假单胞菌属的一个新种

从桔汁矿化水饮料中分离到一株编号为9191的革兰氏阴性短杆菌,其形态、生理生化特性与假单胞菌属已报道的种均不相同.该菌株具极生单鞭毛,氧化酶和触酶均阳性;O-F葡萄糖为非发酵型,不产生荧光色素,不产生脓菌素,不产生类胡萝卜色素,对葡萄糖呈碱反应.经类脂粒染色后细胞内可见PHB颗粒积聚.脲酶阳性,硝酸盐还原阳性,反硝化阴性;不水解淀粉和明胶,精氨酸双水解酶阴性;41℃下不生长,在麦康凯和SS平板上不生长.在0.85%CaCl_2的牛肉汤中不生长;利用β-羟基丁酸作为唯一碳源;DNA中G+C含量为65.15mol%.因此定为假单胞菌属中的一个新种.根据其对生理盐水敏感的特性,命名为盐敏假单胞菌(Pseudomonas halosensibilis Zou ＆ Cai nov.sp.).     

Mechanism of cyanide and thiocyanate decomposition by an association of Pseudomonas putida and Pseudomonas stutzeri strains

The intermediate and terminal products of cyanide and thiocyanate decomposition by individual strains of the genus Pseudomonas, P. putida strain 21 and P. stutzeri strain 18, and by their association were analyzed. The activity of the enzymes of nitrogen and sulfur metabolism in these strains was compared with that of the collection strains P. putida VKM B-2187^T and P. stutzeri VKM B-975^T. Upon the introduction of CN⁻ and SCN⁻ into cell suspensions of strains 18 and 21 in phosphate buffer (pH 8.8), the production of NH ₄ ⁺ was observed. Due to the high rate of their utilization, NH₃, NH ₄ ⁺ , and CNO⁻ were absent from the culture liquids of P. putida strain 21 and P. stutzeri strain 18 grown with CN⁻ or SCN⁻. Both Pseudomonas strains decomposed SCN⁻ via cyanate production. The cyanase activity was 0.75 μmol/(min mg protein) for P. putida strain 21 and 1.26 μmol/(min mg protein) for P. stutzeri strain 18. The cyanase activity was present in the cells grown with SCN⁻ but absent in cells grown with NH ₄ ⁺ . Strain 21 of P. putida was a more active CN⁻ decomposer than strain 18 of P. stutzeri. Ammonium and CO₂ were the terminal nitrogen and carbon products of CN⁻ and SCN⁻ decomposition. The terminal sulfur products of SCN⁻ decomposition by P. stutzeri strain 18 and P. putida strain 21 were thiosulfate and tetrathionate, respectively. The strains utilized the toxic compounds in the anabolism only, as sources of nitrogen (CN⁻ and SCN⁻) and sulfur (SCN⁻). The pathway of thiocyanate decomposition by the association of bacteria of the genus Pseudomonas is proposed based on the results obtained. Original Russian Text ? N.V. Grigor’eva, T.F. Kondrat’eva, E.N. Krasil’nikova, G.I. Karavaiko, 2006, published in Mikrobiologiya, 2006, Vol. 75, No. 3, pp. 320–328.     

The Application of Adsorption Modeling and Fourier Transform Infrared Spectroscopy to the Comparison of Two Species of Plant Growth–Promoting Rhizobacteria as Biosorbents of Cadmium in Different pH Solutions

Batch experiments were performed to determine the cadmium absorption capacity of two plant growth–promoting rhizobacteria at different pH levels and in different cadmium concentrations. Comparison of the mean metal removal from two species of bacteria studied showed that Pseudomonas florescence is the superior species for removing cadmium at all cadmium concentrations. The maximum cadmium absorption by P. florescence and P. putida were at 5 mg/L of cadmium concentration in pH 6 and 7, respectively. The applicability of the Langmuir and Freundlich isotherm models was surveyed. Comparison of two isotherm parameters (Q _m and a) further confirmed that P. fluorescence was better at binding cadmium ions (52.6 and 7.7 mg/g, respectively). Adsorption reaction also was considered by Fourier transform infrared (FTIR) spectroscopy. The FTIR analysis implied that the principal functional sites in the bacterial cell walls were phosphoryl and hydroxyl, carboxyl, amide I, amide II, and amine groups.     

