Directional Modifications of Resibufogenin by Mucor subtilissimus and Pseudomonas aeruginosa

Directional modifications of resibufogenin 1 by Mucor subtilissimus and Pseudomonas aeruginosa were carried out. The substrate was hydroxylated at C-12 by M. subtilissimus AS 3.2454, from which a major product 12-hydroxyresibufogenin 2 was obtained. Then product 2 was dehydrogenated by P. aeruginosa AS 1.860, which resulted in a new compound 12β-hydroxy-3-keto-resibufogenin 3.     

The Use of New Methylene Blue in Pseudomonas aeruginosa Biofilm Destruction

A Pseudomonas aeruginosa biofilm was produced in a model system using the bacterial strain NCIMB 8295, grown on silicone tubing (bore size 0.75 cm). Destruction of the biofilm was attempted using either ampicillin or a combination of white light (light dose=7.2 J cm^{m 2}) and the phenothiazinium photosensitiser new methylene blue, and damage, both to extra-cellular polymeric substance (EPS) and to the organism, was monitored. It was found that although little damage to the EPS occurred with ampicillin, NMB caused both cell death and breakdown of the EPS, suggesting the use of photodynamic antimicrobial chemotherapy (PACT) in the disinfection of pathogenic biofilms, e.g. at external catheter surfaces.     

Characterization of Competitive Inhibitors for the Transferase Activity of Pseudomonas aeruginosa Exotoxin A

A series of small, nonpolar compounds were tested for their ability to inhibit the ADP-ribosyl transferase activity of Pseudomonas aeruginosa exotoxin A. The IC 50 values for the compounds tested ranged from 87 nM to 484 μM for NAP and CMP12, respectively. It was demonstrated that NAP was a competitive inhibitor of the ADPRT reaction for the NAD + substrate with a K i of 45 ± 5 nM, which was in good agreement with the dissociation constant determined independently (K D =56 ± 6 nM). The IC 50 value for NAP was 87 ± 12 nM, which strongly correlated with the K i and K D values. Furthermore, NAP was shown to noncovalently associate with the exotoxin A active site using exhaustive dialysis, NMR, and electrospray mass spectrometry. Finally, a computer molecular model using the X-ray structure of the substrate-bound toxin was generated with NAP bound to the active site of exotoxin A at the nicotinamide-binding site. This model is consistent with the X-ray structure of the catalytic domain of poly-ADP-ribose polymerase complexed with 4-amino-naphthalimide (Compound 4) that was included in this study.     

Mutual Influences of Pseudomonas aeruginosa and Desulfovibrio desulfuricans on their Adhesion to Stainless Steel

The mutual influences of Pseudomonas aeruginosa PAO1 and Desulfovibrio desulfuricans subsp. desulfuricans (ATCC 29577) on their adhesion to stainless steel were investigated in batch and column experiments. It was found that P. aeruginosa promoted the adhesion of D. desulfuricans under conditions of turbulence, but not under quiescent conditions. The enhancement involved the alignment of most D. desulfuricans along P. aeruginosa cells and was attributed to the additional interaction surface area provided by adhered P. aeruginosa to aligning D. desulfuricans cells. A slightly positive effect of pre-adhered D. desulfuricans on the adhesion of P. aeruginosa was found. Under condition of laminar flow, substantially better adhesion of D. desulfuricans to confluent P. aeruginosa biofilms than to steel was observed. The mutual influences are discussed in terms of more favorable adhesion energies and the influence of changed hydraulic conditions due to the roughness of P. aeruginosa biofilms.     

Microbial transformation of the sesquiterpenoid (-)-maalioxide by Mucor plumbeus

Microbial transformation of the sesquiterpenoid (-)-maalioxide by the fungus Mucor plumbeus gave three metabolites, 9beta-hydroxymaalioxide, 1beta-hydroxymaalioxide and 7beta-hydroxymaalioxide. 9beta-hydroxymaalioxide and its structure was established on the basis of its spectroscopic properties and chemical reactions.     

