实时荧光定量PCR法检测环境假单胞菌属细菌丰度

应用实时荧光定量PCR法,建立了总细菌及假单胞菌属的标准曲线,并以环境样本中假单胞菌属与总细菌的比值反映其细菌丰度.应用该方法评价人工除藻反应器中组合填料、无纺布、弹性填料等介质对太湖水中溶藻细菌之一的假单胞菌属的富集情况.结果显示:该方法对总细菌的检测范围为103~108个基因拷贝/μL(R2=0.997);假单胞菌属的检测范围为1~105个基因拷贝/μL(R2=0.994).对除藻反应器溶藻细菌富集效果的评价显示,所建立的方法能有效检测反应器中填料对该类溶藻细菌的富集程度.初步证明所建立的方法可有效定量假单胞菌属在环境样本中的丰度.     

Accurate localization and excision of genomic islands in four strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Mobile genomic islands (GIs) can be excised from the chromosome, then form a circular intermediate and be reintegrated into the chromosome by the GI internal integrase. Some mobile GIs can also be transferred into a new receptor cell by transformation, conjugation, or transduction. The action sites of the integrase are usually flanked direct repeats (DRs) of the GIs. Accurate localization of the flanking sequences is a precondition for determining the mobility of the GI. Mobile GIs are generally associated with transfer RNAs (tRNAs). Based on the correlation between flanking sequences and tRNA sequences, the flanking sequences of 11 putative mobile GIs in Pseudomonas aeruginosa PAO1, P. aeruginosa PA14, P. fluorescens Pf-5 and P. fluorescens Pf0-1 were identified. Among the 11 GIs, Pf0-1GI-1 is responsible for benzoate degradation. PAO1GI-1, Pf5GI-2, Pf5GI-3, and Pf5GI-4 were confirmed experimentally to be excised from a chromosome to form a circular intermediate. The action sites of the integrases are these GIs direct repeats. Due to distinct DRs, cutting sites for the internal integrase of PAO1GI-1, Pf5GI-2, Pf5GI-3 and Pf5GI-4 were determined outside the T-loop of the tRNA^Gly gene, outside the anticodon loop of the tRNA^Ser gene and tRNA^Lys gene, and at the asymmetric 3′-end of the tRNA^Leu gene, respectively. PAO1GI-1 and other mobile GIs may be transferred into many different strains that belong to different phyla because of the clear flanking sequences. This study describes basic information about the action sites of the integrases, assesses the mobility of GIs, and can help design and transfer mobile GIs to candidate strains.     

假单胞菌DM11的二氯甲烷脱卤素酶研究──Ⅱ．动力学性质和抑制剂

对来自假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓｓｐ．）ＤＭ１１菌株的二氯甲烷脱卤素酶（简称ＤＭ１１脱卤素酶）的动力学性质和抑制剂进行了研究。用１／Ｖ对１／［ｓ］作图法，测出ＤＭ１１脱卤素酶以ＣＨ２Ｃｌ２、ＣＨ２Ｂｒ２、ＣＨ２Ｉ２、ＣｌＣＨ２Ｂｒ和ＣｌＣＨ２Ｉ等５种化合物为底物时的Ｋｍ值分别是６７、２０、５５、７和９μｍｏｌ／Ｌ，比活力分别是７７．８、１２３．３、７８．８、５６．１和６０．３ｍｋａｔ／ｋｇ蛋白。测定了１４种抑制剂对ＤＭ１１脱卤素酶的５０％抑制浓度（Ｉ５０）。甲基谷胱甘肽（ＧＳＣＨ３）是ＤＭ１１脱卤素酶的竞争性抑制剂，四硝基甲烷（ＴＮＭ）是非竞争性抑制剂，当甲基谷胱甘肽和四硝基甲烷与ＤＭ１１脱卤素酶一起保温时，甲基谷胱甘肽对酶有保护作用。     

荧光假单胞菌噬铁素的性质研究

本文报道了荧光假单胞菌Ｐ３２产生的噬铁素的特性．噬铁素在紫外光下发出黄绿色荧光，与Ｆｅ３＋有高度亲和性，在２３０ｎｍ和４００ｎｍ附近各有一吸收峰，氨基酸组成为苏氨酸：甘氨酸：赖氨酸：胱氨酸：亮氨酸：天冬氨酸＝２：４：４：４：１：１，生物合成需较低浓度Ｆｅ３＋为条件，与Ｆｅ３＋的复合物无荧光，但可用连二亚硫酸钠，８－羟基喹啉和抗坏血酸粉末恢复荧光．     

假单胞菌中抗汞质粒pBH33的分离与鉴定

从污染环境中分离出一株抗100μM HgCl_2的假单胞菌B-33.经试验证实,假单胞菌B-33具有汞盐抗性质粒,定名为pBH33质粒,其分子量约为7.24×10~6道尔顿.     

