Isolation of a Specific Potato Tuber-Inducing Substance from Potato Leaves

Potato tuberization is induced by an unidentified "tuberizationstimulus" which is produced in the leaves. Recently, we confirmedthe occurrence of two acidic substances in the leaves whichappear to be the stimulus (Koda and Okazawa 1988). We reporthere the isolation of one of the substances from potato leaves.The molecular weight of the substance is 388. The substanceis active in inducing tuberization in vitro at a concentrationof 0.01 mg- liter^-11 (ca. 3 ? 10^-8 M). (Received April 15, 1988; Accepted June 28, 1988)     

Isolation and Identification of Novel Microcystin-Degrading Bacteria

Of 31 freshwater bacterial isolates screened using the Biolog MT2 assay to determine their metabolism of the microcystin LR, 10 were positive. Phylogenetic analysis (16S rRNA) identified them as Arthrobacter spp., Brevibacterium sp., and Rhodococcus sp. This is the first report of microcystin degraders that do not belong to the Proteobacteria.A number of studies have reported biological degradation of microcystin in samples from lakes and sediments (3, 4, 12, 15), but only a few bacterial strains with the ability to degrade microcystins have been isolated and characterized (6). Previously identified bacteria belonged to the Proteobacteria, and with the exception of one isolate (Sphingomonas sp. strain CB4), they were all shown to degrade microcystin LR (MC-LR) via the same degradation pathway: formation of linear MC-LR following cleavage at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg bond and hydrolysis at Ala-Leu to yield a tetrapeptide with Adda as the final product detected (1, 10, 11). Genes encoding enzymes involved in this pathway have been characterized and shown to have similarity in all bacterial isolates currently reported to degrade microcystins (2).Recent work indicated a greater diversity of microbes capable of degrading microcystins and nodularin, with the tentative identification of several novel degradation intermediates (5). In the present study, enrichment was used to isolate bacteria from three Scottish water bodies previously shown to contain microflora capable of microcystin degradation (5). The Biolog MT2 assay was used to screen the ability of the isolated bacteria to metabolize MC-LR, since this had previously been shown to be an effective means of demonstrating metabolism of microcystin by Paucibacter toxinivorans (6).The ability to metabolize MC-LR was determined in the Biolog MT screen, with 10 of the bacterial isolates giving a positive result. We subsequently confirmed that they could all degrade MC-LR in batch degradation studies, as evidenced by liquid chromatography-mass spectrometry (LC-MS) analysis. The microcystin-degrading bacteria were identified by using 16S rRNA gene analysis and investigated to determine the presence of mlrA, mlrB, mlrC, and mlrD, the genes previously reported to be involved in the degradation of MC-LR by Sphingomonas sp. strain ACM-3962 (2). We report here isolates identified as Arthrobacter spp., Brevibacterium sp., and Rhodococcus sp. which have the ability to degrade MC-LR, although none of the previously characterized mlr genes were detected.Surface water samples were collected in sterile Pyrex glass bottles on 26 September 2007 from Loch Rescobie (Ordinance Survey grid reference number NO 52505159), Forfar Loch (NO 293458), and the River Carron (NO 877857), Scotland, United Kingdom. Samples were stored at 4°C overnight and filtered as previously described (5). Aliquots from each water sample (2 × 500 ml) were processed and analyzed by high-performance LC to determine the presence of naturally occurring microcystins (13). Enrichment and shake flask die-away kinetics were monitored in triplicate for each water type (50 ml in sterile 100-ml Erlenmeyer flasks). To enrich bacteria with the ability to degrade a range of different microcystins, three microcystins, selected for their differing polarities, and the pentapeptide nodularin were added to each water sample. MC-LR, MC-RR, MC-LF, and nodularin (Enzo Life Sciences, Lausen, Switzerland) were resuspended in a small volume (100 μl) of methanol and diluted with Milli-Q to a total concentration of 0.4 mg ml⁻¹. The toxin cocktail was sterilized (0.2-μm Dynaguard filter; Fisher, United Kingdom) and added to each flask under aseptic conditions to give a final concentration of 1 μg ml⁻¹of each toxin (i.e., 4 μg ml⁻¹ total concentration). All flasks were incubated at 25°C ± 1°C with shaking at 100 rpm. Aliquots (2 ml) were removed from each flask under sterile conditions every 2 days, transferred into 4-ml glass vials, and frozen (−20°C) immediately. Die-away kinetics were monitored for 14 days. The frozen samples were freeze-dried, reconstituted in 200 μl of 50% aqueous methanol, and centrifuged at 15,000 × g for 10 minutes. The supernatant (100 μl) was removed for LC-MS analysis (5). Sterile controls (3 × 50 ml) were prepared, incubated, and sampled as described above to confirm whether loss of toxin was a result of microbial activity.After 14 days of enrichment, 1 ml of sample was removed aseptically from each flask, namely, the Loch Rescobie (R), Forfar Loch (F), and River Carron (C) samples. Serial dilutions (to 10⁻⁵) were made using Ringer''s solution (Oxoid Ltd., United Kingdom), and 1 ml of each dilution was removed and mixed with 20 to 25 ml of molten LB agar, poured onto sterile petri dishes, and incubated in the dark at 25°C for 5 days. Colonies with differing morphologies were resuspended in liquid LB medium, and pure cultures were obtained by repeated streaking onto LB agar plates. For the Biolog MT2 assay, a