Contribution of the Fenton reaction to the degradation of carbon nanotubes by enzymes

The widespread use of carbon nanotubes (CNTs) has raised concerns about the human health and ecological effects of CNTs released into the environment. Bacteria play an important role in bioremediation and waste treatment, and their enzymes are mostly responsible for the degradation of contaminants. However, there are still only a few reports about the bacterial degradation of CNTs, and evidence showing the involvement of bacterial enzymes in CNT degradation with their mechanisms has never been reported. The purpose of this study is to clarify whether CNTs can be degraded by bacterial enzymes. In this study, the degradation of oxidized (carboxylated) single-walled CNTs (O-SWCNTs) by mt2DyP, a dye-decolorizing peroxidase of Pseudomonas putida mt-2, a common soil bacterium, was investigated. After incubation of O-SWCNTs with recombinant mt2DyP and its substrate H2O2 for 30 d, the optical absorbance and Raman spectra revealed the degradation of O-SWCNTs. However, inactivation of the enzyme was observed within 60 min of the start of incubation, suggesting that the degradation of O-SWCNTs occurred nonenzymatically. The inactivation of mt2DyP was accompanied by the release of iron, the active center metal, and degradation of O-SWCNTs was significantly inhibited in the presence of diethylenetriamine pentaacetic acid, a chelating agent, indicating that O-SWCNTs were degraded by the Fenton reaction with iron released from mt2DyP and H2O2. The same phenomenon was observed with P450, which is also a heme enzyme. Furthermore, we investigated the contribution of the Fenton reaction to the O-SWCNT degradation by horseradish peroxidase (HRP), which was reported to enzymatically and rapidly degrade O-SWCNTs. Our results revealed that the degradation of O-SWCNTs in the presence of HRP is also mainly due to the Fenton reaction, with negligible enzymatic degradation. This contradicts the report showing enzymatic degradation of O-SWCNTs by HRP but supports the subsequent report quantitatively showing very slow transformation of O-SWCNTs by HRP. The current results emphasize that the Fenton reaction, which has received little attention in CNT degradation by heme enzymes, must be taken into consideration and will contribute to the development of a simple disposal method for CNTs, utilizing the Fenton reaction with bacteria/bacterial enzymes and H2O2

Effects of glycosylation on swimming ability and flagellar polymorphic transformation in Pseudomonas syringae pv. tabaci 6605

The role of flagellin glycosylation on motility was investigated in Pseudomonas syringae pv. tabaci. The swimming activity of glycosylation-defective mutants was prominently decreased in a highly viscous medium. The mutants showed differences in polymorphic transitions and in the bundle formation of flagella, indicating that glycosylation stabilizes the filament structure and lubricates the rotation of the bundle.</p

PsyR, a transcriptional regulator in quorum sensing system, binds lux box-like sequence in psyI promoter without AHL quorum sensing molecule and activates psyI transcription with AHL in Pseudomonas syringae pv. tabaci 6605

Quorum sensing (QS) is a mechanism for bacterial cell-cell communication using QS signals. N-acyl-homoserine lactones (AHLs), QS signals in Pseudomonas syringae pv. tabaci (Pta) 6605, are synthesized by an AHL synthase (PsyI) and recognized by the cognate transcription factor PsyR. To reveal the role of PsyR in virulence, we generated a psyR mutant and complemented strains of Pta 6605 and found that the psyR mutant is remarkably reduced in AHL production and ability to cause disease and propagate in host tobacco leaves. The phenotypes of complemented strains were restored to that of the wild type (WT). Because the psyR mutant lost nearly all AHL production, we investigated the function of PsyR in the transcription of psyI and production of AHL. Electrophoretic mobility shift assays suggested that the recombinant PsyR protein binds the promoter region of psyI but not psyR without AHL. The addition of AHL did not significantly affect this binding. The binding core sequence of this region was identified as a 20-bp lux box-like sequence. To reveal the function of PsyR and AHL on psyI transcription, we constructed a psyI promoter::lacZYA chimeric reporter gene, and inserted it into the WT and psyI mutant of Pta 6605. beta-galactosidase activity increased in a bacterial density-dependent manner in the WT and also in a psyI mutant after the addition of exogenous AHL. These results indicate that the solo PsyR binds the lux box in the psyI promoter and activates transcription in the concomitant presence of AHL

Cluster II che genes of Pseudomonas syringae pv. tabaci 6605, orthologs of cluster I in Pseudomonas aeruginosa, are required for chemotaxis and virulence

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a causal agent of wildfire disease in host tobacco plants and is highly motile. Pta6605 has multiple clusters of chemotaxis genes including cheA, a gene encoding a histidine kinase, cheY, a gene encoding a response regulator, mcp, a gene for a methyl-accepting chemotaxis protein, as well as flagellar and pili biogenesis genes. However, only two major chemotaxis gene clusters, cluster I and cluster II, possess cheA and cheY. Deletion mutants of cheA or cheY were constructed to evaluate their possible role in Pta6605 chemotaxis and virulence. Motility tests and a chemotaxis assay to known attractant demonstrated that cheA2 and cheY2 mutants were unable to swarm and to perform chemotaxis, whereas cheA1 and cheY1 mutants retained chemotaxis ability almost equal to that of the wild-type (WT) strain. Although WT and cheY1 mutants of Pta6605 caused severe disease symptoms on host tobacco leaves, the cheA2 and cheY2 mutants did not, and symptom development with cheA1 depended on the inoculation method. These results indicate that chemotaxis genes located in cluster II are required for optimal chemotaxis and host plant infection by Pta6605 and that cluster I may partially contribute to these phenotypes

Gac two-component system in Pseudomonas syringae pv. tabaci is required for virulence but not for hypersensitive reaction

Pseudomonas syringae pv. tabaci 6605 causes wildfire disease on host tobacco plants. To investigate the regulatory mechanism of the expression of virulence, Gac two-Component system-defective mutants, Delta gacA and Delta gacS, and a double mutant, Delta gacA Delta gacS, were generated. These mutants produced smaller amounts of N-acyl homoserine lactones required for quorum sensing, had lost swarming motility, and had reduced expression of virulence-related hrp genes and the algT gene required for exopolysaccharide production. The ability of the mutants to cause disease symptoms in their host tobacco plant was remarkably reduced, while they retained the ability to induce hypersensitive reaction (HR) in the nonhost plants. These results indicated that the Gac two-component system of P. syringae pv. tabaci 6605 is indispensable for virulence on the host plant, but not for HR induction in the nonhost plants.</p

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Delta orf1 and Delta orf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Delta orf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Delta orf1, Delta orf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule

MexEF-OprN multidrug efflux pump transporter negatively controls N-acyl-homoserine lactone accumulation in pseudomonas syringae pv. Tabaci 6605

Our previous studies revealed that flagellar-motility-defective mutants such as ∆fliC of Pseudomonas syringae pv. tabaci 6605 (Pta6605) have remarkably reduced production of N-acyl-homoserine lactones (AHL), quorum-sensing molecules. To investigate the reason of loss of AHL production in ∆fliC mutant, we carried out transposon mutagenesis. Among approximately 14,000 transconjugants, we found 11 AHL production-recovered (APR) strains. In these APR strains, a transposon was inserted into either mexE or mexF, genes encoding for the multidrug efflux pump transporter MexEF-OprN, and mexT, a gene encoding a putative transcriptional activator for mexEF-oprN. These results suggest that MexEF-OprN is a negative regulator of AHL production. To confirm the negative effect of MexEF-OprN on AHL production, loss- and gain-of-function experiments for mexEF-oprN were carried out. The ∆fliC∆mexF and ∆fliC∆mexT double mutant strains recovered AHL production, whereas the mexT overexpressing strain abolished AHL production, although the psyI, a gene encoding AHL synthase, is transcribed as wild type. Introduction of a mexF or mexT mutation into another flagellar-motility- and AHL production-defective mutant strain, ∆motCD, also recovered the ability to produce AHL. Furthermore, introduction of the mexF mutation into other AHL production-defective mutant strains such as ∆gacA and ∆aefR also recovered AHL production but not to the ∆psyI mutant. These results indicate that MexEF-OprN is a decisive negative determinant of AHL production and accumulation

Contribution of the Fenton reaction to the degradation of carbon nanotubes by enzymes

The widespread use of carbon nanotubes (CNTs) has raised concerns about the human health and ecological effects of CNTs released into the environment. Bacteria play an important role in bioremediation and waste treatment, and their enzymes are mostly responsible for the degradation of contaminants. However, there are still only a few reports about the bacterial degradation of CNTs, and evidence showing the involvement of bacterial enzymes in CNT degradation with their mechanisms has never been reported. The purpose of this study is to clarify whether CNTs can be degraded by bacterial enzymes. In this study, the degradation of oxidized (carboxylated) single-walled CNTs (O-SWCNTs) by mt2DyP, a dye-decolorizing peroxidase of Pseudomonas putida mt-2, a common soil bacterium, was investigated. After incubation of O-SWCNTs with recombinant mt2DyP and its substrate H2O2 for 30 d, the optical absorbance and Raman spectra revealed the degradation of O-SWCNTs. However, inactivation of the enzyme was observed within 60 min of the start of incubation, suggesting that the degradation of O-SWCNTs occurred nonenzymatically. The inactivation of mt2DyP was accompanied by the release of iron, the active center metal, and degradation of O-SWCNTs was significantly inhibited in the presence of diethylenetriamine pentaacetic acid, a chelating agent, indicating that O-SWCNTs were degraded by the Fenton reaction with iron released from mt2DyP and H2O2. The same phenomenon was observed with P450, which is also a heme enzyme. Furthermore, we investigated the contribution of the Fenton reaction to the O-SWCNT degradation by horseradish peroxidase (HRP), which was reported to enzymatically and rapidly degrade O-SWCNTs. Our results revealed that the degradation of O-SWCNTs in the presence of HRP is also mainly due to the Fenton reaction, with negligible enzymatic degradation. This contradicts the report showing enzymatic degradation of O-SWCNTs by HRP but supports the subsequent report quantitatively showing very slow transformation of O-SWCNTs by HRP. The current results emphasize that the Fenton reaction, which has received little attention in CNT degradation by heme enzymes, must be taken into consideration and will contribute to the development of a simple disposal method for CNTs, utilizing the Fenton reaction with bacteria/bacterial enzymes and H2O2

Efficacy of RAD001 (everolimus) in peritoneal dissemination of gastric cancer

Peritoneal dissemination occurs frequently in patients with unresectable advanced stage gastric cancer. In this study, we tested mTOR inhibitor RAD001 (everolimus) for efficacy on peritoneal dissemination of gastric cancer. Using the two cell lines 58As1, a highly peritoneal metastatic cell line, and its parental HSC58, a human scirrhous gastric cancer cell line, we first examined the growth inhibition activity of everolimus in vitro. Methylene blue assay demonstrated a moderate inhibitory effect on both cell lines under normal culture condition. When cells were maintained in hypoxic (1% O2) conditions, growth inhibition by everolimus was greatly reduced in HSC58, whereas the reduction was much smaller in 58As1. In western blotting, phosphorylation of mTOR, and its down-stream signaling molecules, P70S6K and 4E-BP1, were decreased under hypoxic conditions in HSC58. However, in 58As1, phospho-P70S6K and -4E-BP1 remained active state in hypoxic conditions and was suppressed by treatment with everolimus. Cell-cycle analysis showed that the hypoxia-induced G1 arrest was not manifested in 58As1 cells as compared to HSC58 cells. Separately, an in vivo orthotopic mouse model of 58As1 revealed that everolimus significantly reduces peritoneal dissemination as evaluated by quantitative photon counting method. Taken together, our results suggest that everolimus may have activity against gastric cancer, particularly in cases with peritoneal dissemination