Pentachlorophenol degradation by Pseudomonas aeruginosa

Five Pseudomonas species were tested for ability to degrade pentachlorophenol (PCP). Pseudomonas aeruginosa completely degraded PCP up to 800 mg/l in 6 days with glucose as co-substrate. With 1000 mg PCP/l, 53% was degraded. NH₄ ⁺ salts were better at enhancing degradation than organic nitrogen sources and shake-cultures promoted PCP degradation compared with surface cultures. Degradation was maximal at pH 7.6 to 8.0 and at 30 to 37°C. Only PCP induced enzymes that degraded PCP and chloramphenicol inhibited this process. The PCP was degraded to CO₂, with release of Cl^-.The authors are with the Bacteriology Laboratory, Central Leather Research Institute, Madras-600 020, India.     

铜绿假单胞菌产蛋白酶的发酵条件优化

【目的】鉴定一株来源于酱油曲能够分泌蛋白酶的铜绿假单胞菌CAU342A,优化其产蛋白酶的发酵条件。【方法】采用形态学观察、16S r RNA基因序列比对和生理生化方法鉴定菌株CAU342A;通过碳源、氮源、初始pH、温度、表面活性剂及发酵时间的单因素优化和正交试验获得最适发酵条件。【结果】菌株CAU342A被鉴定为铜绿假单胞菌(Pseudomonas aeruginosa),其最适发酵产酶条件为(质量体积比):3%酒糟,1.5%酵母浸提物,0.05%吐温-80,0.5%NaCl,0.7%K_2HPO_4,0.3%KH_2PO_4,0.04%MnSO_4,培养基初始pH 7.5,30°C培养72 h。在最适发酵条件下,该菌株最大产酶水平达到2 653.5 U/m L。蛋白酶酶谱分析表明该菌株能够产生至少4种具有蛋白酶活性的同工酶,其中两个主要酶谱带对应分子量分别为32 k D和50 k D。【结论】铜绿假单胞菌CAU342A高产蛋白酶,具有很大的工业应用潜力。     

The Combined Structural and Kinetic Characterization of a Bacterial Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes NMO Class I and II

Nitronate monooxygenase (NMO) oxidizes the mitochondrial toxin propionate 3-nitronate (P3N) to malonate semialdehyde. The enzyme has been previously characterized biochemically in fungi, but no structural information is available. Based on amino acid similarity 4,985 genes are annotated in the GenBank^TM as NMO. Of these, 4,424 (i.e. 89%) are bacterial genes, including several Pseudomonads that have been shown to use P3N as growth substrate. Here, we have cloned and expressed the gene pa4202 of Pseudomonas aeruginosa PAO1, purified the resulting protein, and characterized it. The enzyme is active on P3N and other alkyl nitronates, but cannot oxidize nitroalkanes. P3N is the best substrate at pH 7.5 and atmospheric oxygen with k_cat^app/K_m^app of 12 × 10⁶ m⁻¹ s⁻¹, k_cat^app of 1300 s⁻¹, and K_m^app of 110 μm. Anerobic reduction of the enzyme with P3N yields a flavosemiquinone, which is formed within 7.5 ms, consistent with this species being a catalytic intermediate. Absorption spectroscopy, mass spectrometry, and x-ray crystallography demonstrate a tightly, non-covalently bound FMN in the active site of the enzyme. Thus, PA4202 is the first NMO identified and characterized in bacteria. The x-ray crystal structure of the enzyme was solved at 1.44 Å, showing a TIM barrel-fold. Four motifs in common with the biochemically characterized NMO from Cyberlindnera saturnus are identified in the structure of bacterial NMO, defining Class I NMO, which includes bacterial, fungal, and two animal NMOs. Notably, the only other NMO from Neurospora crassa for which biochemical evidence is available lacks the four motifs, defining Class II NMO.     