loop of each isolated bacterial strain was transferred to 5 ml of liquid LB medium and incubated overnight in the dark at 25°C. The exponentially growing cultures were then washed twice by centrifugation at 1,000 × g for 15 min, the bacterial pellets were resuspended in sterile 0.01 M phosphate-buffered saline, and the cultures were incubated at 25°C for 24 h to deplete residual carbon. The turbidity of all bacterial suspensions was an A₅₉₀ of 0.35. MC-LR was added to Biolog MT2 plates (Technopath, Limerick, Ireland) in triplicate to give final concentrations of 10, 1, 0.1, and 0 μg ml⁻¹. Wells were inoculated with bacterial suspension (150 μl), and plates were incubated at 25°C. Absorbance at 595 nm was recorded by using a Dynex microplate reader (Jencons, Leighton Buzzard, United Kingdom) immediately after inoculation (0 h) and at 3, 6, 15, 18, 24, and 48 h. Metabolism of MC-LR results in the reduction of tetrazolium violet, giving a color reaction that can be quantified spectroscopically (8). Bacterial isolates found by using the Biolog MT screen to metabolize MC-LR were evaluated for their ability to degrade MC-LR. Isolates C1, C3, and C6 (from the River Carron), F3, F7, and F10 (from Forfar Loch), and R1, R4, R6, and R9 (from Loch Rescobie) were grown overnight in LB liquid medium at 25°C. Bacterial isolates were washed and carbon depleted as described above (0.5 ml) and then added to glass universal bottles containing 9 ml of 0.2-μm-filter-sterilized water from their original locations. Aqueous MC-LR (0.5 ml) was added to each bottle under aseptic conditions at a final concentration of 5 μg ml⁻¹. Triplicate samples were prepared for each isolate and incubated at 25°C ± 1°C with shaking at 100 rpm. Aliquots (0.5 ml) were removed at 24-h intervals under sterile conditions, freeze-dried, reconstituted in 200 μl of 50% aqueous methanol, and centrifuged at 15,000 × g for 10 min. The supernatant (100 μl) was removed for LC-MS analysis performed as previously described (6). Experiments with sterile controls were performed for each water sample. Paucibacter toxinivorans DSMZ-16998 (Braunschweig, Germany) was used as a positive control as it has been reported to degrade MC-LR, MC-YR, and nodularin (16).To identify selected isolates, total DNA was extracted from the pellet by using an UltraClean DNA isolation kit (Mo Bio Laboratories, CA). Sequencing was performed with a BigDye Terminator cycle sequencing reaction kit (202 instrument; Applied Biosystems, United Kingdom) using 8F, 1492, and various other internal primers (518R and 1087R) on an automated DNA sequencer (ABI, United Kingdom) (7, 17). The quality of the sequence was checked by using the sequence analysis software (ABI), and the products of the forward and reverse primers were aligned using Kodon (Applied Maths, Saint-Martens-Latem, Belgium). The analyzed sequences were compared to DNA sequences in public databases using the BLAST function of NCBI (http://www.ncbi.nlm.nih.gov). Individual isolates were classified according to their similarity to sequences in the database. DNA sequences of all isolates, along with those of related bacteria and some known microcystin-degrading bacteria, were used to construct a phylogenetic tree using MEGA4 (18). The sequences were first aligned using Clustal W, and then a phylogenetic tree was constructed by performing neighbor-joining tree analysis with 1,000 bootstrap replicates. Each microcystin-degrading isolate was assayed to determine if mlr genes for the degradation of microcystin could be detected. The PCR method used primers specific for mlrA, mlrB, mlrC, and mlrD with conditions as described before (9). A positive control for these genes was used (Sphingopyxis sp. strain LH21).The Biolog MT2 plates, used to screen 31 isolates, were shown to be an effective means of rapidly identifying bacteria with the ability to metabolize MC-LR (Fig. ​(Fig.1).1). Ten isolates which demonstrated respiration in the presence of MC-LR using the Biolog format were subsequently proven to be microcystin-degrading bacteria in batch studies where MC-LR almost or totally disappeared after 3 days of incubation (Table ​(Table1).1). The results of LC-MS analysis indicated that MC-LR disappeared with no obvious biotransformation or intermediate degradation products. This may be because degradation resulted in only very low concentrations of these compounds and MC-LR is readily utilized by the isolates, as evidenced by respiration in the Biolog assay. Employing the Biolog MT plate enabled rapid (approximately 24 h) selection of bacteria in a high-throughput format (96-well plates) using considerably less microcystin or nodularin, i.e., 5 μg per isolate in the Biolog MT plate compared to 300 μg to follow degradation in die-away kinetics as described herein). Furthermore, following degradation by the latter method requires sample processing and high-performance LC analysis, increasing the time and cost. While the Biolog plates have been widely used for community profiling and bacterial identification, they have yet to be fully exploited in biodegradation studies, where they may facilitate rapid, cost-effective screening of many more bacterial isolates for the ability to utilize a wide range of environmental pollutants.Open in a separate windowFIG. 1.Results of Biolog screen for MC-LR metabolism by bacteria isolated from Loch Rescobie (A), Forfar Loch (B), and the River Carron (C) after 24 h of incubation. Control samples (black bars) contained no additional carbon source. MC-LR was added as the carbon source at 0.1 (open bars), 1 (hatched bars), and 10 (shaded bars) μg ml⁻¹. Error bars represent 1 standard deviation (n = 3).

