Cytochrome oxidase from Pseudomonas aeruginosa. IV. Reaction with oxygen and carbon monoxide

The reaction between a cytochrome oxidase from Pseudomonas aeruginosa and oxygen has been studied by a rapid mixing technique. The data indicate that the heme d₁ moiety of the ascorbate-reduced enzyme is oxidized faster than the heme c component. The oxidation of heme d₁ is accurately second order with respect to oxygen and has a rate constant of 5.7 · 10⁴ M⁻¹ · s⁻¹ at 20 °C. The oxidation of the heme c has a first-order rate constant of about 8 s⁻¹ at infinite concentration of O₂. The results indicate that the rate-limiting step is the internal transfer of electrons from heme c to heme d₁. These more rapid reactions are followed by more complicated but smaller absorbance changes whose origin is still not clear.

The reaction of ascorbate-reduced oxidase with CO has also been studied and is second order with a rate constant of 1.8 · 10⁴ M⁻¹ · s⁻¹. The initial reaction with CO is followed by a slower reaction of significantly less magnitude. The equilibrium constant for the reaction with CO, calculated as a dissociation constant from titrimetric experiments with dithionite-reduced oxidase, is about 2.3 · 10⁻⁶ M. From these data a rate constant of 0.041 s⁻¹ can be calculated for the dissociation of CO from the enzyme.     

Pseudomonas aeruginosa ExoS and ExoT

ExoS and ExoT are bi-functional type-III cytotoxins of Pseudomonas aeruginosa that share 76% primary amino acid homology and contain N-terminal RhoGAP domains and C-terminal ADP-ribosylation domains. The Rho GAP activities of ExoS and ExoT appear to be biochemically and biologically identical, targeting Rho, Rac, and Cdc42. Expression of the RhoGAP domain in mammalian cells results in the disruption of the actin cytoskeleton and interference of phagocytosis. Expression of the ADP-ribosyltransferase domain of ExoS elicits a cytotoxic phenotype in cultured cells, while expression of ExoT appears to interfere with host cell phagocytic activity. Recent studies showed that ExoS and ExoT ADP-ribosylate different substrates. While ExoS has poly-substrate specificity and can ADP-ribosylate numerous host proteins, ExoT ADP-ribosylates a more restricted subset of host proteins including the Crk proteins. Protein modeling predicts that electrostatic interactions contribute to the substrate specificity of the ADP-ribosyltransferase domains of ExoS and ExoT.     

Pseudomonas aeruginosa lipopolysaccharides and pathogenesis

Pseudomonas aeruginosa lipopolysaccharide (LPS) plays a key role in pathogenesis. In acute infections, a smooth LPS protects the organism from complement-mediated killing and, during chronic lung infections, an altered rough LPS helps the organism evade host defense mechanisms.     

Cytochrome oxidase from Pseudomonas aeruginosa. III. Reduction of hydroxylamine

Pseudomonas aeruginosa cytochrome oxidase, which reduces nitrite and oxygen, is also capable of reducing hydroxylamine to ammonia.The K_m for hydroxylamine reduction is 6 · 10^?4M compared to 5 · 10^?5M for nitrite reduction. NADH, NADPH, reduced P. aeruginosa cytochrome c₅₅₁, and reduced P. aeruginosa copper protein were ineffective as electron donors for hydroxylamine reduction whereas reduced pyocyanine and methylene blue acted as electron mediators.Hydroxylamine reduction did not require the presence of Mn²⁺ of FAD and was not inhibited by prolonged dialysis versus sodium diethyldithiocarbamate. Cyanide, nitrite, and CO were very effective inhibitors.Removal of heme d and its reconstitution, as well as inhibition by CO, suggest that the reduction of hydroxylamine, like the reduction of nitrite or oxygen, proceeds via the heme d.     

铜绿假单胞菌锌离子摄取系统的研究进展

锌作为一种结构、催化和信号的成分,在许多生理过程中起着关键的作用.它也是病原微生物生长所必需的,不但参与病原微生物代谢和各种毒力因子的调控,而且是病原微生物在宿主中感染和定殖所必需的.铜绿假单胞菌侵染宿主发挥毒力时,宿主会采取营养免疫的策略来限制体内环境中游离的锌离子浓度而抑制该病原菌的感染和定殖.反过来,铜绿假单胞菌...     

