海洋真菌产抗菌物质培养基及其性质研究

目的:海洋真菌Fy-04产抗菌物质培养基及性质研究.方法:管碟法测定抑菌效果确定海洋真菌Fy-04产抗菌物质的培养基及其性质.结果:海洋真菌Fy-04产抗菌物质对金黄色葡萄球菌抑菌最佳培养基组成为:马铃薯海水浸出液60%,葡萄糖2%,蛋白胨1%,酵母膏0.1%,抑菌圈直径34.6mm;对大肠杆菌抑菌最佳培养基组成为:葡萄糖2%,蛋白胨0.7%,酵母膏0.1%,FePO4 0.04%,抑菌圈直径21.6mm;抗菌物质80℃处理1h,残留活性60%以上,pH稳定范围在pH 6.0～10.0.结论:该菌株代谢产物对细菌有较好的抑菌效果,并能溶于乙醚、三氯甲烷中等极性的有机溶剂中.     

Inhibition of Vancomycin and High-Level Aminoglycoside-Resistant Enterococci Strains and Listeria monocytogenes by Bacteriocin-Like Substance Produced by Enterococcus faecium E86

Three hundred and thirty nine lactic bacteria strains isolated from food samples were screened for antimicrobial activity. Only one strain isolated from meat pie and identified as Enterococcus faecium produced a bacteriocin-like inhibitory substance (BLIS) showing activity against Enterococcus, Leuconostoc, Lactobacillus, Listeria, Corynebacterium and Staphylococcus aureus. The BLIS produced was resistant to acid and alkali treatment and 121oC for 15 min. The addition of BLIS in BHI contaminated with Listeria monocytogenes decreased the contamination in 4.8 log cycles in 24 h. The inhibition of listeria was also obtained in milk. Forty multiresistant enterococci strains were inhibited in the well-diffusion test. Two vancomycin resistant strains tested in liquid with BLIS were also inhibited. The BLIS producer showed no pathogenicity marker.     

Characterization and Identification of a Novel Marine Streptomyces sp. Produced Antibacterial Substance

Strain GB-2 is a marine microbe with broad-spectrum antimicrobial activity, isolated from soil taken from the coastal city Lianyungang in the JiangSu province of China. Analysis of its morphological, physiological, and biochemical characteristics as well as chemical components of the cell wall strongly suggested that the strain GB-2 belonged to the Streptomyces sp. Analysis of the nucleotide sequence of the 16S rRNA gene of Streptomyces sp. GB-2 strain showed a strong similarity (98%) with the 16 rRNA gene of Streptomyces fradiae. Application to antibacterial substance of strain Streptomyces sp. GB-2 by various separation steps led to isolation of one active molecule having a retention time of 9.495 min, P _9.495 min, which possessed antibacterial activity against Bacillus cereus and Escherichia coli. Through analysis by liquid chromatography–mass spectrometry and mass/mass spectrometry of the peak, the molecular weight of the antibacterial substance (P _9.495 min sample) was 447.5 Da and it was determined to be sisomicin according to the analysis of ion fragments. Nuclear magnetic resonance spectrum of the peak also demonstrated that the antibacterial substance was sisomicin. This study is the first to introduce the finding of sisomicin produced from marine Streptomyces sp. This work provides a preference for the production of sisomicin in pharmaceutical industries and a probability for studying the biodiversity of marine microbe.     

Culture Conditions Affect the Molecular Weight Properties of Hyaluronic Acid Produced by Streptococcus zooepidemicus

The effect of five culture variables on the molecular weight properties of hyaluronic acid (HA) produced by Streptococcus zooepidemicus was studied in batch culture with a complex medium containing glucose and 10 g of yeast extract per liter. Neither the culture pH (pH 6.3 to 8.0) nor the agitation speed (300 to 1,000 rpm) affected the weight-average molecular weight (M(infw)) of HA under anaerobic conditions at 37(deg)C when 20 g of glucose per liter was used initially. M(infw) was in the narrow range of 1.5 x 10(sup6) to 2.3 x 10(sup6), and polydispersity (P) was between 1.8 and 2.5. When S. zooepidemicus was grown at lower temperatures or with aeration, higher-molecular-weight polymer and increased yields were observed. The polydispersity, however, remained unaffected. Anaerobically, the mean M(infw) (based on three samples taken within 4 h of glucose exhaustion) was (2.40 (plusmn) 0.10) x 10(sup6) and (1.90 (plusmn) 0.05) x 10(sup6) at 32 and 40(deg)C respectively. Aeration of the culture at 1 vol/vol/min produced HA with mean M(infw) of (2.65 (plusmn) 0.05) x 10(sup6) compared with (2.10 (plusmn) 0.10) x 10(sup6) under equivalent anaerobic conditions. The initial glucose concentration had the most pronounced effect on polymer characteristics. Increasing this concentration from 20 to 40 g/liter produced HA with mean M(infw) of (3.1 (plusmn) 0.1) x 10(sup6) at 1-vol/vol/min aeration. The molecular weight of HA also exhibited time dependency, with smaller chains (M(infw), ca. 2.5 x 10(sup6)) detected early in the culture time course, rising to a maximum (M(infw), 3.2 x 10(sup6)) in the late exponential phase of growth. The mean polydispersity was also greater (2.7 (plusmn) 0.1) under these conditions. Replicate experiments performed under conditions resulting in the lowest (40(deg)C, anaerobic) and highest (40 g of glucose per liter, 1-vol/vol/min aeration)-M(infw) polymer demonstrated excellent experimental reproducibility.     

An Autoinhibitory Substance Produced by Platydorina caudata Kofoid

An inhibitory substance was obtained from culture filtrates of Platydorina caudata Kofoid, (a colonial, green flagellate), in which death of the culture had occurred. The cessation of growth occurs before, and is seemingly independent of, medium depletion. The substance can be detected as early as the 15th day, and by the 25th day growth is completely suppressed. A bioassay method for quantitating the activity of the substance was devised. The substance is heat-labile, nondialyzable, acid-labile, and apparently specific for Platydorina. The substance resists short periods of freezing and bacterial contamination. Preliminary results suggest that trypsin destroys activity of the substance.     

Induction of autolysis in Streptococcus faecium

Autolysis of exponential-phase Streptococcus faecium cells was promoted by pretreating the bacteria (freezing-thawing; -70 degrees C) in Tris buffer, followed by incubation at 37 degrees C in the same buffer. The effect was dependent on Tris concentration. The pretreatment provoked ultrastructurally visible damage with extensive loss of K+ and leakage of UV-absorbing components. No autolysis was observed when the bacteria frozen-thawed in Tris were incubated in the presence of the autolysin inhibitor N-bromosuccinimide nor when they had been grown in the presence of chloramphenicol or tetracycline. Furthermore, two autolytic-defective mutants, EC31 and EC78, isolated from S. faecium, did not autolyse when frozen-thawed and incubated in Tris. Freezing-thawing in Tris, however, imparted extensive cell damage to the mutants and to the antibiotic-treated bacteria as well as considerable leakage of K+ and UV-absorbing materials. These observations indicate that the lysis of S. faecium reported above is due to the activity of the endogenous bacterial autolysin. Induction of autolysis of S. faecium by freezing-thawing was also observed, although to a lesser extent, when Tris was replaced by imidazole.     

微生物源抗菌肽研究概况

抗菌肽是广泛存在于生物体内的一种小分子多肽,具有分子量小、高效、稳定、作用机制独特和不易产生耐药性等特点,对细菌、真菌、寄生虫、病毒以及肿瘤细胞均有抑制作用。介绍了微生物来源,尤其是细菌源抗菌肽的结构特点、生物活性、作用机制及其在感染性疾病中的应用情况。     

Analysis of Streptococcus mutans Proteins Modulated by Culture under Acidic Conditions

Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.     

Alpha-glycerophosphate oxidase in Streptococcus faecium F 24

alpha-Glycerophosphate oxidase, in a strain of Streptococcus faecium, was found to be adaptive to aerated conditions of growth. The enzyme was purified and found to mediate electron transfer from alpha-glycerophosphate to O(2), with the production of stoichiometric concentrations of H(2)O(2) and dihydroxyacetone phosphate. The enzyme is an anionic flavoprotein, with flavine adenine dinucleotide as the apparent prosthetic group. By manometric methods, a K(m) of 6 x 10(-3)m, with reference to substrate concentration, was obtained. An active reduced nicotinamide adenine dinucleotide diaphorase was closely associated with this enzyme in chromatographic mobility on ECTEOLA-cellulose. The purified alpha-glycerophosphate oxidase was not inhibited by KCN, azide, or sulfhydryl reagents, nor was it stimulated by alpha-lipoate, yeast extract, or other supplements.     

Enzymatic deacylation of lipoteichoic acid by protoplasts of Streptococcus faecium (Streptococcus faecalis ATCC 9790).

High-molecular-weight, micellar lipoteichoic acid (LTA) was converted to a lower-molecular-weight, apparently deacylated polymer when the former was incubated in the presence of growing protoplasts of Streptococcus faecium (S. faecalis ATCC 9790), but not when incubated in fresh or spent protoplast medium. The mobility of the low-molecular-weight polymer upon agarose gel electrophoresis was indistinguishable from that of native extracellular lipoteichoic acid LTA(X) from this organism or from chemically deacylated LTA. Native LTA(X) was shown to contain less than one fatty acid equivalent per 18 LTA(X) molecules, in contrast to the 4:1 ratio of fatty acids to polyglycerolphosphate chains in micellar LTA.     

A New Toxic Substance,Teleocidin, Produced by Streptomyces

The production and isolation of a new toxic substance, Teleocidin, and its biological properties were previously reported^1,2). Thereafter it has been found that an other strain of Streptomyces produced such specific toxic substance as Teleocidin in its cultured mycellium. Comparative tests of these two purified crystalline powders showed the new toxic substance resembles Teleocidin closely though differs in certain chemical properties. Therefore, the original Teleocidin is designated Teleocidin A, whereas that produced by a new strain of Streptomyces is named Teleocidin B, which had been tentatively called as the SK-toxic substance.

From the results of the chemical studies of Teleocidin B and its hydrogenated derivative, which was easily obtained as a crystalline form by the catalytic hydrogenation of Teleocidin B with Adam’s catalyst, molecular formula, C₂₈H_39~41N₃O₂ was postulated for Teleocidin B.

It was also recognized that an alcoholic hydroxyl, a lactam ring and a heterocyclic ring like indole or pyrrole structure existed as the functional groups of Teleocidin B.     

Activities of Arginine Dihydrolase and Phosphatase in Streptococcus faecalis and Streptococcus faecium

Strains of Streptococcus faecalis and S. faecium are known to produce ammonia from arginine, but only S. faecalis couples the adenosine triphosphate (ATP) produced through the arginine dihydrolase pathway to growth processes. The specific activities of the arginine dihydrolase enzymes were found to be much lower in S. faecium (0.01 to 0.10) than in S. faecalis (0.24 to 1.60). Phosphatase activities in both strains were similar (up to 0.11), but equaled or exceeded the activities of the arginine dihydrolase enzymes in S. faecium. The failure of S. faecium to show increased growth in arginine media is explained on the basis of low activities of the arginine dihydrolase enzymes coupled with sufficient phosphatase activity to negate any benefit from ATP formed.