TABLE 1.

Batch degradation of MC-LR by bacterial isolates in source

临床厌氧菌的分离和鉴定

我们从口腔、胸部、腹部和盆腔等处采集了110份临床感染标本,以自制的输送培养基立即送实验室,在厌氧手套箱(霍尔玛厌氧系统1029型)或Gas-pak罐中,行厌氧菌的分离和培养,其中64份标本检出厌氧菌,阳性率为58％。临床标本中牙周炎标本厌氧菌检出率高达100％,牙髓炎标本为85％,阑尾脓肿和腹膜炎标本为83％,胆道标本为39％,脓胸标本为57％,盆腔标本为33％,早期单纯性阑尾炎和甲状腺囊肿合并感染的标本各5份,都未检出厌氧菌。从64份阳性分离的标本中共分离到厌氧菌370株,经鉴定分别属于11个菌属32个菌种(未定种的有74株),其中类杆菌最多占45.9％(类杆菌属中脆弱类杆菌占37.6％),次为梭杆菌属和消化链球菌属,各占15.1％。革兰氏阳性无芽胞厌氧菌占8％左右,而梭菌属为8.9％,其余是二氧化碳噬纤维菌属(2.9％)、韦荣氏球菌属(1.3％)、链球菌属和纤毛菌属(1.5％)等。在厌氧菌鉴定中,我们使用了微量生化直接酶测定技术和代谢产物的气相色谱分析技术,这些方法在厌氧菌鉴定中比常规方法敏感且有较大的价值。     

亚硝化细菌的分离及初步鉴定

目的:为了使短程硝化-反硝化生物脱氮工艺能更有效地应用于含氮污废水的处理,需分离出高效的亚硝化细菌.方法:采用亚硝化细菌富集培养基选择培养和硅胶平板分离法,通过初筛、复筛,从广西大学农场菜园土中分离到一株亚硝化速率较高的菌株N4.结果:菌株N4革兰氏染色阴性,不产芽孢,细胞短杆状,呈单个排列,有一根亚极端鞭毛.结论:初步鉴定菌株N4为亚硝化单胞菌属(Nitrosomonas sp).     

常温土壤中耐冷菌的分离、鉴定及产酶分析

目的:从常温土壤中筛选冷适应微生物,并进行初步鉴定和产低温酶分析。方法:采集吉首大学校园内土壤样品,通过低温富集培养筛选冷适应微生物;通过形态观察、生理生化特性检测和基于16S rRNA基因序列的系统发育分析,对分离的菌株进行初步鉴定;利用平板筛选法检测其产低温酶特性。结果:分离获得6株耐冷细菌JSBP-1～JSBP-6,初步鉴定其分属假单胞菌属(Pseudomonas)、紫色杆菌属(Janthinobacterium)和节杆菌属(Arthrobacter);在5℃和15℃培养条件下,菌株JSBP-1产蛋白酶能力较强,JSBP-2和JSBP-6产淀粉酶能力较强,JSBP-5仅在5℃条件下有较强的产脂肪酶特性。结论:常温土壤中存在一定数量的冷适应微生物,其中假单胞菌是其优势菌群之一。这类适冷微生物菌群具有潜在的生产低温酶能力。     