An anion-selective channel from the Pseudomonas aeruginosa outer membrane

Protein P from Pseudomonas aeruginosa outer membrane was reconstituted in lipid bilayer membranes from diphytanoylphosphatidylcholine. The reconstitution resulted in the formation of anion-selective channels with a conductance of 160 pS for 0.1 M chloride solution. The channels were at least 100-times more selective for anions than for cations as judged from zero-current membrane potentials. The single-channel conductance was dependent on the size of the different anions and saturated at higher salt concentrations suggesting single ion occupancy of the protein P channel.     

Degradation of pyrimidine ribonucleosides by Pseudomonas aeruginosa

Pyrimidine ribonucleoside degradation in the human pathogen Pseudomonas aeruginosa ATCC 15692 was investigated. Either uracil, cytosine, 5-methylcytosine, thymine, uridine or cytidine supported P. aeruginosa growth as a nitrogen source when glucose served as the carbon source. Using thin-layer chromatographic analysis, the enzymes nucleoside hydrolase and cytosine deaninase were shown to be active in ATCC 15692. Compared to (NH₄)₂SO₄-grown cells, nucleoside hydrolase activity in ATCC 15692 approximately doubled after growth on 5-methylcytosine as a nitrogen source while its cytosine deaminase activity increased several-fold after growth on the pyrimidine bases and ribonucleosides examined as nitrogen sources. Regulation at the level of protein synthesis by 5-methylcytosine was indicated for nucleoside hydrolase and cytosine deaminase in P. aeruginosa.     

Tn501 insertion mutagenesis in Pseudomonas aeruginosa PAO

Summary Transposon insertion mutagenesis of the Pseudomonas aeruginosa PAO chromosome with Tn1 and Tn501 was carried out using a mutant plasmid of R68::Tn501 temperature-sensitive for replication and maintenance. This method consists of three steps. Firstly, the temperature-independent, drug-resistant clones were selected from the strain carrying this plasmid. In the temperature-indepent clones, the plasmid was integrated into the chromosome by Tn1- or Tn501-mediated cointegrate formation. Secondly, such clones were cultivated at a permissive temperature to provoke the excision of the integrated plasmid from the chromosome. Excision occurred by the reciprocal recombination between the two copies of Tn1 or Tn501 flanking the integrated plasmid, leaving one Tn1 or Tn501 insertion on the chromosome. Thirdly, the excised plasmid was cured by cultivating these isolates at a non-permissive temperature without selection for the drug resistance. Using this method, we isolated 1 Tn1-induced and 43 Tn501-induced auxotropic mutations in this organism. Genetic mapping allowed us to identify two new genes, pur-8001 and met-8003. The Tn501-induced auxotrophic mutations were distributed non-randomly among auxotrophic genes, and the reversion of the mutations by precise excision of the Tn501 insertion occurred very rarely.     

铜绿假单胞菌二鸟苷酸环化酶SiaD突变体的功能研究

【目的】铜绿假单胞菌(Pseudomonas aerugionsa)二鸟苷酸环化酶SiaD调控着铜绿假单胞菌的生物被膜形成等表型。在研究过表达siaD对生物被膜的调控作用时发现,与野生型siaD基因回补菌株相比,一株回补菌株的生物被膜产量显著升高。本文的目的即是探究该菌株生物被膜产量升高的原因,并对该菌株的其他表型进行研究。【方法】通过测序确定突变位点;利用生物被膜定性和定量实验对发生点突变的菌株表型进行分析;利用Western blotting实验检测SiaD^R119M蛋白表达水平;利用GST-pulldown实验检测SiaC蛋白与SiaD^R119M蛋白在体外的结合能力;针对siaD^R119M点突变基因进行融合蛋白表达载体的构建,表达并纯化该蛋白,利用高效液相色谱检测SiaD^R119M的酶活;为了进一步研究c-di-GMP与细菌运动能力的关系,对细菌的运动能力进行检测。【结果】测序比对结果显示,序列的第119个氨基酸发生了突变,由精氨酸突变成了甲硫氨酸。生物被膜定性和定量实验显示,与野生型siaD...     

