艾丁湖可培养嗜盐菌多样性及功能酶、抗菌活性筛选

【目的】探究艾丁湖可培养嗜盐菌的多样性、功能酶活性以及抗菌活性,进一步了解其次级代谢产物情况,为新型生物活性物质的发掘提供依据。【方法】选用以20种糖及糖的衍生物作为唯一碳源的寡营养培养基从艾丁湖5个样点中分离得到298株嗜盐菌,根据形态特征去重复后,选取62株菌运用16S r RNA基因系统发育分析的方法研究样品中嗜盐菌的多样性;从不同类群选取22株代表菌株,采用点接法进行3种功能酶的筛选,运用平板对峙法检测代表菌株对12种病原菌的抗菌活性。【结果】从5%、10%和15%3个盐浓度中分别分得221、54和23株嗜盐菌,获得的嗜盐菌分布在9个科18个属;其中放线菌分布于4个属,细菌分布于14个属;Nocardiopsis和Pontibacillus属为艾丁湖可培养嗜盐菌的优势类群,分别占17.7%和16.1%;有15株嗜盐菌相似性低于98.5%,可能为潜在新种。所选取的22株代表菌株中,分别有68.2%、22.7%和72.7%的实验菌株具有蛋白酶、淀粉酶和酯酶活性;45.5%的代表菌株对12种病原菌表现出了抗至少1种病原菌的活性,其中一株Nocardiopsis属放线菌能抗9种病原菌,表现出了广谱的抗菌活性。【结论】新疆艾丁湖土样中嗜盐菌的多样性较丰富,而且具有较好的生物活性,为后续进一步研究其次级代谢产物提供了依据。     

Inhibition and Activation of Bacterial Luciferase Synthesis

Luciferase synthesis is repressed when bioluminescent bacteria are inoculated into fresh medium but is induced after the cells have grown in the medium for some time. In minimal medium, an activator which leads to induction of the enzyme is released into the medium by the bacteria. Complete medium contains a dialyzable and quite stable inhibitor which leads to repression of luciferase. The bacteria remove the inhibitor from the medium and also produce activator, thus allowing synthesis of the enzyme. Two unidentified nonluminescent strains of bacteria were unable to remove the inhibitor. Two different bioluminescent strains, Photobacterium fischeri and P. fischeri strain MAV, produce specific activators that are ineffective with cells of the other strain. The two activators are different with respect to heat stability, but both are small molecules. The activators can be assayed on the basis of their ability to counteract the inhibitor. Identification of the inhibitor and the activators may allow the bioluminescent system to be linked to other metabolic processes of the cells.     

Factors Related to the Oxygen Tolerance of Anaerobic Bacteria

The effect of atmospheric oxygen on the viability of 13 strains of anaerobic bacteria, two strains of facultative bacteria, and one aerobic organism was examined. There were great variations in oxygen tolerance among the bacteria. All facultative bacteria survived more than 72 h of exposure to atmospheric oxygen. The survival time for anaerobes ranged from less than 45 min for Peptostreptococcus anaerobius to more than 72 h for two Clostridium perfringens strains. An effort was made to relate the degree of oxygen tolerance to the activities of superoxide dismutase, catalase, and peroxidases in cell-free extracts of the bacteria. All facultative bacteria and a number of anaerobic bacteria possessed superoxide dismutase. There was a correlation between superoxide dismutase activity and oxygen tolerance, but there were notable exceptions. Polyacrylamide gel electropherograms stained for superoxide dismutase indicated that many of the anaerobic bacteria contained at least two electrophoretically distinct enzymes with superoxide dismutase activity. All facultative bacteria contained peroxidase, whereas none of the anaerobic bacteria possessed measurable amounts of this enzyme. Catalase activity was variable among the bacteria and showed no relationship to oxygen tolerance. The ability of the bacteria to reduce oxygen was also examined and related to enzyme content and oxygen tolerance. In general, organisms that survived for relatively long periods of time in the presence of oxygen but demonstrated little superoxide dismutase activity reduced little oxygen. The effects of medium composition and conditions of growth were examined for their influence on the level of the three enzymes. Bacteria grown on the surface of an enriched blood agar medium generally had more enzyme activity than bacteria grown in a liquid medium. The data indicate that superoxide dismutase activity and oxygen reduction rates are important determinants related to the tolerance of anaerobic bacteria to oxygen.     