Rifampin-Blood-Agar as a Selective Medium for the Isolation of Certain Anaerobic Bacteria

A selective medium which allows detection of relatively small numbers of Fusobacterium varium in fecal specimens is described. Blood-agar containing 50 mug of rifampin per ml inhibits the growth of many species of Bacteriodes and of F. fusi-forme/nucleatum but allows good growth of F. varium and most strains of F. mortiferum. Quantitative cultures of 11 fecal specimens were done on rifampin and other selective and nonselective media. F. varium was recovered in counts of 10(6) and 10(7) per gram from two specimens on rifampin only. A third specimen yielded 10(10)F. varium on several media, including rifampin. Some Eubacterium and Clostridium species also grew on rifampin, and these ordinarily were distinguished from the Fusobacterium by colony morphology. This medium is of value in fecal flora studies and should be useful with other kinds of specimens where mixtures of organisms are common.     

一株甲烷氧化菌的分离鉴定与特性

甲烷氧化菌是一类能以甲烷作为唯一碳源和能源进行同化和异化代谢的微生物。从若尔盖高原不同地点采集的样品中筛选得到一株名为XN1的甲烷氧化菌,根据此菌株的形态与16SrRNA序列同源性分析,证实该菌株属于Methylomonas属。对该菌株的培养条件进行研究的结果表明,以甲烷与甲醇共同作为碳源,硝酸钾和氯化铵共同为氮源时菌生长最好,最适生长温度为25℃,最适生长pH为6.5,培养基中CuSO4·5H2O和FeSO4·7H2O的浓度以0.03mg/L和0.4mg/L为宜。     

New Method for the Isolation and Identification of Methanogenic Bacteria

A new technique is reported for the rapid growth and detection of methanogenic bacteria by using petri plates. The method employs an anaerobic glove box containing an inner chamber with separate gas-flushing facilities. The numbers of methanogenic bacteria recovered from domestic sewage sludge are comparable to those recovered by other methods. The methanogenic organisms isolated from sludge include Methanosarcina, Methanospirillum, Methanobacterium strain M. o. H., and Methanobacterium formicicum. Identification of colonies containing methanogenic bacteria is facilitated by taking advantage of the unique fluorescence properties of these organisms. Colonies as small as 0.5 mm can be detected by exposing them to long-wave ultraviolet light.     

大蒜内生细菌的分离及拮抗菌筛选与鉴定

利用常规分离方法对大蒜鳞茎进行内生细菌的分离,采用对峙法和平板涂布法对分离的内生菌进行拮抗试验研究,并对菌株DSP6进行16S rDNA全序列鉴定。结果表明:分离得到19株内生细菌,其中10株菌对2种以上植物病原真菌有不同程度的抑制作用,占分离菌总数的52.6%,DSN7对番茄早疫病的抑菌圈半径最大,为13mm;17株菌对5种病原细菌中至少1种有抑制作用,占分离菌总数的89.5%,其中菌株DSP3对大肠杆菌的抑菌圈半径最大,达到10 mm;菌株DSP6对供试的9种病原菌有较强的抑菌作用,且抑菌圈平均半径最大,为6.88mm;16S rDNA全序列鉴定显示,菌株DSP6与芽孢杆菌属Bacillus axarquiensis相似性为100%,表明菌株DSP6为Bacillus axarquiensis。     

薯蔓多糖的分离纯化和性质鉴定

以我国资源量巨大的薯蔓为原料, 采用中试设备, 利用水提醇沉法提取多糖。研究了活性炭脱色工艺, 经初步纯化获得薯蔓多糖(PSPV), 并研究了其理化性质。DEAE-纤维素柱对来自于二个季节的PSPV进行分离、纯化, 分别得到PSPVⅠ和PSPVⅡ、PSPVⅢ三个多糖组分, 高效凝胶过滤色谱法测定三个组分的分子量分别为6.278×104 D, 3.801×104 D和1.418×104 D, 气相色谱结合标样测定单糖组成。研究结果为充分高效利用薯蔓资源提供了理论依据。     