Association of the biocide 5-chloro-2-methyl-isothiazol-3-one with Pseudomonas aeruginosa and Pseudomonas fluorescens

The biocide 5-chloro-2-methyl-isothiazol-3-one (CMI) associated rapidly with cells of Pseudomonas aeruginosa and Pseudomonas fluorescens with association being nearly complete within 10–15 min. The cells serve as a sink for CMI, concentrating it up to 400-fold. Kinetics of association are very similar amongst the strains examined. Examination of the relation of CMI concentration to the rate of association indicates that there are two kinetically distinguishable processes, with the breakpoint occurring around the transition from inhibitory to suprainhibitory levels of CMI. This suggests the rapid onset of toxic effects at suprainhibitory CMI concentrations affects the associative process. Discharging the proton motive force by treatment with uncoupling agents or selectively depleting it by treatment with inhibitors reduces the amount of CMI which associates with the cells. Selective depletion of the ATP pool has no effect. These results suggest that either the proton motive force (pmf) is involved directly in CMI association in an active transport process, or that an intact pmf is required for some facet of the cells metabolism which maintains the cells as a sink for CMI. The nonchlorinated analogue 2-methyl-isothiazol-3-one is a poor competitor for CMI association.     

铜绿假单胞菌中鼠李糖脂生物合成的研究进展*

鼠李糖脂因其具有环境友好和卓越的物理化学特性,而有望成为化学合成表面活性剂的替代物。近年来鼠李糖脂得到了广泛的研究,其目的是利用低价的可再生资源进行大规模生产,但目前的研究成果仍不足以选育出更具商业竞争力的鼠李糖脂过量合成菌株。为此,进一步理解鼠李糖脂生物合成的复杂基因调控网络,探索降低生产成本的发酵工艺势在必行。综述了铜绿假单胞菌中鼠李糖脂的生物合成途径、群体感应对主要基因的调控、鼠李糖脂在生物膜形成中所发挥的作用,以及发酵优化对鼠李糖脂产量的影响。有助于加深对鼠李糖脂生物合成的认识,为提高鼠李糖脂产量提供重要参考信息。     

铜绿假单胞菌中鼠李糖脂生物合成的研究进展*

鼠李糖脂因其具有环境友好和卓越的物理化学特性,而有望成为化学合成表面活性剂的替代物。近年来鼠李糖脂得到了广泛的研究,其目的是利用低价的可再生资源进行大规模生产,但目前的研究成果仍不足以选育出更具商业竞争力的鼠李糖脂过量合成菌株。为此,进一步理解鼠李糖脂生物合成的复杂基因调控网络,探索降低生产成本的发酵工艺势在必行。综述了铜绿假单胞菌中鼠李糖脂的生物合成途径、群体感应对主要基因的调控、鼠李糖脂在生物膜形成中所发挥的作用,以及发酵优化对鼠李糖脂产量的影响。有助于加深对鼠李糖脂生物合成的认识,为提高鼠李糖脂产量提供重要参考信息。     

维拉帕米对铜绿假单胞菌群体感应抑制作用的体外研究

考察维拉帕米对铜绿假单胞菌（Pseudomonas aeruginosa, PA）群体感应（Quorum sensing, QS）的抑制作用。测定维拉帕米最小抑菌浓度(Minimal inhibit concentration,MIC) ;构建培养环境,考察维拉帕米对PA生长的影响,绘制生长曲线;毒力因子表达实验中,分别考察维拉帕米对PA弹性蛋白酶表达、蛋白水解酶表达和绿脓毒素表达的影响。结果表明,维拉帕米MIC₅₀为128 μg/mL,MIC₉₀为512 μg/mL,最低抑菌质量浓度范围为128~512 μg/mL时,具有较好抑菌活性;生长曲线表明,维拉帕米质量浓度为16 μg/mL时,开始抑制PA的生长,随着浓度的增加,抑制作用逐渐增大;当维拉帕米质量浓度为512、256、128、64、32、16 μg/mL时,明显抑制弹性蛋白酶表达（P<0.01）,质量浓度为8 μg/mL时,对弹性蛋白酶有一定抑制作用（P<0.05）,维拉帕米质量浓度为512、256、128、64、32 μg/mL时,明显抑制蛋白水解酶的活性和绿脓毒素的表达（P<0.01）,当质量浓度为16 μg/mL时,对蛋白水解酶活性和绿脓毒素的表达有一定抑制作用（P<0.05）;维拉帕米对QS的抑制有浓度依赖。维拉帕米对PA的QS有明显抑制作用,体外可明显抑制PA生长,可作为抗菌增效的潜在开发药物。     