海洋低温蛋白酶菌株选育及发酵培养基的研究(Ⅰ)

以渤海和黄海分离出400多株在低温条件下生长良好的菌株为出发菌株,利用常规筛选方法选出2株低温蛋白酶产生菌（Pseudorrtortas alcaligenes）。经UV、DES、NTG、EMS、LiCl单独及复合诱变,选育出一株（Pa040523）蛋白酶高产突变株。通过单因素实验,确定了Pa040523菌株蛋白酶发酵培养基为：玉米淀粉糖1．8％,尿素0．6％,磷酸氢二钾0．6％,磷酸二氖钾0．3％。该突变株低温蛋白酶产量为940．8U／mg。     

城市污水处理系统的菌群分析和除氮基因工程出发菌的选择

目的:对脱氮污水处理工艺的活性污泥的菌群组成进行分析,以期获得适合于脱氮基因工程改良的出发菌.方法:首先采用平板稀释法对活性污泥进行菌落计数,并对分离到的菌落进行详细的生化鉴定,对其中的优势菌-假单胞菌进行脱氮能力测定,并分析其对常作为筛选标志的抗生素的药物敏感性.结果:发现在采用该工艺的活性污泥中,优势菌为假单胞菌、肠杆菌、莫拉菌和不动杆菌,分别占总菌数的23%、16%、16%和12%.根据菌群分析的结果,从中选择了两株耐药性弱、脱氮能力强的菌作为基因改良的出发菌.结论:本研究阐明了活性污泥的菌群构成,获得了两株适合基因工程改良的菌株,为日后脱氮基因工程菌的构建奠定基础.     

甲烷利用菌培养条件的优化及其初步应用

利用统计学实验设计（RSM）对能够利用甲烷的假单胞菌菌株ME16主要培养条件进行了优化。以液体无机盐和甲烷气体作为培养基分别进行了温度、接种量、甲烷含量和培养pH对细菌生长影响的研究,并在此基础上,利用响应面法分析优化了ME16菌株的主要培养条件,得到最佳培养条件为：温度29.4℃,接种量1.8%,甲烷含量25%。采用优化培养条件进行培养,细菌生物量增大0.8倍,达到稳定期的培养时间缩短了50h。该菌株初步应用于甲烷气体的脱除,脱除率达65.7 %,表明该菌株能良好的脱除空气中甲烷。     

Pseudomonas sp. RT-1低温脂肪酶的纯化及酶学特性

Pseudomonas sp. RT-1是从低温环境下分离的低温脂肪酶产生菌,对该菌产生的胞外脂肪酶(PL-1)进行纯化,并对其酶学特性进行初步研究。Pseudomonas sp. RT-1的发酵上清液经60%(NH4)2SO4沉淀、12~14000截留相对分子质量(MWCO)透析袋透析、Sephadex G75分子筛和超滤浓缩后,得到了电泳纯的P-L1。SDS-PAGE电泳估算其表观相对分子质量为4.43×104。对其酶学特性研究表明:PL-1是低温碱性脂肪酶且对有机溶剂的耐受性较好。10~40℃内有较好的催化活性,最适作用温度为18℃;0~50℃该酶的稳定性较好,当温度超过50℃时则容易失活;最适作用pH为10.2,且pH在9~11时较稳定;该酶对有机溶剂的耐受性较好,10mmol/L的Ca2+、K+、Na+和Fe3+对PL1的酶活力有促进作用,其中Ca2+促进作用最大,提高了146.07%,而10mmol/L的Cu2+、Co2+、Mn2+、Mg2+、Zn2+、Ba2+和Al3+对酶活力具有不同程度的抑制作用,其中Al3+抑制作用最强,抑制了98.55%;PL-1对C链长度小于或等于12的短链脂肪酸形成的甘油三酯具有较强的水解能力;1mmol/L的去氧胆酸盐(desoxycholate)和0.01%的Triton X100对酶活力具有提高作用,分别提高了30.74%和11.83%;0.01%的SDS和Tween-80、1mmol/L的EDTA和尿素对酶活都有抑制作用,其中EDTA的抑制作用最大,抑制了80%。     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid.

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Optimization of the hide powder azure assay for quantitating the protease of Pseudomonas fluorescens

The extracellular protease of Pseudomonas fluorescens NC 3 was optimally active at 40°C in a reaction mixture containing: 50 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer (pH 6.6), 0.5 mM CaCl₂, and 25 mg hide powder azure in 5 ml total volume. Divalent cation chelators, i.e., EDTA, o-phenanthroline, citrate or phosphate, inhibited the enzyme. Protease production by P. fluorescens NC 3 was initiated during late-logarithmic-growth phase in a sodium caseinate medium and reached its maximum at the onset of the stationary phase.     

A bacterial cell to cell signal in the lungs of cystic fibrosis patients

Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of mortality in cystic fibrosis (CF) patients. This bacterium has numerous genes controlled by cell to cell signaling, which occurs through a complex circuitry of interconnected regulatory systems. One of the signals is the Pseudomonas Quinolone Signal (PQS), which was identified as 2-heptyl-3-hydroxy-4-quinolone. This intercellular signal controls the expression of multiple virulence factors and is required for virulence in an insect model of P. aeruginosa infection. Previous studies have implied that the intercellular signals of P. aeruginosa are important for human disease, and our goal was to determine whether PQS was produced during human infections. In this report, three types of samples from CF patients infected with P. aeruginosa were analyzed for the presence of PQS. Sputum, bronchoalveolar lavage fluid, and mucopurulent fluid from distal airways of end-stage lungs removed at transplant, all contained PQS, indicating that this cell to cell signal is produced in vivo by P. aeruginosa infecting the lungs of CF patients.     

