The Identification of 2,4-diacetylphloroglucinol as an Antifungal Metabolite Produced by Cutaneous Bacteria of the Salamander <Emphasis Type="Italic">Plethodon cinereus</Emphasis>

Beneficial bacteria that live on salamander skins have the ability to inhibit pathogenic fungi. Our study aimed to identify the specific chemical agent(s) of this process and asked if any of the antifungal compounds known to operate in analogous plant–bacteria–fungi systems were present. Crude extracts of bacteria isolated from salamander skin were exposed to HPLC, UV-Vis, GC-MS, and HR-MS analyses. These investigations show that 2,4-diacetylphloroglucinol is produced by the bacteria isolate Lysobacter gummosus (AB161361), which was found on the red-backed salamander, Plethodon cinereus. Furthermore, exposure of the amphibian fungal pathogen, Batrachochytrium dendrobatidis (isolate JEL 215), to different concentrations of 2,4-diacetylphloroglucinol resulted in an IC₅₀ value of 8.73 μM, comparable to crude extract concentrations. This study is the first to show that an epibiotic bacterium on an amphibian species produces a chemical that inhibits pathogenic fungi.     

GH-16 Type β-1,3-Glucanase from Lysobacter sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase,and Its Glucan-binding Domain Binds to Fungal Cell-wall

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type β-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with β-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using β-glucans.     

Ammonia accumulation in culture broth by the novel nitrogen-fixing bacterium, Lysobacter sp. E4

This is the first report that Lysobacter fixes nitrogen under free-living conditions, as shown by its ability to grow on nitrogen-free medium and accumulate relatively high amounts of ammonia in the culture broth. Growth of the E4 Lysobacter strain, isolated in a screen for nitrogen-fixing and ammonia-producing bacteria, resulted in higher ammonia accumulation (0.53 mM ammonium ion concentration) in media containing glucose rather than other tested carbon sources. The optimum glucose concentration was 0.30% at an initial medium pH of 7.0 and incubation temperature of 30 °C. From time-course experiments, when the glucose in the culture was exhausted, ammonia began to be accumulated, and maximum ammonia accumulation (∼ 1.60 mM) was reached after 8 days of incubation. Ammonia accumulation by this strain required molybdenum, manganese, and iron.     

利用响应面法优化溶杆菌UCo1产溶菌酶的发酵培养基

采用响应面法对溶杆菌UCo1产溶菌酶的培养基进行了优化。首先对溶杆菌UCo1产溶菌酶的发酵培养基进行了单因素试验,确定了影响产酶的3个显著因素,即碳源麦芽糖,氮源大豆蛋白胨和表面活性剂Tween-20。采用Box-Behnken响应面法对溶菌酶的发酵培养基组成进行了优化,确定了最佳条件。结果表明,麦芽糖,大豆蛋白胨和Tween-20 3因素的最佳浓度分别为1.725%,3.25%,0.048%时,溶菌酶酶活达到6973.5U/mL,与模型所得到的最大预测值6990.99U/mL基本吻合,且较优化前的酶活力(6230.4U/mL)提高了11.93%。     

产酶溶杆菌L-43的筛选及其抑菌物质的初步鉴定

采用透明圈法和琼脂扩散法筛选具有抑菌活性的菌株,通过形态观察、理化分析及16S rDNA序列比对鉴定菌株,采用胞外酶活测定、蛋白酶水解、硫酸铵沉淀与膜分析、稳定性研究鉴定菌株所产抑菌物质。结果表明,从农田土壤中获得一株抑菌活性较好且没有溶血性的产酶溶杆菌（Lysobacter enzymogenes）L-43。该菌所产抑菌物质抑菌谱较广,对G+细菌具有良好的抑制作用,抑菌圈直径大于10 mm。对抑菌物质进行分析,其不具有溶菌酶活性、不含过氧化氢;对蛋白酶E、蛋白酶K、胃蛋白酶、胰蛋白酶敏感,抑菌活性回收率为73.43%~79.00%;经过膜分析,透析袋截留分子量为3 500 u和10 000 u时,抑菌活性回收率分别为93.43%和59.82%,初步判定抑菌物质主要是分子量在3 500~10 000 u之间的多肽类物质;抑菌物质具有良好的热、酸碱、金属离子、化学试剂和储存稳定性,121 ℃处理30 min活性回收率为85.66%,pH值2~10时活性回收率高于80%,进一步确定抑菌物质为多肽。综上,L. enzymogenes L-43所产抑菌物质为抑菌谱广且稳定性好的多肽。该研究为抗菌菌株及其代谢物的开发奠定了基础。     

The First Homologous Expression System for the β-Lytic Protease of Lysobacter capsici VKM B-2533T,a Promising Antimicrobial Agent

A successful homologous expression system based on Lysobacter capsici VKM B-2533^T and the plasmid pBBR1-MCS5 was first developed for a promising bacteriolytic enzyme of this bacterium, β-lytic protease (Blp). In the expression strains, blp gene expression under the regulation of the GroEL(A) and T5 promoters increased by 247- and 667-fold, respectively, as compared with the wild-type strain. After the cultivation of the expression strains L. capsici P_GroEL(A)-blp and L. capsici P_T5-blp, the Blp yield increased by 6.7- and 8.5-fold, respectively, with respect to the wild-type strain. The cultivation of the expression strain L. capsici P_T5-blp was successfully scaled up. Under fermentation conditions the yield of the enzyme increased by 1.6-fold. The developed homologous system was used to express the gene of the bacteriolytic serine protease (Serp) of L. capsici VKM B-2533^T. The expression of the serp gene in L. capsici P_T5-serp increased by 585-fold. The developed homologous system for the gene expression of bacteriolytic Lysobacter enzymes is potentially biotechnologically valuable, and is promising for creating highly efficient expression strains.     

Structural and Functional Characterization of β−lytic Protease from Lysobacter capsici VKM B−2533T

The crystal structure of the Lysobacter capsici VKM B−2533^T β-lytic protease (Blp), a medicinally promising antimicrobial enzyme, was first solved. Blp was established to possess a folding characteristic of the M23 protease family. The groove of the Blp active site, as compared with that of the LasA structural homologue from Pseudomonas aeruginosa, was found to have amino acid differences. Biochemical analysis revealed no differences in the optimal reaction conditions for manifesting Blp and LasA bacteriolytic activities. At the same time, Blp had a broader range of action against living and autoclaved target cells. The results suggest that the distinction in the geometry of the active site and the charge of amino acid residues that form the active site groove can be important for the hydrolysis of different peptidoglycan types in target cells.