Expression and activation of an esterase from Pseudomonas aeruginosa 1001 in Escherichia coli

An expression vector was constructed to overproduce a maltose binding protein (MBP)-esterase fusion protein in Escherichia coli. Soluble fusion protein was separated by centrifugation after cell disruption. The fusion protein was partially purified with amylose resin. The higher concentration of fusion protein (above 2 mg/ml) did not show any activity but about 0.3 mg/ml of fusion protein had the highest activity (142 U/ml). It is due to the difficulty of contact between substrate and active site of enzyme in compact form at high concentration. The fusion protein over-expressed could not be separated into MBP and esterase by the action of protease ‘Factor Xa’. The esterase could be cleaved from MBP fusion protein by the treatment of SDS with the Factor Xa, and the resulting esterase activity was increased to 34% after cleavage.     

The hierarchy quorum sensing network in Pseudomonas aeruginosa

Pseudomonas aeruginosa causes severe and persistent infections in immune compromised individuals and cystic fibrosis sufferers. The infection is hard to eradicate as P. aeruginosa has developed strong resistance to most conventional antibiotics. The problem is further compounded by the ability of the pathogen to form biofilm matrix, which provides bacterial cells a protected environment withstanding various stresses including antibiotics. Quorum sensing (QS), a cell density-based intercellular communication system, which plays a key role in regulation of the bacterial virulence and biofilm formation, could be a promising target for developing new strategies against P. aeruginosa infection. The QS network of P. aeruginosa is organized in a multi-layered hierarchy consisting of at least four interconnected signaling mechanisms. Evidence is accumulating that the QS regulatory network not only responds to bacterial population changes but also could react to environmental stress cues. This plasticity should be taken into consideration during exploration and development of anti-QS therapeutics.     

绿脓杆菌对鲎素耐受性特点及耐受性机制的研究

【目的】为了探讨鲎素作为抗菌药物在临床使用中的安全性问题,通过鲎素连续增高浓度法对绿脓杆菌进行耐受性诱导,并对其耐受性机制进行初步研究,以期为鲎素的广泛应用提供理论依据。【方法】绿脓杆菌ATCC27853为试验菌株,通过连续增高浓度诱导法筛选抗药菌株,并通过抗药稳定性、交叉抗药性、抗药性代偿测定来探究其耐受性特点,通过对其胞外蛋白酶活性、生物膜形成、胞外多糖含量的变化来探讨其抗药性机制。【结果】通过连续增高鲎素浓度法对原始菌株进行30多代诱导后,绿脓杆菌ATCC27853对鲎素的MIC值逐渐增高,80多代时产生了明显抗药性。抗药菌株对丁胺卡那以及pexiganan、鲎素同源肽tachyplesin III、polyphemusin I均能产生不同程度的抗药性。在无药培养基中抗药菌株以更长的延滞期作为抗药性代偿,但在有药培养基中具有更短的延滞期和更大的生长速率。抗药菌株较原始菌株分泌的胞外蛋白酶活性增高,并能降低鲎素的抗菌活性。在同样条件下抗药菌株较原始菌株胞外多糖含量增高,更易形成生物膜。【结论】在长期选择压力下绿脓杆菌ATCC27853对鲎素能产生抗药性,其抗药性机制可能与生物膜形成、胞外蛋白酶失活鲎素有关。关于细菌对鲎素的抗药性机制,有待进一步研究。     

Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia

Background  

Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia.