Engineering Escherichia coli for high-yielding 2,5-Dimethylpyrazine synthesis from L-Threonine by reconstructing metabolic pathways and enhancing cofactors regeneration.

2,5-Dimethylpyrazine (2,5-DMP) is important pharmaceutical raw material and food flavoring agent. Recently, engineering microbes to produce 2,5-DMP has become an attractive alternative to chemical synthesis approach. In this study, metabolic engineering strategies were used to optimize the modified Escherichia coli BL21 (DE3) strain for efficient synthesis of 2,5-DMP using L-threonine dehydrogenase (EcTDH) from Escherichia coli BL21, NADH oxidase (EhNOX) from Enterococcus hirae, aminoacetone oxidase (ScAAO) from Streptococcus cristatus and L-threonine transporter protein (EcSstT) from Escherichia coli BL21, respectively. We further optimized the reaction conditions for synthesizing 2,5-DMP. In optimized conditions, the modified strain can convert L-threonine to obtain 2,5-DMP with a yield of 2897.30 mg/L. Therefore, the strategies used in this study contribute to the development of high-level cell factories for 2,5-DMP.

Biosynthesis and Characterization of Medium-Chain-Length Polyhydroxyalkanoate with an Enriched 3-Hydroxydodecanoate Monomer from a Pseudomonas chlororaphis Cell Factory.

Polyhydroxyalkanoates (PHAs) have been reported with agricultural and medical applications in virtue of their biodegradable and biocompatible properties. Here, we systematically engineered three modules for the enhanced biosynthesis of medium-chain-length polyhydroxyalkanoate (mcl-PHA) in Pseudomonas chlororaphis HT66. The phzE, fadA, and fadB genes were deleted to block the native phenazine pathway and weaken the fatty acid ß-oxidation pathway. Additionally, a PHA depolymerase gene phaZ was knocked out to prevent the degradation of mcl-PHA. Three genes involved in the mcl-PHA biosynthesis pathway were co-overexpressed to increase carbon flux. The engineered strain HT4Δ::C1C2J exhibited an 18.2 g/L cell dry weight with 84.9 wt % of mcl-PHA in a shake-flask culture, and the 3-hydroxydodecanoate (3HDD) monomer was increased to 71.6 mol %. Thermophysical and mechanical properties of mcl-PHA were improved with an enriched ratio of 3HDD. This study demonstrated a rational metabolic engineering approach to enhance the production of mcl-PHA with the enriched dominant monomer and improved material properties.

Rapid monitoring of the target protein expression with a fluorescent signal based on a dicistronic construct in Escherichia coli.

Real-time quantification of recombinant proteins is important in studies on fermentation engineering, cell engineering, etc. Measurement of the expression level of heterologous proteins in bacterial fermentation broth has traditionally relied on time-consuming and labor-intensive procedures, such as polyacrylamide gel electrophoresis, immunoblot analysis, and biological activity assays. We describe a simple, fast, and high sensitive assay for detecting heterologous proteins production in bacteria either at the overall level (fluorescence spectrophotometry) or at the individual level (fluorescence microscopic image) in this study. Based on a dicistronic model, the translation of target gene in the upstream open reading frame (ORF) was coupled with the synthesis of the mCherry reporter in the downstream ORF in E. coli cells, and subsequently this demonstrated a positive correlation between the expression of target gene and mCherry. Although a time lag exists between the expression of target protein and mCherry reporter, the method described here allows facile monitoring of dynamic changes in target protein expression, relying on indirect determination of the fluorescence intensity of mCherry during fermentation in real-time models. Additionally, the performance of a single bacterial cell factory could be checked under the fluorescence microscope field.

Surface display of metal binding domain derived from PbrR on Escherichia coli specifically increases lead(II) adsorption.