Asymmetric bioreduction of p-haloacetophenones by Mucor

Abstract

A number of p-haloacetophenones were asymmetrically bioreduced to their corresponding (S)-alcohols by Mucor sp. CG10 with good conversion and excellent enantioselectivity. The results showed that the electronic effects of the halogen substituent (X-group) affected the conversion of the substrates and the enantioselectivity of the reaction. The trend observed was as the X-group at the para-position became more electron donating from F, to Cl, Br and I, the conversion of substrates decreased, while the enantioselectivity increased.     

光声光谱技术用于绿脓杆菌的鉴定

【目的】探索一种无创、快速、可靠、经济地鉴定绿脓杆菌的新方法。【方法】利用光声光谱技术分别对大肠杆菌、绿脓杆菌在35°C下培养24 h后产生的挥发性代谢产物(Bacterial volatile compounds,BVCs)进行连续检测,获得各细菌挥发性气体的光声光谱图谱,并用“逆向思维”的方法对其检测结果进行分析。【结果】利用光声光谱技术对绿脓杆菌、大肠杆菌的挥发性代谢产物进行检测分析,发现绿脓杆菌产生了较高浓度的氰化氢(HCN),而大肠杆菌并未检测出HCN,据此可以对绿脓杆菌进行初步鉴定。【结论】光声光谱技术为绿脓杆菌的鉴定提供了一种简单、快速、经济的方法。为加速其在临床中的应用,提出了“三步走”方案:建立“大数据”、完善“比对算法”、创建“自动检测-比对-校对-鉴定”系统。     

提高毛霉菌转化合成腺三磷产率的研究

采用毛霉菌由腺嘌呤转化合成腺三磷（ATP）,在综合研究其转化条件的基础上,进一步研究添加剂对提高ATP产率的影响。经过筛选获得较佳的添加剂新洁尔灭,在含有3g/L腺嘌呤的反应液中,加入50mg/L新洁尔灭,经33℃转化反应5h,能产生6g/L以上的腺三磷,比对照提高75％以上。     

Uptake and metabolism of methylammonium by Pseudomonas aeruginosa

The mechanism of ammonium uptake was studied in Pseudomonas aeruginosa, measuring the uptake (transport and metabolism) of [14C]methylammonium (MA). This ammonium analogue was not utilized for growth, but unmetabolized MA was accumulated to intracellular concentrations about 30 times higher than those in the medium. Most of the MA taken up, however, was rapidly metabolized to gamma-N-methylglutamine, which could be removed from the cells by the addition of ammonium. Uptake of MA exhibited distinct optima at pH 7.0 and 35 to 40 degrees C and depended on metabolic energy, as indicated by the inhibitory effect of various metabolic poisons. Growth with ammonium as nitrogen source resulted in the repression of MA uptake, whereas high uptake rates were observed with nitrate or after incubation without nitrogen source. These results suggested that the ammonium/MA uptake system is subject to nitrogen control in P. aeruginosa.     

钌化合物对铜绿假单胞菌的光动力杀伤作用

【摘 要】 目的 探讨光动力疗法（PDT）对体外培养的铜绿假单胞杆菌的杀伤效应。方法 以多重耐药铜绿假单胞杆菌（P. aeruginosa）为对象,用457 nm和532 nm的激光作为光源,对不同浓度钌化合物孵育的细菌悬液进行光照,用菌落计数法观测PDT对铜绿假单胞杆菌的杀伤作用;同时观察了铜绿假单胞菌临床耐药菌与标准菌株对相同PDT作用的敏感性差异。结果 PDT处理组具有剂量－效应关系,在光照剂量相同的情况下,457 nm激光作为光源有更好的PDT杀伤效果;对多重耐药的铜绿假单胞菌,光动力同样有效,其作用甚至强于标准株。结论 钌化合物介导的光动力作用对体外培养的铜绿假单胞杆菌具有很强的杀伤作用,其效果和剂量关系密切。     