Evaluation of three decarboxylating agar media to detect histamine and tyramine-producing bacteria in ripened sausages

Histidine- and tyrosine-decarboxylase activity of 175 strains of bacteria isolated from eight retail samples of Spanish ripened sausages was tested in three decarboxylating agars (Niven medium, Joosten and Northolt medium and modified decarboxylating agar of Maijala) and confirmed by an enzymic method (histamine) and thin-layer chromatography (tyramine). Enterobacteria and pseudomonads showed the highest percentage of positive responses to histamine and tyramine in the three decarboxylating agars, but only enterobacteria were subsequently confirmed as histamine-producing. Confirmed tyramine-producing strains were all identified as enterococci or lactic acid bacteria. The medium described by Joosten and Northolt was more sensitive and faster at detecting tyramine-producing micro-organisms. However, all three media failed to detect one histamine-positive strain of lactic acid bacteria used as a control.     

Studies on regulatory functions of malic enzymes. V. Comparative studies of malic enzymes in bacteria.

Screening of four malic enzymes--NAD-linked enzyme [EC 1.1.1.38], NAD, NADP-linked enzyme [EC 1.1.1.39], NADP-linked enzyme [EC 1.1.1.40], and D-malic enzyme--was carried out with cell-free extracts of the following 16 strains of bacteria by the aid of Sepharose 6B column chromatography: 9 strains of enteric bacteria, 3 strains of Pseudomonas, Alcaligenes faecalis, Agrobacterium tumefaciens, Rhodospirillum rubrum, and Clostridium tetanomorphum. All the strains tested contained at least one malic enzyme. The NADP-linked enzyme activity was found in all the strains except C. tetanomorphum, the NAD-linked enzyme activity in 12 strains--8 strains of enteric bacteria, 2 strains of Pseudomonas, Ag. tumefaciens, and C. tetanomorphum--and D-malic enzyme activity in 4 strains--A, aerogenes (IFO 3319 and 12059), Ps. fluorescens, and R. rubrum. The NADP-linked and NAD-linked enzyme activities of two strains of Pseudomonas were not separated by the chromatography. The available evidence suggested that the NAD, NADP-linked enzyme was not present in these 16 strains. The comparative studies of molecular, enzymatic, and serological properties of the malic enzymes in these 16 strains revealed a close similarity of the same types of malic enzymes among enteric bacteria.     

伊犁河流域春季可培养细菌多样性及其胞外酶检测

利用可培养法对新疆伊犁河流域水体和沉积物中细菌多样性进行分析，以期初步阐明流域河流可培养细菌群落结构。采用5种琼脂培养基分离纯化可培养细菌，依据其16S rRNA基因序列进行系统发育分析，并运用平板法对纯化菌株的胞外酶产生情况进行检测。序列分析结果表明，225株细菌分别属于变形菌门γ亚群(Gamma-pseudomonadota, 56.44%)、放线菌门(Actinomycetota, 18.22%)、厚壁菌门(Firmicutes, 14.22%)、变形菌门α亚群(Alpha-pseudomonadota, 4.89%)、变形菌门β亚群(Beta-pseudomonadota, 4%)、拟杆菌门(Bacteroidota, 0.44%)和异常球菌-栖热菌门(Deinococcota, 0.44%)等7个大的系统发育类群，41个属84个种。其中假单胞菌属(Pseudomonas,42.22%)、不动杆菌属(Acinetobacter,9.33%)和芽胞杆菌属(Bacillus,9.33%)为优势菌属。菌种分布结果显示，伊犁河流域主要支流和干流中可培养细菌地域分布性强。分离菌株产胞外酶...     