OBJECTIVES: To improve the Pb2+ biosorption capacity of the potential E. coli biosorbent, a putative Pb2+ binding domain (PbBD) derived from PbrR was efficiently displayed on to the E. coli cell surface. RESULTS: The PbBD was obtained by truncating the N-terminal DNA-binding domain and C-terminal redundant amino acid residues of the Pb2+-sensing transcriptional factor PbrR. Whole-cell sorbents were constructed with the full-length PbrR and PbBD of PbrR genetically engineered onto the surface of E. coli cells using Lpp-OmpA as the anchor. Followed by a 1.71-fold higher display of PbBD than PbrR, the presence of PbBD on the surface of E. coli cells enabled a 1.92-fold higher Pb2+ biosorption than that found in PbrR-displayed cells. Specific Pb2+ binding via PbBD was the same as Pb2+ binding via the full-length PbrR, with no observable decline even in the presence of Zn2+ and Cd2+. CONCLUSIONS: Since surface-engineered E. coli cells with PbBD increased the Pb2+ binding capacity and did not affect the adsorption selectivity, this suggests that surface display of the metal binding domain derived from MerR-like proteins may be used for the bioremediation of specific toxic heavy metals.

Novel Three-Component Phenazine-1-Carboxylic Acid 1,2-Dioxygenase in Sphingomonas wittichii DP58.

Phenazine-1-carboxylic acid, the main component of shenqinmycin, is widely used in southern China for the prevention of rice sheath blight. However, the fate of phenazine-1-carboxylic acid in soil remains uncertain. Sphingomonas wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources for growth. In this study, dioxygenase-encoding genes, pcaA1A2, were found using transcriptome analysis to be highly upregulated upon phenazine-1-carboxylic acid biodegradation. PcaA1 shares 68% amino acid sequence identity with the large oxygenase subunit of anthranilate 1,2-dioxygenase from Rhodococcus maanshanensis DSM 44675. The dioxygenase was coexpressed in Escherichia coli with its adjacent reductase-encoding gene, pcaA3, and ferredoxin-encoding gene, pcaA4, and showed phenazine-1-carboxylic acid consumption. The dioxygenase-, ferredoxin-, and reductase-encoding genes were expressed in Pseudomonas putida KT2440 or E. coli BL21, and the three recombinant proteins were purified. A phenazine-1-carboxylic acid conversion capability occurred in vitro only when all three components were present. However, P. putida KT2440 transformed with pcaA1A2 obtained phenazine-1-carboxylic acid degradation ability, suggesting that phenazine-1-carboxylic acid 1,2-dioxygenase has low specificities for its ferredoxin and reductase. This was verified by replacing PcaA3 with RedA2 in the in vitro enzyme assay. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) analysis showed that phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation, indicating that PcaA1A2A3A4 constitutes the initial phenazine-1-carboxylic acid 1,2-dioxygenase. This study fills a gap in our understanding of the biodegradation of phenazine-1-carboxylic acid and illustrates a new dioxygenase for decarboxylation.IMPORTANCE Phenazine-1-carboxylic acid is widely used in southern China as a key fungicide to prevent rice sheath blight. However, the degradation characteristics of phenazine-1-carboxylic acid and the environmental consequences of the long-term application are not clear. S. wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources. In this study, a three-component dioxygenase, PcaA1A2A3A4, was determined to be the initial dioxygenase for phenazine-1-carboxylic acid degradation in S. wittichii DP58. Phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation. This finding may help us discover the pathway for phenazine-1-carboxylic acid degradation.

iTRAQ-based quantitative proteomic analysis reveals potential factors associated with the enhancement of phenazine-1-carboxamide production in Pseudomonas chlororaphis P3.