薯蔓多糖的分离纯化和性质鉴定

以我国资源量巨大的薯蔓为原料, 采用中试设备, 利用水提醇沉法提取多糖。研究了活性炭脱色工艺, 经初步纯化获得薯蔓多糖(PSPV), 并研究了其理化性质。DEAE-纤维素柱对来自于二个季节的PSPV进行分离、纯化, 分别得到PSPVⅠ和PSPVⅡ、PSPVⅢ三个多糖组分, 高效凝胶过滤色谱法测定三个组分的分子量分别为6.278×104 D, 3.801×104 D和1.418×104 D, 气相色谱结合标样测定单糖组成。研究结果为充分高效利用薯蔓资源提供了理论依据。     

脂肪酶产生菌的筛选、鉴定及其产酶条件优化

目的:寻找合适的产酶菌。方法:从富油土壤中分离到一株脂肪酶产生菌,并通过16S rRNA部分序列分析和系统发育分析将其鉴定为假单胞菌属,定名为:Pseudomonas sp.26-2。本研究进一步通过正交试验设计对该菌株的产脂肪酶条件进行了优化。结果:在摇瓶培养条件下,其最适产酶条件为:淀粉1.5%,酵母提取物3%,硫酸镁0.05%,K2HPO40.2%,橄榄油0.2%;反应起始pH值为7.0,发酵温度为30℃。在此条件下,发酵脂肪酶活力可达15.5U/ml。结论:所获得的假单胞菌26-2具有一定的脂肪酶生产能力,并为该菌株的菌种改良以及脂肪酶的高效基因工程菌的构建奠定了基础。     

具有抑菌活性的海洋细菌的分离与鉴定

分离并筛选具有抑菌活性的海洋细菌对于开发和利用海洋微生物具有重要意义,该研究从6份海泥样品中共分离到78株海洋细菌,以6种细菌作为敏感指示菌,采用覆盖技术对分离菌株进行拮抗试验,17株海洋细菌具有抑菌活性,对其中2株具有较强抑菌活性的海洋细菌进行革兰氏染色,耐盐试验,运动性观察,过氧化氢酶测定,明胶液化试验,硫化氢产生试验,石蕊牛奶试验,糖类发酵,硝酸盐还原等特性分析,依据《伯杰氏细菌鉴定手册》进行分类鉴定,它们分别应归属为气单孢菌属（Aeromonas sp.）和假单孢菌属（Pseudomonas sp.）。     

好氧反硝化菌的分离鉴定及特性研究

从土壤中分离到1株能以硝酸钠为氮源进行好氧反硝化作用的细菌,命名为Rhodococcussp.DN,分离菌株革兰氏染色为阳性,球状或杆状,菌落颜色为橙红色。该细菌能以乙酰胺为惟一碳源和氮源,能进行氨化和硝化作用并产生亚硝酸。部分长度的16S rDNA序列分析表明,所分离的细菌与Rhodococcus ruber的16S rDNA序列具有99%相似性。     

Isolation, Crystallization, and Partial Identification of Potato Factor II from Potato Tubers

The isolation, crystallization, and partial identification of potato factor II, a stimulator from the chemically neutral fraction of potato extract, is described. The compound was originally found to stimulate elongation of dwarf peas grown under red light, a gibberellin bioassay. It melts between 137° and 139°. In paper chromatography it migrates to R_F 0.62 in isopropyl alcohol: ammonium hydroxide: H₂O (10:1:1, v/v). Based on infrared and NMR data, it does not contain a lactone ring and possibly possesses an amide radical and an OH⁻ group, as well as many methylene radicals. Potato factor II may be similar to certain of the fatty acid derivatives previously reported to stimulate growth of excised sections, but it is unique in that it stimulates growth of intact plants. This effect points to the need for completely separating neutral from acid gibberellin-like substances when the latter are assayed on dwarf peas.     

芦笋老茎堆肥中嗜热细菌的分离与鉴定

利用稀释涂布法对芦笋老茎堆肥不同发酵阶段6个样品中的嗜热细菌进行分离,并采用16S rDNA序列分析方法对分离得到的菌落形态有明显区别的22株细菌进行鉴定.根据16S rDNA序列分析结果,22株细菌菌株中13株属于芽胞杆菌属(Bacillus),1株属于类芽胞杆菌属(Paenibacillus),4株属于假黄色单胞菌属(Pseudoxanthomonas),1株属于肠杆菌属(Enterobacter),1株属于副球菌属(Paracoccus),1株属于短芽胞杆菌属(Brevibacillus),菌株D-b2在GenBank数据库中未找到与其相似的已知细菌属的序列,分类地位待定.从以上鉴定结果可以看出,芦笋老茎堆肥中的优势嗜热细菌主要是芽胞杆菌(Bacillus spp.)和假黄色单胞菌(Pseudoxanthomonas spp.).