The selection of the composition of the medium for optimizing the amine-synthetic activity of aerobic bacilli]

Nutrient medium chosen as a basic one after preliminary test of several media known from literature has been optimized to intensify biosynthesis and amine nitrogen production by three strains of aerobic sporulating bacteria to culture liquid. The method of mathematical planning used in the experiments has permitted obtaining the components ratio for the medium on which production of amine nitrogen to the environment increased 2.3-3.2 times. The best variants of the optimized medium promoted an increase of the aminosynthetic activity of the studied bacteria by more than 320%. The obtained nutrient medium is appropriate for a wide screening of aerobic bacilli for their ability to synthesize amino compounds.     

一株抗小鼠乳腺癌和一株有抑菌活性的北极细菌的初步研究

从采自北级的海泥、海水样品中共分离得到101株低温细菌,采用小鼠温敏型乳腺癌tsFT210细胞株和纸片扩散法对其进行了抗肿瘤和抑菌活性筛选,得到抗肿瘤活性细菌1株,抑茼活性菌株8株,并对其中抑菌活性较强的一株细菌AR084的适宜发酵条件、活性物质的稳定性进行了初步研究,确定了其培养条件。报道了北极海洋微生物的抗肿瘤和抑菌活性,且抑菌活性尚未见有报道。由此表明,极地微生物是潜在的活性物质的来源,在基础研究和开发应用方面具有广阔的前景。     

Studies on the Metabolism of D-amino acid in Microorganisms

Several strains of bacteria belonging to genus Aerobacter were found to oxidize D-glutamate rapidly, while tbey show feeble oxidative activity toward the L-form even when they were grown in the medium containing DL-glutamate.

The isolation of L-glutamate, a natural amino acid, from its DL-form was achieved by the degradation of D-glutamic acid using one of these strains.

This may be the first observation on a natural amino acid obtained from the racemic one by the metabolic action of the organism.

A new enzyme, D-glutamic acid oxidase, which is responsible for D-glutamate degradation in this organism and differs from Krebs’ D-amino acid oxidase, has been postulated.     

Isolation and characterization of an acidic lethal phospholipase Az from Malayan cobra (Naja naja sputatrix) venom

An acidic, lethal phospholipase Az was purified to electrophoretic homogeneity from the venom of the Malayan cobra (Naja naja sputatrix). The enzyme has an isoelectric point of 5.58, a molecular weight of 12000, and a medium lethal dose (LD50) of 0.86 micrograms/g in mice by intravenous injection. The enzyme also exhibited weak anticoagulant and edema-forming activities. The amino acid composition of the enzyme is similar to those of other cobra venom phospholipases Az.     

Penicillinase production by Neisseria gonorrhoeae strains isolated from the patients of Dermatology and Wenerology Clinic, Warsaw Medical University in 2006 - 2009

In recent years the resistance of Neisseria gonorrhoeae to antibiotics is increasing in many countries. The aim of the study was to investigate penicillinase production by Neisseria gonorrhoeae strains isolated from the patients of Clinic of Dermatology and Wenerology WUM in a period between 2006 - 2009. We cultured the bacteria on Roiron medium and we used the iodometric test or BBL Cefinase discs to detect penicillinase. The enzyme was produced by 1,1% of 183, 0,9% of 111, 1,1% of 94 and 0% of 91 of investigated strains, respectively in 2006, 2007, 2008 and 2009 year - on average by 0,8%. This is the lowest result in Europe and one of the lowest in the world.     