Phenazine-1-carboxamide (PCN), a phenazine derivative, is strongly antagonistic to fungal phytopathogens. Pseudomonas chlororaphis HT66 is a PCN-producing, non-pathogenic biocontrol strain, and we obtained the mutant P. chlororaphis P3, which produces 4.7 times more PCN than the wild-type HT66 strain. To reveal the cause of PCN production enhancement in P3 and find potential factors related to PCN biosynthesis, an iTRAQ-based quantitative proteomic analysis was used to study the expression changes between the two strains. Of the 452 differentially expressed proteins, most were functionally mapped into PCN biosynthesis pathway or other related metabolisms. The upregulation of proteins, including PhzA/B, PhzD, PhzF, PhzG, and PhzH, involved in PCN biosynthesis was in agreement with the efficient production of PCN in P3. A number of proteins that function primarily in energy production, amino acid metabolism, and secondary metabolism played important roles in PCN biosynthesis. Notably, proteins involved in the uptake and conversion of phosphate, inorganic nitrogen sources, and iron improved the PCN production. Furthermore, the type VI secretion system may participate in the secretion or/and indirect biosynthetic regulation of PCN in P. chlororaphis. This study provides valuable clues to better understand the biosynthesis, excretion and regulation of PCN in Pseudomonas and also provides potential gene targets for further engineering high-yield strains.

[Association between cytokines and trichloroethylene-induced hypersensitivity dermatitis].

OBJECTIVE: To detect the cytokines levels in serums of patients with trichloroethylene-induced hypersensitivity dermatitis and explore the effect biomarkers associated with this disease. METHODS: Twenty-two patients with TCE-induced hypersensitivity dermatitis, twenty-two healthy TCE-exposed workers from the same workshops with patients and twenty-two comparable unexposed controls were recruited in this study. Eight cytokines in serums from all subjects were detected using Liquid Suspended Biochip; the correlation among the eight cytokines including interleukin (IL)-1ß (IL-1ß), IL-5, IL-8, IL-10, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), macrophage chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1ß (MIP-1ß) and the correlation between IL-5 and eosinophil count were analyzed. RESULTS: The medians of levels of IL-1ß, IFN-γ, IL-5, IL-10, MCP-1, MIP-1ß, IL-8 among patients were 0.15, 80.13, 2.95, 6.45, 83.83, 1057.90, 440.22 pg/ml, respectively, which were higher than those among the TCE-exposed workers (0.09, 16.93, 0.11, 0.07, 28.75, 241.07, 28.26 pg/ml, respectively, all P values < 0.01) and unexposed controls (0.09, 3.14, 0.11, 0.07, 25.27, 209.64, 207.34 pg/ml, respectively, all P values < 0.01). The median of level of TNF-α among the patients was 13.26 pg/ml, which was significantly higher than that among TCE-exposed workers (4.87 pg/ml, P < 0.01) but not among unexposed controls; the median of level of IL-5 among the TCE-exposed workers was 0.11 pg/ml, which was significantly higher than that among the unexposed controls (0.11 pg/ml, P < 0.01). The median of levels of IL-8 among the unexposed controls was 207.34 pg/ml, which was significantly higher than that among the TCE-exposed workers (28.26 pg/ml, P < 0.01). In case group, except for correlation of TNF-α and IFN-γ, TNF-α and IL-5, the significant positive correlations were found among any two cytokines (r(IL-1ß,IFN-γ) = 0.500, r(IL-1ß,TNF-α) = 0.348, r(IL-1ß,MCP-1) = 0.537, r(IL-1ß,MIP-1ß) = 0.477, r(IL-1ß,IL-8) = 0.466, r(IL-1ß,IL-5) = 0.610, r(IL-1ß,IL-10) = 0.626, r(IFN-γ,MCP-1) = 0.460, r(IFN-γ,MIP-1ß) = 0.491, r(IFN-γ,IL-8) = 0.322, r(IFN-γ,IL-5) = 0.532, r(IFN-γ,IL-10) = 0.511, r(TNF-α,MCP-1) = 0.325, r(TNF-α,MIP-1ß) = 0.283, r(TNF-α,IL-8) = 0.430, r(TNF-α,IL-10) = 0.271, r(MCP-1,MIP-1ß) = 0.659, r(MCP-1,IL-8) = 0.526, r(MCP-1,IL-5) = 0.504, r(MCP-1,IL-10) = 0.614, r(MIP-1ß,IL-8) = 0.601, r(MIP-1ß,IL-5) = 0.451, r(MIP-1ß,IL-10) = 0.579, r(IL-8,IL-5) = 0.255, r(IL-8,IL-10) = 0.403, r(IL-5,IL-10) = 0.798, all P values < 0.05). The median of level of IL-5 among the patients with high eosinophils counts was 8.92 pg/ml, which was significantly higher than that among the patients with low eosinophils counts (1.04 pg/ml, P < 0.05). CONCLUSION: The abnormal production of IL-1ß, IFN-γ, TNF-α, IL-8, MCP-1, MIP-1ß, IL-5 and IL-10 was related with the pathogenesis of hypersensitivity dermatitis induced by TCE. These cytokines could be used as referential indexes in the early health surveillance and clinic disease treatment.

Quantitative investigation of resolution increase of free-flow electrophoresis via simple interval sample injection and separation.

Interval free-flow zone electrophoresis (FFZE) has been used to suppress sample band broadening greatly hindering the development of free-flow electrophoresis (FFE). However, there has been still no quantitative study on the resolution increase of interval FFZE. Herein, we tried to make a comparison between bandwidths in interval FFZE and continuous one. A commercial dye with methyl green and crystal violet was well chosen to show the bandwidth. The comparative experiments were conducted under the same sample loading of the model dye (viz. 3.49, 1.75, 1.17, and 0.88 mg/h), the same running time (viz. 5, 10, 15, and 20 min), and the same flux ratio between sample and background buffer (= 10.64 × 10⁻³). Under the given conditions, the experiments demonstrated that (i) the band broadening was evidently caused by hydrodynamic factor in continuous mode, and (ii) the interval mode could clearly eliminate the hydrodynamic broadening existing in continuous mode, greatly increasing the resolution of dye separation. Finally, the interval FFZE was successfully used for the complete separation of two-model antibiotics (herein pyoluteorin and phenazine-1-carboxylic acid coexisting in fermentation broth of a new strain Pseudomonas aeruginosa M18), demonstrating the feasibility of interval FFZE mode for separation of biomolecules.

[Effects of n-hexane exposure on human serum myelin basic proteins].

OBJECTIVE: To explore the effects of n-hexane on expression of serum myelin proteins (MBP) in workers occupationally exposed to n-hexane. METHODS: In this study, 269 workers exposed to n-hexane for more than one year and 104 subjects not exposed to n-hexane served as the exposure group and the control group, respectively. The urinary 2,5-hexanedione levels in all subjects were detected. On the basis of urinary 2,5-hexanedione levels, the exposure group was divided into the high exposure sub-group and low exposure sub-group. The serum myelin basic protein (MBP) levels were measured by ELISA kit. RESULTS: The mean concentration of urinary 2,5-hexanedione in the exposed group was (3.10 +/- 1.35) mg/L. The concentration of urinary 2,5-hexanedione in the control group was undetectable. The levels of serum MBP in the high exposure sub-group and low exposure sub-group were (2.43 +/- 0.24) and (1.62 +/- 0.23) microg/L, respectively, which were significantly higher than that (0.78 +/- 0.12) microg/L in the controls (P < 0.01). Pearson correlation analysis showed the positive correlation between serum MBP levels and urinary 2,5-hexanedione levels (r = 0.781, P < 0.01). CONCLUSION: The results of present study showed that the serum MBP levels of workers occupationally exposed to n-hexane significantly elevated, and the serum MBP can serve as the effective biomarker of n-hexane exposure.

[Effects on serum myelin proteins of n-hexane exposure].