毛霉的产蛋白酶发酵条件优化

从发酵豆制品中分离出1株产蛋白酶性质优良的毛霉M2, 并研究M2菌株的产蛋白酶条件。研究优化得出该毛霉菌株的产酶培养基条件为：氮源为大豆分离蛋白, 碳源为葡萄糖, 无机盐为磷酸二氢钾、氯化钙和氯化镁; 它的适宜的产酶发酵条件为：培养温度为28°C, 接种量为2%, 300 mL三角瓶的装液量为100 mL, 初始pH为5, 摇床转速为150 r/min, 培养时间为48 h; 发酵液蛋白酶酶活力可达4.35 U/mL。凝胶电泳法测定出毛霉所分泌蛋白酶的分子量为36.4 kD。     

铜绿假单胞菌分泌性蛋白的抗肿瘤效应

目的 探讨3株不同来源的铜绿假单胞菌分泌性蛋白的抗肿瘤活性。方法 将3株不同来源的铜绿假单胞菌经LB培养基对其过夜静置培养,离心获得上清液,采用硫酸铵盐析沉淀蛋白质,再经PBS透析除盐。然后将蛋白作用于人肝癌细胞Hep-G2、宫颈癌细胞HeLa、肺癌细胞A549和人永生化表皮细胞HaCaT,CCK-8检测其对细胞的毒性作用,吉姆萨染色观察凋亡细胞形态变化。结果 SDS-PAGE证实成功获得不同分子量的分泌蛋白,CCK-8结果显示混合蛋白对3种不同肿瘤细胞的生长均有不同程度的抑制作用,且其抑制作用存在一定的时间和浓度依赖性,但对人正常细胞无明显抑制作用。吉姆萨染色初步显示细胞形态为凋亡状态。结论 初步证实该3株铜绿假单胞菌产生的分泌性蛋白具有不同程度的抗肿瘤作用,为进一步分离纯化具有抗肿瘤活性的单一蛋白质及研究其抗肿瘤机制提供依据。     

哈尔滨市112株绿脓杆菌的血清学分型和耐药性测定

对从临床分离的112株绿脓杆菌进行系统鉴定后,血清学分型表明:6、2和3型分别占32.14％、15.18％、15.18％,为主要流行型,共占总分离株的62.50％。耐药性测定结果为:对10种抗生素5耐以上者占69.6％。其中对多粘菌素、妥布霉素、丁胺卡那霉素三种抗生素最为敏感,敏感率分别为100％、70.6％、86.5％。     

铜绿假单胞菌产酰化高丝氨酸内酯信号分子抑制剂的生产及分离

目的：通过自体诱导信号分子抑制剂的生产获得部分分离纯化的酰化高丝氨酸内酯（AHL）抑制剂。方法：病原菌铜绿假单胞菌经摇床培养后获得AHL抑制剂,采用溶解度差异性和树脂进行分离纯化。结果：铜绿假单胞菌PAO1不仅分泌自体诱导信号分子,而且在生长的后期还合成一种信号分子抑制剂,该信号分子抑制剂对群体感应中的AHL类信号分子有明显的抑制作用;该抑制剂具有醇溶性和水溶性,采用乙醇溶解可以除去糖类和无机小分子等不溶于醇的物质;大孔吸附树脂不具有吸附抑制剂的能力,但可以除去醇溶性糖类物质;阴离子交换树脂能够吸附信号分子抑制剂,具有较好的分离效率。结论：获得了除去大部分杂质,得到部分分离纯化的AHL抑制剂。     

Specific and non-specific mouse protection induced by different chemotypes of the Pseudomonas aeruginosa lipopolysaccharides

Abstract Lipopolysaccharides (LPS) of Pseudomonas aeruginosa were studied by the mouse active, cross-protection test. The primary structure of O-specific polysaccharides (O-repeating units) of different chemotypes was determined and their cross-protective activity demonstrated. Low doses of LPS (0.1–1 μg) stimulated chemotype-specific protection against P. aeruginosa in mice. This immunity was associated with the primary structure of the LPS and it lasted for 14 days after the first or second immunization. High doses of LPS (10–100 μg) induced cross-protection against P. aeruginosa in mice. The cross-protective capacity was caused evidently by the secondary structure or conformation of LPS molecule, i.e. by the common conformational protective determinant. This cross-protection lasted for only 5 days after the first or second immunization.