Enzymic detection of adhesion of enteropathogenic Escherichia coli to HEp-2 cells

We established a new method for detecting enteropathogenic Escherichia coli adhering to HEp-2 cells. An essential part of the method is an assay of beta-galactosidase activity of adhered bacterial cells. It consisted of the following steps: (1) culture of bacterial cells in a medium containing isopropyl-thio-beta-D-galactoside, an inducer of beta-galactosidase; (2) incubation of a bacterial culture with monolayered HEp-2 cells in a 96-well culture plate; (3) washing wells to remove bacterial cells which did not adhere to HEp-2 cells, and (4) enzymic reaction for beta-galactosidase activities. However, a calibration curve for the enzyme activity, obtained from each bacterial sample, showed that 10(5) bacteria per well permitted an accurate estimation. The enzyme activity of adhered bacteria to the monolayered cells showed that 10(7) bacteria were appropriate for the adherence assay. The number of adhered bacteria thus obtained was in good agreement with a viable cell count. The result indicates that the new method is more reliable than a widely used method, counting the number of bacteria under a microscope. The present method also makes it easy to detect adherent strains of E. coli in large numbers of specimens.     

Purification,characterization, and identification of a sphingomyelin synthase from Pseudomonas aeruginosa. PlcH is a multifunctional enzyme

Sphingomyelin synthase is the enzyme that synthesizes sphingomyelin (SM) in mammalian cells by transferring a phosphorylcholine moiety from phosphatidylcholine to ceramide. Despite its importance, the gene and/or the protein responsible for this activity has not yet been identified. Here we report the purification, identification, and biochemical characterization of an enzymatic activity that synthesizes SM in Pseudomonas aeruginosa. SM synthase-like activity was found secreted in the culture medium of P. aeruginosa, strains PA01 and PAK, whereas it could not be detected in cultures of Escherichia coli. From the medium of PAK cultures, SM synthase was purified through sequential chromatographic columns. After separation on polyacrylamide-SDS gels and visualization by silver staining, the purified enzyme showed two bands, one of approximately 75 kDa and one of 30-35 kDa. Interestingly, the highly purified SM synthase preparation also showed neutral sphingomyelinase activity. We therefore investigated whether the protein we purified as SM synthase could actually be the previously identified PlcH, a 78-kDa phospholipase C known to hydrolyze phosphatidylcholine and SM in P. aeruginosa. First, the purified SM synthase preparation contained a 78-kDa protein that reacted with monoclonal antibodies raised against purified PlcH. Second, purified PlcH showed SM synthase activity. Third, using different knockout mutant strains for the PlcH operon, PlcH was found to be necessary for SM synthase activity in P. aeruginosa. Interestingly, SM synthase activity was specific to the Pseudomonas PlcH as other bacterial phospholipases did not display SM synthase activity. Biochemical studies on the Pseudomonas SM synthase confirmed that it is a transferase, similar to the mammalian enzyme, that specifically recognizes the choline head-group and the primary hydroxyl on ceramide. This SM synthase did not have reverse transferase activity. In conclusion, the Pseudomonas PlcH also exerts SM synthase activity; therefore, for the first time, we have identified a structural gene for a SM synthase.     

近江牡蛎肠道细菌及其产酶能力的研究

从近江牡蛎(Jinjiang Ostrea vivularisGould)肠道中分离出21株菌,其中9株革兰氏阳性菌,12株革兰氏阴性菌。并检测其蛋白酶、脂肪酶、淀粉酶、纤维素酶的产酶能力。结果表明,13株菌(61.9%)能分泌蛋白酶,13株(61.9%)分泌淀粉酶;11株(52.4%)产脂肪酶;7株(33.3%)产纤维素酶。产4种酶的有5株,产3种酶的3株,产2种酶的5株,产1种酶的3株,不产酶的仅有5株。产酶菌株的比例高达76.2%(16/21),可见近江牡蛎肠道细菌对食饵消化有重要作用。     

Mechanism of Isolated Hemicellulose and Xylan Degradation by Cellulolytic Rumen Bacteria

Although certain strains of cellulolytic rumen bacteria cannot utilize isolated hemicelluloses or xylan as a source of energy, all strains examined can degrade or solubilize these materials from an 80% ethyl alcohol insoluble to a soluble form. Centrifugation and washing of the cellobiose-grown bacterial cells did not affect the rate or extent of utilization or degradation or both. When the level of a nonutilizing culture inoculum (either normal or washed) was doubled, a corresponding increase in the initial rate of degradation was observed. With a nitrogen-free medium, utilization of xylan was almost completely inhibited for a utilizing strain, whereas degradation by either type of organism was not markedly affected. Cellobiose medium cell-free culture filtrates from a nonutilizing strain were able to degrade or solubilize xylan. The percentage of degradation increased with the volume of cell-free filtrate, and all activity was lost when the filtrate was boiled. No utilization (loss in total pentose) was observed with cell-free filtrates from utilizing or nonutilizing strains. The release of free hexose from insoluble cellulose by culture filtrates from a nonutilizing strain was very limited. On the other hand, carboxymethylcellulose (CMC-70L) and cellulodextrins were degraded to an 80% ethyl alcohol soluble form by filtrates from both types of organisms. Similar enzyme activity was obtained in cell-free culture filtrates from four additional strains of cellulolytic rumen bacteria (one xylan utilizer and three nonutilizers). When the assays were carried out aerobically, CMC-70L solubilization was reduced to a much greater extent than xylan or cellulodextrin solubilization. The enzyme or enzymes responsible for the degradation of hemicellulose by cellololytic rumen bacteria unable to utilize the hemicellulose as an energy source appear to be constitutive in nature, and this activity may be a nonspecific action of a beta-1, 4-glucosidase or -cellulase.     

Secreted phospholipases of the dimorphic fungus, Candida albicans; separation of three enzymes and some biological properties

Several phospholipases are secreted into the culture medium by growing yeast cells of Candida albicans 3125. DEAE-Sephadex column chromatography of concentrated culture filtrate revealed three separable fractions with phospholipase activities. Analysis of products of hydrolysis showed that the enzyme activities were lysophospholipase, lysophospholipase-transacylase and a phospholipase B.     

Novel phospholipase A activity secreted by Legionella species

Bacterial phospholipases are regarded as a major virulence factor in infection. In bacteria associated with pneumonia, destruction of lung surfactant and host cell membranes by bacterial phospholipases secreted during infection is thought to contribute to the disease. Phospholipase C (PLC) activity has been described in several Legionella species (W. B. Baine, J. Gen. Microbiol. 134:489-498, 1988; W. B. Baine, J. Gen. Microbiol. 131:1383-1391, 1985). By using detection methods such as thin-layer chromatography and mass spectrometry, PLC activity could not be detected in several strains of Legionella pneumophila. Instead, phospholipid degradation was identified to be caused by a novel PLA activity. We could demonstrate that PLA secretion starts at the mid-exponential-growth phase when bacteria were grown in liquid culture. Several Legionella species secreted different amounts of PLA. Legionella PLA may act as a powerful agent in the mediation of pathogenicity due to destruction of lung surfactant and epithelial cells.     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.     

The hydrogen peroxide microvolumetric method of the OF test: the rapid determination of glucose oxidation and fermentation by gram-negative bacteria

For the first time the method of the rapid (1 hour) screening of groups of gram-negative bacteria by the OF test with glucose, carried out with the use of microvolumetric techniques, has been developed. The method is based on the use of hydrogen peroxide at non-bactericidal concentration as a component of a liquid buffer medium containing indicator and glucose and intended for the oxidation of glucose. Catalase of bacteria introduced into the medium for study ensures the rapid saturation of the medium with oxygen and the completion of the oxidation of glucose in 10-60 minutes. An equal period is necessary to achieve the complete fermentation of glucose in the same medium without hydrogen peroxide. The method has been proved to yield significant results in the joint study of the OF test on 502 strains, belonging to 21 genera of fermenting and nonfermenting gram-negative bacteria, by the hydrogen peroxide method and in Hugh-Leifson medium.