OBJECTIVE: Exploring the effects of n-hexane on expression of serum myelin proteins in occupational exposure workers, and finding the early biomarker of n-hexane exposure. METHODS: In the study, 373 subjects were recruited, 269 exposure workers (work experience of more than1 year) and 104 non-exposure workers were selected. Firstly examined the level of urinary 2,5-hexanedione in the two groups, based on urinary 2,5-hexanedione biological limit value (4 mg/L), the exposed group was divided into high-exposed group and low-exposed group. And then collected blood samples and extracted serum. Human peripheral myelin protein zero (P0) antibody (IgG, IgM) and human peripheral myelin protein two (P2) antibody (IgG, IgM) analysis was performed according to ELISA kit. RESULTS: The concentration of urinary 2,5-hexanedione in the exposed group was (3.10 ± 1.35) mg/L. The level of P0 antibody (IgG, IgM) and P2 antibody (IgG, IgM) in the high-exposed group and low-exposed group were both higher than that in the controls (P < 0.01). CONCLUSION: P0 antibody and P2 antibody could be used as the early biomarkers of n-hexane exposure, which not only evaluate the occupational hazards in the early, but also provide the policy maker with scientific evidence.

[Analysis of subgroups of lymphocyte in peripheral blood among dermatitis medicamentosa-like of trichloroethylene patients and healthy exposed workers].

OBJECTIVE: To study the effects of trichloroethylene (TCE) to lymphocyte subsets among exposed workers, and explore the early immunological effect biomarkers for prevention of hypersensitivity dermatitis induced by TCE. METHODS: Twenty-eight patients with TCE-induced hypersensitivity dermatitis, 56 healthy TCE-exposed workers from the same workshops with patients, and 28 comparable unexposed controls were recruited in this study. The total lymphocyte count and the major lymphocyte subsets including T cell, CD4(+) T cell, CD8(+) T cell, B cell, NK cell in peripheral blood were measured by Flow Cytometer analysis and Standard blood count analysis. RESULTS: The total lymphocyte count and T cell, CD4(+) T cell, CD8(+) T cell among patients (median at 2810.00, 1846.17, 831.87, 904.05 cell counts/µl blood) were significantly increased compared with TCE-exposed workers (median at 2101.00, 1218.59, 643.87, 482.81 cell counts/µl blood, Z = -3.19, -4.96, -3.22, -4.99, P < 0.001) and unexposed controls (median at 1900.00, 1223.60, 558.60, 325.80 cell counts/µl blood, Z = -3.30, -4.46, -3.45, -5.03, P < 0.001), the NK cell and CD3(+)CD4(+)/CD3(+)CD8(+) ratio among patients (median at 255.50 cell counts/µl blood and 1.11) were significantly decreased compared with the unexposed controls (median at 642.60 cell counts/µl blood and 1.96, Z = -3.56 and -3.11, P < 0.01). Meanwhile, for the exposed workers, the CD8(+) T cell (median at 482.81 cell counts/µl blood) was significantly increased and the NK cell and CD3(+)CD4(+)/CD3(+)CD8(+) ratio (median at 318.76 cell counts/µl blood and 1.27) were significantly decreased compared with unexposed controls (median at 325.80 and 642.60 cell counts/µl blood and 1.96, Z = -2.63, -3.52, -2.29, P < 0.05). CONCLUSION: Occupational exposure to TCE could affect the lymphocyte subsets, especially T cell and NK cell. The total lymphocyte count, T cell and CD4(+) T cell might be effect biomarkers for subjects with hypersensitivity dermatitis among TCE-exposed workers.

Medium optimization for phenazine-1-carboxylic acid production by a gacA qscR double mutant of Pseudomonas sp. M18 using response surface methodology.

