma longibrachiatum SMF2 secretes a large quantity of peptaibols that have been shown to have a range of biological activities and therefore great application values. However, the mechanism of the regulatory expression of peptaibols is still unclear. The putative methyltransferase LaeA/LAE1 is a global regulator involved in the biosynthesis of some secondary metabolites in filamentous fungi. In this study, we demonstrated that the ortholog of LaeA/LAE1 in the biocontrol fungus T. longibrachiatum SMF2, TlLAE1, plays an important role in the regulation of peptaibols production. Deletion of Tllae1 resulted in a slight negative impact on mycelial growth, and a significant defect in conidial production. Deletion of Tllae1 also compromised the production of peptaibols to a large degree. Further analyses indicated that this defect occurred at the transcriptional level of the two synthetases-encoding genes, tlx1 and tlx2, which are responsible for peptaibols production. By contrast, constitutive expression of Tllae1 in T. longibrachiatum SMF2 led to 2-fold increased peptaibols production, suggesting that this is a strategy to improve peptaibols production in Trichoderma fungi. These results demonstrate the important role of LAE1 in the regulation of peptaibols production in T. longibrachiatum SMF2.

Enhancing peptaibols production in the biocontrol fungus Trichoderma longibrachiatum SMF2 by elimination of a putative glucose sensor.

Trichoderma spp. are main producers of peptide antibiotics known as peptaibols. While peptaibols have been shown to possess a range of biological activities, molecular understanding of the regulation of their production is largely unclear, which hampers the production improvement through genetic engineering. Here, we demonstrated that the orthologue of glucose sensors in the outstanding biocontrol fungus Trichoderma longibrachiatum SMF2, TlSTP1, participates in the regulation of peptaibols production. Deletion of Tlstp1 markedly impaired hyphal growth and conidiation, but significantly increased peptaibols yield by 5-fold for Trichokonins A and 2.6-fold for Trichokonins B. Quantitative real-time polymerase chain reaction analyses showed that the increased peptaibols production occurs at the transcriptional levels of the two nonribosomal peptide synthetase encoding genes, tlx1 and tlx2. Transcriptome analyses of the wild type and the Tlstp1 mutant strains indicated that TlSTP1 exerts a regulatory effect on a set of genes that are involved in a number of metabolic and cellular processes, including synthesis of several other secondary metabolites. These results suggest an important role of TlSTP1 in the regulation of vegetative growth and peptaibols production in T. longibrachiatum SMF2 and provide insights into construction of peptaibol-hyperproducing strains through genetic engineering.

Mechanistic insight into 3-methylmercaptopropionate metabolism and kinetical regulation of demethylation pathway in marine dimethylsulfoniopropionate-catabolizing bacteria.

The vast majority of oceanic dimethylsulfoniopropionate (DMSP) is thought to be catabolized by bacteria via the DMSP demethylation pathway. This pathway contains four enzymes termed DmdA, DmdB, DmdC and DmdD/AcuH, which together catabolize DMSP to acetylaldehyde and methanethiol as carbon and sulfur sources respectively. While molecular mechanisms for DmdA and DmdD have been proposed, little is known of the catalytic mechanisms of DmdB and DmdC, which are central to this pathway. Here, we undertake physiological, structural and biochemical analyses to elucidate the catalytic mechanisms of DmdB and DmdC. DmdB, a 3-methylmercaptopropionate (MMPA)-coenzyme A (CoA) ligase, undergoes two sequential conformational changes to catalyze the ligation of MMPA and CoA. DmdC, a MMPA-CoA dehydrogenase, catalyzes the dehydrogenation of MMPA-CoA to generate MTA-CoA with Glu435 as the catalytic base. Sequence alignment suggests that the proposed catalytic mechanisms of DmdB and DmdC are likely widely adopted by bacteria using the DMSP demethylation pathway. Analysis of the substrate affinities of involved enzymes indicates that Roseobacters kinetically regulate the DMSP demethylation pathway to ensure DMSP functioning and catabolism in their cells. Altogether, this study sheds novel lights on the catalytic and regulative mechanisms of bacterial DMSP demethylation, leading to a better understanding of bacterial DMSP catabolism.

Mechanistic Insights into Dimethylsulfoniopropionate Lyase DddY, a New Member of the Cupin Superfamily.

The marine osmolyte dimethylsulfoniopropionate (DMSP) is one of Earth's most abundant organosulfur molecules. Bacterial DMSP lyases cleave DMSP, producing acrylate and dimethyl sulfide (DMS), a climate-active gas with roles in global sulfur cycling and atmospheric chemistry. DddY is the only known periplasmic DMSP lyase and is present in ß-, γ-, δ- and ε-proteobacteria. Unlike other known DMSP lyases, DddY has not been classified into a protein superfamily, and its structure and catalytic mechanism are unknown. Here, we determined the crystal structure of DddY from the γ-proteobacterium Acinetobacter bereziniae originally isolated from human clinical specimens. This structure revealed that DddY contains a cap domain and a catalytic domain with a Zn2+ bound at its active site. We also observed that the DddY catalytic domain adopts a typical ß-barrel fold and contains two conserved cupin motifs. Therefore, we concluded that DddY should belong to the cupin superfamily. Using structural and mutational analyses, we identified key residues involved in Zn2+ coordination, DMSP binding and the catalysis of DMSP cleavage, enabling elucidation of the catalytic mechanism, in which the residue Tyr271 of DddY acts as a general base to attack DMSP. Moreover, sequence analysis suggested that this proposed mechanism is common to DddY proteins from ß-, γ-, δ- and ε-proteobacteria. The DddY structure and proposed catalytic mechanism provide a better understanding of how DMSP is catabolized to generate the important climate-active gas DMS.

Cellular and molecular insight into the inhibition of primary root growth of Arabidopsis induced by peptaibols, a class of linear peptide antibiotics mainly produced by Trichoderma spp.

Trichoderma spp. are well known biocontrol agents that produce a variety of antibiotics. Peptaibols are a class of linear peptide antibiotics mainly produced by Trichoderma Alamethicin, the most studied peptaibol, is reported as toxic to plants at certain concentrations, while the mechanisms involved are unclear. We illustrated the toxic mechanisms of peptaibols by studying the growth-inhibitory effect of Trichokonin VI (TK VI), a peptaibol from Trichoderma longibrachiatum SMF2, on Arabidopsis primary roots. TK VI inhibited root growth by suppressing cell division and cell elongation, and disrupting root stem cell niche maintenance. TK VI increased auxin content and disrupted auxin response gradients in root tips. Further, we screened the Arabidopsis TK VI-resistant mutant tkr1. tkr1 harbors a point mutation in GORK, which encodes gated outwardly rectifying K(+)channel proteins. This mutation alleviated TK VI-induced suppression of K(+)efflux in roots, thereby stabilizing the auxin gradient. The tkr1 mutant also resisted the phytotoxicity of alamethicin. Our results indicate that GORK channels play a key role in peptaibol-plant interaction and that there is an inter-relationship between GORK channels and maintenance of auxin homeostasis. The cellular and molecular insight into the peptaibol-induced inhibition of plant root growth advances our understanding of Trichoderma-plant interactions.

Deep RNA sequencing reveals a high frequency of alternative splicing events in the fungus Trichoderma longibrachiatum.

BACKGROUND: Alternative splicing is crucial for proteome diversity and functional complexity in higher organisms. However, the alternative splicing landscape in fungi is still elusive. RESULTS: The transcriptome of the filamentous fungus Trichoderma longibrachiatum was deep sequenced using Illumina Solexa technology. A total of 14305 splice junctions were discovered. Analyses of alternative splicing events revealed that the number of all alternative splicing events (10034), intron retentions (IR, 9369), alternative 5' splice sites (A5SS, 167), and alternative 3' splice sites (A3SS, 302) is 7.3, 7.4, 5.1, and 5.9-fold higher, respectively, than those observed in the fungus Aspergillus oryzae using Illumina Solexa technology. This unexpectedly high ratio of alternative splicing suggests that alternative splicing is important to the transcriptome diversity of T. longibrachiatum. Alternatively spliced introns had longer lengths, higher GC contents, and lower splice site scores than constitutive introns. Further analysis demonstrated that the isoform relative frequencies were correlated with the splice site scores of the isoforms. Moreover, comparative transcriptomics determined that most enzymes related to glycolysis and the citrate cycle and glyoxylate cycle as well as a few carbohydrate-active enzymes are transcriptionally regulated. CONCLUSIONS: This study, consisting of a comprehensive analysis of the alternative splicing landscape in the filamentous fungus T. longibrachiatum, revealed an unexpectedly high ratio of alternative splicing events and provided new insights into transcriptome diversity in fungi.

Mercury distribution in sediment along urban-rural gradient around Shanghai (China): implication for pollution history.

The increasing levels of heavy metals in the environment generally related with the rapid industrialization and urbanization. Mercury (Hg) is a global toxin with wide concerns, and China gradually becomes the main producer, consumer, and emitter of Hg in the world. However, few historical data are available on the occurrence of Hg in Chinese urban areas. Here, we collected 35 lake surface sediment samples from 35 public parks and 1 sediment core in the Luxun Park in Shanghai, a hyper-urbanization city in China, to determine the spatial and vertical distributions of total mercury (THg) and methylmercury (MeHg) and to explore the Hg pollution history with the industrial development. Higher concentrations of Hg and MeHg and greater Hg enrichment were found in urban areas compared with suburban area with the following order: central urban core area > developed urban area > developing urban area > suburban area. The THg concentration in the sediment core showed an increasing trend from 1876 to 2000 and a decreasing trend from 2000 to 2012, coinciding with the process of industrialization and urbanization in Shanghai. However, THg fluxes unceasingly increased from 1876 to present probably attributed to coal consumption in the suburban area and transportation agglomeration in the central urban core area. Unlike THg, no significant variations for MeHg with time and the maximum value (0.17 µg/kg) appeared in 1947. The methylation ratio of MeHg to THg in the sediment is pretty low, and more studies are needed to further understand the fate of Hg in the environment.

Trichokonins from Trichoderma pseudokoningii SMF2 induce resistance against Gram-negative Pectobacterium carotovorum subsp. carotovorum in Chinese cabbage.

Peptaibols, mainly produced by Trichoderma, play a pivotal role in controlling plant disease caused by fungi, virus, and Gram-positive bacteria. In the current study, we evaluated the control effect of Trichokonins, antimicrobial peptaibols from Trichoderma pseudokoningii SMF2, on soft rot disease of Chinese cabbage caused by a Gram-negative bacterium Pectobacterium carotovorum subsp. carotovorum and analyzed the mechanism involved. Trichokonins treatment (0.3 mg L(-1) ) enhanced the resistance of Chinese cabbage against Pcc infection. However, Trichokonins could hardly inhibit the growth of Pcc in vitro, even at high concentration (500 mg L(-1) ). Therefore, the direct effect of Trichokonins on Pcc may not the main reason why Trichokonins could control soft rot of Chinese cabbage. Trichokonin treatment led to an obvious increase in the production of reactive oxygen species hydrogen peroxide and superoxide radical, a significant enhance of the activities of pathogenesis-related enzymes catalase, polyphenoloxidase and peroxidase, and upregulation of the expression of salicylic acid - responsive pathogenesis-related protein gene acidic PR-1a in Chinese cabbage. These results indicate that Trichokonins induce resistance in Chinese cabbage against Pcc infection through the activation of salicylic acid signaling pathway, which imply the potential of Trichoderma and peptaibols in controlling plant disease caused by Gram-negative bacteria.

Comprehensive risk assessment of heavy metals in lake sediment from public parks in Shanghai.

Rapid urbanization has caused potential pollution of heavy metal in Shanghai. A comprehensive pollution study of heavy metals (Cd, Cu, Pb, and Zn) in 35 lake surface sediments from city parks in the four different urbanized areas of Shanghai was conducted. Intensive human activities caused moderate enrichment of the four metals in highly urbanized areas, especially Cd with the significant enrichment in the central urban core area. However, the levels of the four metals in all the sediments were lower than the corresponding consensus-based Probable Effect Concentration, indicating adverse effects not to occur frequently. The integrated pollution assessments of multiple heavy metals also suggested low ecological risk and 15-29 percent probability of toxicity in most of sediments. The metal speciation analysis showed that Cd, Cu, Pb, and Zn were dominated by the non-residual fractions and thus they have high mobility and bioavailability, indicating significant anthropogenic sources. According to the Risk Assessment Code, Cd had the highest bioavailable fraction and represented high or very high risk, followed by Zn with medium or high risks in most of samples, while no or low risk was found for Cu and Pb at most sites because they were dominated by reducible and residual fractions. Correlation analysis showed that chemical fractions of heavy metals were prone to transform among each other if environmental conditions changed. Therefore, in view of anthropogenic inputs and speciation distribution, heavy metals with very high bioavailability at very low total levels and those with low bioavailability at very high total levels should not be ignored.

The effects of P6 electrical acustimulation on postoperative nausea and vomiting in patients after infratentorial craniotomy.

BACKGROUND: Postoperative nausea and vomiting (PONV) are frequent and harmful complications after neurosurgery. Current pharmacy-based treatment is the standard of care; it, however, lacks efficiency. Invasive and noninvasive acupuncture at the P6 meridian point has been shown to be effective in the prevention of PONV. We evaluated the effectiveness of transcutaneous electrical acupoint stimulation (TEAS) at P6 for the prophylaxis of PONV in patients undergoing infratentorial craniotomy. METHODS: In this prospective, blind, and randomized study, patients received TEAS at P6 on the dominant side starting 30 minutes before the induction of anesthesia and up to 24 hours after surgery or sham acustimulation at P6. The anesthesia was maintained with sevoflurane/remifentanil and intermittent fentanyl/cisatracurium. Antiemetics with 4 mg ondansetron and 10 mg dexamethasone were administered intraoperatively. Data documenting postoperative episodes of nausea and vomiting and the need for antiemetic rescue (10 mg metoclopramide intramuscularly) were collected. Statistical analysis was performed using the χ test. P<0.05 was considered to be significant. RESULTS: Of the 130 patients enrolled, 119 patients completed the study. The 24-hour cumulative incidence of vomiting was significantly lower in the TEAS group than in the control group (22% vs. 41%, P=0.025). The cumulative incidences of nausea at 6 hours (27% vs. 47%, P=0.019) and 24 hours (33% vs. 58%, P=0.008) after surgery were also significantly lower in the TEAS group compared with the control group. The overall requirements of rescue antiemetics were similar between the groups. CONCLUSION: Perioperative TEAS at P6 may be an effective adjunct to the standard antiemetic drug therapy for the prevention of PONV after infratentorial craniotomy.