一株原油降黏细菌的筛选

自环境中分离出一株假单胞菌，在厌氧条件下能显著降低高黏度原油黏度，降黏率最高可达68％，气相色谱显示，降解前后原油中各组份含量发生显著变化。     

抑制姜瘟青枯假单胞菌的木霉菌株的筛选及其抑菌机理

筛选得到一株可有效抑制姜瘟青枯假单胞菌的木霉菌株SMF2(Tricoderma spp SMF2)，证实其胞外分泌的抗生素--木霉素是抑制姜瘟菌生长的主要物质，该物质对热稳定，固体发酵是培养木霉制剂的有效方法，并对培养基的组成、培养条件进行了初步优化。     

Isolation,purification and characterization of a new organphosphorus hydrolase OPHC2

A bacterium with the capability of degrading organphosphorus, identified as Pseudomonas pseudoaicaligenes, is isolated from OP-treated soil. The organphosphorus hydrolase OPHC2 from this bacterium has been purified and characterized. OPHC2 has optimum activity for the reaction at 65℃ and pH 9.0 with methyl parathion as a substrate, it also shows good thermal and pH stability. Most metal ions and chemicals have no effect on the activity of OPHC2. The analyses of nucleotide sequence encoding OPHC2 and amino acid sequence of OPHC2 show that there are lower homologies with those of organphosphorus hydrolase reported in GenBank.     

A novel salicylaldehyde dehydrosenase-NahV involved in catabolism of naphthalene from Pseudomonas putida ND6

A novel salicylaldehyde dehydrogenase involved in catabolism of naphthalene from Pseudomonas putida ND6, NahV, has been identified. NahV exhibited lower identity in amino acid sequence with the classic salicylaldehyde dehydrogenase, NahF, from P. putida ND6. This is the first report of an isofunctional enzyme of bacterial salicylaldehyde dehydrogenase. Comparison of Km and Vmax values of NahV and NahF demonstrated that NahF has a more efficient catalytic reaction than NahV, while NahV has much higher affinity for salicylaldehyde and NAD^＋. Both enzymes exhibited broad substrate specificities and catalyzed the oxidation of salicylaldehyde, 5-chlorosalicylaldehyde, formaldehyde, m-nitrobenzaldehyde, o-nitrobenzaldehyde, o-methoxybenxaldehyde, glutaraldehyde, caprylic aldehyde, and glyoxal. However, the relative rates at which the substituted analogs are transformed differ considerably. NahV activity could be enhanced by Fe^2＋, Cu^2＋ and Zn^2＋; whereas NahF activity could only be stimulated by Fe^2＋, NahF is more stable than NahV at elevated temperatures. Dot-blot hybridization analyses showed that nahF-like genes occurred in all naphthalene-degradation bacteria isolated in this study, whereas nahV-like genes were present in only some naphthalene-degrading bacteria.     

Formation of Polyhydroxyalkanoate Blends by Pseudomonas pseudoalcaligenes M1-2 from Various Carbon Sources

ＩｎｔｒｏｄｕｃｔｉｏｎＰｏｌｙｈｙｄｒｏｘｙａｌｋａｎｏａｔｅｓ (ＰＨＡｓ)ａｒｅａｆａｍｉｌｙｏｆｉｎｔｒａｃｅｌｌｕｌａｒｂｉｏｐｏｌｙｍｅｒｓｓｙｎｔｈｅｓｉｚｅｄｂｙｍａｎｙｂａｃｔｅｒｉａａｓｉｎｔｒａｃｅｌｌｕｌａｒｃａｒｂｏｎａｎｄｅｎｅｒｇｙｓｔｏｒａｇｅｃｏｍｐｏｕｎｄｓ[1] .ＰＨＡｓｈａｖｅａｗｉｄｅｒａｎｇｅｏｆｐｈｙｓｉｃａｌｐｒｏｐｅｒｔｉｅｓｒａｎｇｉｎｇｆｒｏｍｂｒｉｔｔｌｅｔｏｆｌｅｘｉｂｌｅｔｏｅｌａｓｔｉｃ ,ｄｅｐｅｎｄｉｎｇｏｎｔｈｅｉｒｍｏｎｏｍｅｒｓｔｒ…