多器官功能衰竭患者检出栖稻假单胞菌1例

栖稻假单胞菌(Pseudomonas oryzihabitans,P. oryzihabitans)作为条件致病菌和院内感染菌,多见于免疫力低下合并基础疾病患者,其抗感染预后良好,甚少出现多器官功能衰竭病例。本例患者无明显诱因出现双下肢水肿半月余,后全身乏力、气短、尿少3 d,继而出现多器官功能衰竭。患者的血培养中检出1株革兰氏阴性杆菌,经生化鉴定、全自动快速微生物质谱检测(MALDI-TOF MS)和16S rRNA测序,鉴定为P. oryzihabitans;运用Mega 7.0软件构建系统发育树,发现其与耐冷假单胞菌(Pseudomonas psychrotolerans,P. psychrotolerans)亲缘关系十分密切;采用纸片扩散法(Kirby-Bauer method, K-B method)进行药物敏感试验(antimicrobial susceptibility test,AST),显示该菌对头孢他啶、庆大霉素、亚胺培南、左氧氟沙星等敏感,对氨曲南、氨苄西林、头孢唑啉等耐药。采用亚胺培南西司他丁联合莫西沙星对患者进行抗感染治疗,但因患者家属要求出院,无法追踪后续治疗结果。本例报告提示,临床微生物工作者应关注栖稻假单胞菌病例报道和相关研究,以获取更多资料为该病的临床诊治提供依据。     

Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions

AIMS: This study investigated the effect of growth conditions on proteolytic activity of a Pseudomonas strain, named Pseudomonas sp. LBSA1, isolated from bulk raw milk. It was compared with three Pseudomonas chlororaphis and one Pseudomonas fluorescens strain from culture collections. METHODS AND RESULTS: Bacteriae were grown in a minimal salt medium. For all the strains, addition of 1% (v/v) skim milk to the growth medium was sufficient to induce protease production in 48-h culture. Addition of 1 mmol l(-1) calcium chloride permitted the detection of proteolytic activity of four strains in 48-h cultures but not for Pseudomonas sp. LBSA1. The five strains presented two patterns of proteolytic activity when grown in the minimal salt medium supplemented with 2% (v/v) skim milk at various temperatures for 48 h. Two electrophoretic protease patterns were also obtained from the zymogram of extracellular medium for the five strains. CONCLUSIONS: The growth conditions permitting protease production are variable and do not depend on the genus of the producing strain. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time a study on proteolytic activity of P. chlororaphis strains is reported. Among the tested criteria, zymograms of extracellular medium were the only ones that permitted distinguishing the P. chlororaphis strains from the P. fluorescens strain.     

Saccharides of seven Pseudomonas aeruginosa immunotypes

Abstract The structures of O-specific polysaccharides obtained by mild acid degredation of lipopolysaccharides (LPS) from seven Pseudomonas aeruginosa Fisher's immunotypes have been studied. The polysaccharides consist mainly of monoamino and diamino sugars, frequently also carrying acidic functions. Some of the sugars were detected in nature for the O-specific polysaccharides of the immunotypes 2, 3, 4, 5 and 6 are identical to those of the polysaccharides of the 011; 0(2a)2c; 01; 010a, 10b and 07a, 7d Lányi-Bergan serological subgroups respectively, whereas no analogues have been found for the immunotypes 1 and 7. Some cross-reactions between the LPS of different immunotypes were observed in passive haemagglutination tests; the results of inhibition of passive haemagglutination and agar gel immunoprecipitation point, however, to a specificity of the LPS. Many of the LPS of the seven Pseudomonas aeruginosa immunotypes manifest rather a high cross-protective activity in active immunization tests in mice. The nature of the cross-protective activity of the LPS is discussed.     

趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷(MP)高效降解菌株,同时对MP具有趋化性。cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异。通过土壤盆钵试验(MP浓度为50mg/kg),发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%~30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用。     

Peptidase N of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa possesses a peptidase N activity analogous to those described in Escherichia coli and Salmonella typhimurium . This activity resides in a protein with an M _r value of 85 000. Part of this peptidase activity appears to be associated with the cytoplasmic membrane. The K _M value for this peptidase bound to the cytoplasmic membrane is in the range of 0.5 mM.     

绿脓杆菌脂多糖疫苗免疫效应的研究

应用绿脓杆菌脂多糖(Lipopolysaccharide简称LPS)疫苗免疫家兔,可剌激家兔产生体液抗体;增强嗜中性白细胞的吞噬功能。免疫剂量对免疫抗体水平有明显的影响,免疫时间对抗体水平也起着重要作用,免疫时间不同,免疫剂量相同,抗体滴度相差悬殊。