Statistics based experimental designs were applied to optimize the culture medium components for enhancing phenazine-1-carboxylic acid (PCA) production by Pseudomonas sp. strain M18GQ, a gacA qscR double mutant of Pseudomonas sp. strain M18. The medium components, including soybean meal, glucose and corn steep liquor, had significant effects on PCA production based on a 2(5-1) fractional factorial design. The concentrations of these three significant factors were subsequently optimized using a central composite design. An optimum concentration of soybean meal 73.3g/L, corn steep liquor 18.1g/L, glucose 17.9g/L, and ethanol 18.0ml/L was obtained by response surface analysis. The predicted maximum PCA yield was as high as 4011.5mg/L, and was confirmed by validation experiments. The double mutant M18GQ produced 4032.2mg/L PCA, which was almost 2 to 3.3-fold that produced by either single mutant M18G and M18Q. The achieved production level is economically useful for industrial application.

Rapid quantitative analysis of phenazine-1-carboxylic acid and 2-hydroxyphenazine from fermentation culture of Pseudomonas chlororaphis GP72 by capillary zone electrophoresis.

Natural phenazines in secondary metabolites of bacteria have been receiving increasing attention in recent years due to their potential usage as antibiotics. In the present study, a rapid and reliable capillary zone electrophoresis (CZE) method was developed and validated for monitoring for the first time dynamic phenazine-1-carboxylic acid (PCA) and the 2-hydroxyphenazine (2-OH-PHZ) production of Pseudomonas chlororaphis GP72 during the entire fermentation cycle. The paper begins with the optimization of separate conditions for 2-OH-PHZ and PCA together with phenazine (PHZ), which is used as internal standard. The optimized conditions are: 10mM, pH 7.3 phosphate buffer, a fused-silica capillary with a total length of 49 cm x 75 microm ID, 375 microm OD with an effective length of 40 cm, 25 kV, 13 mbar 10s pressure sample injection and 25 degrees C air-cooling. The three compounds could be separated within 2 min under optimized conditions. The validation of the newly developed study shows the linear response of 2-OH-PHZ and PCA ranging from 10 to 250 microg mL(-1) with high correlation coefficient (r=0.9997 and 0.9993, n=7), low limits of detection (0.47 and 0.38 microg mL(-1)) and quantification (1.56 and 1.28 microg mL(-1)), respectively. Good precision values for intra- and inter-day detection and acceptable individual recovery ranges for 2-OH-PHZ and PCA are indicated. The newly developed method was also validated through monitoring dynamic PCA and 2-OH-PHZ production of P. chlororaphis GP72 during an 84 h growth cycle.

[Construction of Pseudomonas sp. M18 qscR- mutant and its regulation on biosynthesis of PCA and Plt].

In Gram-negative bacteria, global regulator QscR controls the expression of many virulence determinants, secondary metabolites, stationary phase genes and genes involved in biofilm formation through quorum sensing (QS) systems. QscR binds the promoter region of target genes and regulates the gene expression at the transcriptional level. Using homologous recombination technique a chromosomal qscR inactivated mutant strain M-18Q was constructed in Pseudomonas sp. M-18, a strain of plant-growth-promoting rhizobacteria, which could inhibit several soilborn phytopathogens by producing secondary metabolites including phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt) in one single strain. To further study the effect of QscR on the synthesis of Plt and PCA in the wild type strain M-18, the dynamic curves of Plt and PCA produced respectively by M-18 and M-18Q strains were measured in both KMB and PPM mediums. The synthesis of PCA was much more activated in the mutant than in the wild type both in KMB and PPM mediums. The PCA production in the mutant strain is four-to-six fold over that in the wild type in the PPM medium, reaching 480 pg/mL, and three-to-five fold in the KMB medium, reaching 140 microg/mL. The synthesis of Plt, however, was not detected in PPM medium and was nearly not influenced by the QscR protein in KMB medium. PCA production was inhibited but Plt biosynthesis was not altered after complementation with qscR gene in trans in the strain of M-18Q. The regulation of qscR gene on PCA production was further confirmed by the analysis of beta-galactosidase activities from the translational phzA '-' lacZ fusion, in which phzA is the first enzyme gene of the phenazine biosynthesis pathway. These results indicate that QscR can control PCA production negatively but not Plt production in M-18, and show that QscR functions as a global regulator to differently regulate the synthesis of PCA and Plt on the gene expression level.

Autoinduction of RpoS biosynthesis in the biocontrol strain Pseudomonas sp. M18.

The rpoS gene from Pseudomonas sp. M18, which encodes predicted protein (an alternative sigma factor s, sigma(S), or sigma(38)) with 99.5% sequence identity with RpoS from Pseudomonas aeruginosa PAO1, was first cloned. In order to investigate the mechanism of rpoS expression, an rpoS null mutant, named M18S, was constructed with insertion of aacC1 cassette bearing a gentamycin resistance gene. With introduction of a plasmid containing an rpoS'-'lacZ translational fusion (pMERS) to wild-type strain M18 or M18S, it was first found that beta-galactosidase activity expressed in strain M18S (pMERS) decreased to fourfold of that expressed in the strain M18 (pMERS). When strain M18S (pMERS) was introduced with another plasmid pBBS containing the wild-type rpoS gene, its beta-galactosidase expression level was enhanced and almost restored to that in strain M18 (pMERS). Similarly, expression of beta-galactosidase from a chromosomal fusion of the promoter of the wild-type rpoS gene with lacZ (rpoS-lacZ) was enhanced fivefold in the presence of a plasmid with the wild-type rpoS gene. With these findings, it is suggested that RpoS sigma factor may be involved in autoinducing its own gene expression in Pseudomonas sp. M18.

[Analysis of mechanism and relationship of GacA and RsmA, two regulators of antibiotics production in Pseudomonas sp. M18].

In previous study, it has already been confirmed that the wild type strain of Pseudomonas sp. M18 isolated from the agricultural soil can produce two antifungal agents phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt). Biosynthesis and secretion of these secondary metabolites contribute to its biological control and suppression of soilborne pathogenic fungi. As main regulators, GacA and RsmA differentially exert global regulation on production of PCA and Plt, respectively. In order to study the regulatory mechanism of secondary metabolites production in Pseudomonas sp. M18, a gacArsmA double mutant, designated as M18GR, was constructed with insertional mutation. Then, the mutant M18G, M18R, M18GR and the wild type strain M18 were inoculated into PPM or King's medium B (KMB), respectively. During cultivation of strain M18 and its derivatives, their PCA and Pit were respectively detected with High Performance Liquid Chromatography (HPLC). The results showed that PCA production in the mutant M18GR was lower than that in the mutant M18G and higher than that in the mutant M18R. Plt production in the mutant M18GR was, however, much less than that in the mutant M18R and much more than that in the strain M18 and the mutant M18G. With these observations, it is tempting to suggest that biosynthesis of PCA and Plt regulated by GacA or RsmA seem to occur at posttranscriptional level, not at transcriptional level. This regulation on secondary metabolites seems to be indirectly mediated by other unknown factors. Meanwhile, based on the construction of two translational fusions, gacA'-'lacZ and rsmA'-'lacZ, the assay of beta-galactosidase activities in KMB medium indicated that both GacA and RsmA did not have autoinduction of their own gene expression, respectively. Although GacA did not influence expression of the rsmA gene, RsmA could exert some positive influence on the gacA gene expression in Pseudomonas sp. M18.

[Isolation and identification of lipopeptides produced by Bacillus subtilis fmbJ].

Isolation and idenfication of lipopeptides from Bacillus subtilis fmbJ was carried out in this paper. With HPLC method, it was determined that the antimicrobial substance was composed of many components, and one of them had the similar retention time similar to surfactin. In addition, the antimicrobial substance was proved to include the closed cycle peptide bind by TLC, and one of them had the migrating rate similar to surfactin. Furthermore, ESI-MS analysis showed that the antimicrobial substance contained five homologues of fengycin, such as m/z1449.9, m/zl1463.8, m/zl1477.8, m/z1491.9 and m/z1505.9, and three homologues of surfactin, such as m/z1008.8, m/z1022.8 and m/z1036.8.

[Negative regulation on PCA production is not correlated with positive regulation on BHL and HHL synthesis by gacA in Pseudomonas sp. M18].

At least 5 kinds of N-acyl-homoserine lactones(AHLs) were identified in Pseudomonas sp. M18. They were: N-butyryl-L-homoserine lactone(C4-HSL, BHL), N-hexanoyl-L-homoserine lactone(C6-HSL, HHL), N-(3-oxohexanoyl)-L-homoserine lactone(3-Oxo-C6-HSL, OHHL), N-(3-oxooctanoyl)-L-homoserine lactone (3-Oxo-C8-HSL, OOHL) and N-(3-oxodecanoyl)-L-homoserine lactone(3-Oxo-C10-HSL, ODHL). Compared with the wide-type strain M18, the variety of the AHLs in the gacA mutant strain M18G reduced to 4 species with the decreased quantity. But the phenazine-1-carboxylic acid (PCA) production was increased by about 2-fold. The product of rhll plays an important role in synthesizing BHL and HHL. The rhll'-'lacZ translational fusion expression plasmid pMEIZ was constructed in vector pME6015 and then was introduced into the wild-type strain M18 and the gacA mutant strain M18G. The galactosidase activity in chromosomal gacA disruption mutant was only 60% of that in the wild-type strain M18. This result suggested that GacA could regulate the rhll expression positively. There was no influence on PCA production by adding exogenous BHL and HHL and both together to the culture of strains M18 and M18G. This result suggested that there was no relation between GacA's negative regulation on PCA production and positive regulation on BHL and HHL synthesis.

[Inhibitory effect of new antimicrobial substance by Bacillus subtilis fmbJ on Newcastle disease virus and infectious Bursal disease virus in vitro].

The resistance effect on Newcastle disease virus (NDV) and Infectious Bursal Disease Virus(IBDV) in vitro of a new antimicrobial substance (AS), which produced by a Bacillus subtilis strain named B. subtilis fmbJ. Results showed that the TD50 and TD0 value of this AS on Chicken Embryo Fibroblasts cell (CEF) were 128.95mg/L and 25.79mg/L, respectively. This AS could strongly inhibit the cytopathic effects of cell induced by NDV as well as IBDV, and increase the survival rate of cell remarkably. This AS could inhibit the function of NDV and IBDV, and it could defend against the infection and inhibit multiplication of NDV and IBDV, and the effect was the same as the antiviral medicine Ribavirin. It had lower toxicity to CEF cell, therefore we would study it further that it was as antiviral medicine.

[Media optimization for the novel antimicrobial peptide by Bacillus sp. fmbJ224].

The novel antimicrobial peptide in submerged fermentation by Bacillus sp. fmbJ224 is strongly influenced by many internal and external factors, namely medium constituents and fermentation conditions. In this study, Plackett-Burman design was undertaken to evaluate the effects of the seventeen factors. By the statistical regression analysis, the significant factors affecting the novel antimicrobial peptide in submerged fermentation by Bacillus sp. fmbJ224 were determined as follows: glucose, NH4NO3, glutamic acid, CaCl2, MnSO4. In the second phase of the optimization process, a response surface methodology (RSM) was used to optimize the above critical internal factors, and to find out the optimization concentraction levels and the relationships between these factors. By solving the quadratic regression model equation using appropriate statistic methods, the optimal concentration of the variables were determined as: 8.13 g/L glucose, 6.14 g/L NH4NO3, 4.2 g/L glutamic acid, 3.98 mg/L CaCl2, 4.87 mg/L MnSO4. The content of the novel antimicrobial peptide was increased from 1304.21 microg/mL to 1487.58 microg/mL. The experimental data under various conditions have validated the theoretical values.