A nontoxicPseudomonas exotoxin a induces active immunity and passive protective antibody againstPseudomonas exotoxin a intoxication

Pseudomonas exotoxin A (PE) is one of the most potent cytotoxic agents produced byPseudomonas aeruginosa. In this study, we examined the possibility of using PE with a deletion of 38 carboxyl-terminal amino acid residues, designated PE(576–613), for active immunization against PE-mediated disease. We first examined the toxic effects of PE and PE(576–613) on 5- and 9-week-old ICR mice. The results show that the subcutaneous administration of PE(576–613) at a dose of 250 µg was still nontoxic to 5- and 9-week-old ICR mice, while native PE was lethal at a dose of 0.5 and 1 µg, respectively. PE(576–613) was then used to immunize ICR mice. The minimum dose of PE(576–613) that could effectively induce anti-PE antibodies in 5- and 9-week-old ICR mice was found to be 250 ng. However, immunization with 250 ng PE(576–613) failed to protect the immunized mice from a lethal dose of PE. The effective immunization dose of PE(576–613) that could protect mice against a 2 µg PE challenge was found to be 15 µg. In addition, sera obtained from PE(576–613)-immunized ICR mice were able to neutralize PE intoxication and effectively protect mice from PE. Thus, PE(576–613) may be used as an alternative route to new PE vaccine development.     

Heterologous expression and regulation of the lysA genes of Pseudomonas aeruginosa and Escherichia coli

Summary Chlorsulfuron-resistant mutants of Arabidopsis thaliana were isolated by screening for growth of seedlings in the presence of the herbicide. Both whole plants and derived tissue cultures were resistant to concentrations of the herbicide approximately 300-fold higher than that required to prevent growth of the wild-type. The resistance is due to a single dominant nuclear mutation at a locus designated csr which has been genetically mapped to chromosome-3. Acetohydroxy acid synthase activity in extracts from chlorsulfuron-resistant plants was much less-susceptible to inhibition by chlorsulfuron and a structurally related inhibitor than the activity in wild-type extracts. This suggests that the csr locus is the structural gene for acetohydroxy acid synthase.     

Purification and structural characterization of glycolipid biosurfactants fromPseudomonas aeruginosa YPJ-80

Glycolipids produced byPseudomonas aeruginosa YPJ-80 were characterized by chromatographic and spectroscopic techniques as a mixture of two rhamnolipids. For recovery of glycolipids from the culture broth, various isolation methods including ultrafiltration, adsorption and solvent extraction were compared. Ultrafiltration showed the best results in terms of glycolipids recovery. Further purification for spectroscopic analysis was carried out by adsorption chromatography and preparation thin layer chromatography. From the spectroscopic analysis, such as IR spectroscopy, FAB-MS,¹H-NMR and¹³C-NMR and hydrolysis analysis, the glycolipids were identified as L-α-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate and 2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate. Monorhamnolipid and dirhamnolipid lowered the surface tension of water to 28.1 mN/m and 29.3 mN/m, respectively.     

Isolation and identification of antialgal substances produced byPseudomonas aeruginosa

Pseudomonas aeruginosa strongly inhibited the growth of green microalgae and cyanobacteria by the release of low molecular weight, thermoresistant factors. The antialgal substances were resistant to various enzymes and remained active in agar after a 3-months storage period at 4 °C in the absence of light. The results indicate that inhibition of algal growth was mediated by the phenazine pigments released by the bacterium. Pyocyanine and an unidentified pale blue pigment had no effect on algal growth, whereas 1-hydroxyphenazine and oxychlororaphine showed strong antialgal activity.Groupe de Recherche en Recyclage Biologique et AquicultureDépartement des Sciences et Technologie des Aliments     

Pseudomonas aeruginosa, Candida albicans, and device-related nosocomial infections: implications, trends, and potential approaches for control

For many years, device-associated infections and particularly device-associated nosocomial infections have been of considerable concern. Recently, this concern was heightened as a result of increased antibiotic resistance among the common causal agents of nosocomial infections, the appearance of new strains which are intrinsically resistant to the antibiotics of choice, and the emerging understanding of the role biofilms may play in device-associated infections and the development of increased antibiotic resistance. Pseudomonas aeruginosa and Candida albicans are consistently identified as some of the more important agents of nosocomial infections. In light of the recent information regarding device-associated nosocomial infections, understanding the nature of P. aeruginosa and C. albicans infections is increasingly important. These two microorganisms demonstrate: (1) an ability to form biofilms on the majority of devices employed currently, (2) increased resistance/tolerance to antibiotics when associated with biofilms, (3) documented infections noted for virtually all indwelling devices, (4) opportunistic pathogenicity, and (5) persistence in the hospital environment. To these five demonstrated characteristics, two additional areas of interest are emerging: (a) the as yet unclear relationship of these two microorganisms to those species of highly resistant Pseudomonas spp and Candida spp that are of increasing concern with device-related infections, and (b) the recent research showing the dynamic interaction of P. aeruginosa and C. albicans in patients with cystic fibrosis. An understanding of these two opportunistic pathogens in the context of their ecosystems/biofilms also has significant potential for the development of novel and effective approaches for the control and treatment of device-associated infections.     

Plasmid-determined resistance to arsenic and antimony inPseudomonas aeruginosa

Resistance to arsenic salts in aPseudomonas aeruginosa clinical isolate was shown to be determined by a 100 kb transferable plasmid. The resistance pattern included arsenate, arsenite, and antimonate ions. Arsenate and arsenite resistances were inducible by previous exposure of cultures to subinhibitory amounts of either of the two ions. Phosphate ions protectedP. aeruginosa cells from the toxic effects of arsenate but did not alter arsenite toxicity.     

The Mechanism of Formation of Pseudomonas aeruginosa Biofilm,a Type of Structured Population

The paper is an attempt to analyze and generalize molecular and cell biology data on the formation of polysaccharide matrix–based biofilms. The conception of biofilms as structured populations sharing the characteristics of uni- and multicellular organisms and population is proposed.     

The effect of inorganic phosphate on cyanogenesis by Pseudomonas aeruginosa

The biosynthesis of hydrogen cyanide (HCN) by a strain of Pseudomonas aeruginosa is found to be significantly influenced by inorganic phosphate. Optimum HCN production occurs when the phosphate concentration is between 1 and 10 mM. Above and below this concentration the amount of HCN produced decreases sharply and at 0.1 and 100 mM phosphate low HCN production occurs. If a culture growing at 0.1 mM phosphate and producing low HCN is shifted to 10 mM phosphate, HCN biosynthesis resumes. Experiments with chloramphenicol indicate that de novo-protein synthesis is required for the process.     

Iron uptake regulation in Pseudomonas aeruginosa

The Pseudomonas genus belongs to the γ division of Proteobacteria and many species produce the characteristic yellow–green siderophore pyoverdine, and often a second siderophore, of lower affinity for iron. These bacteria are known for their ability to colonize different ecological niches and for their versatile metabolism. It is therefore not surprising that they are endowed with the capacity to take up exogenous xenosiderophores via different TonB-dependent receptors. Uptake of iron is controlled by the central regulator Fur, and via extracytoplasmic sigma factors or other types of regulators (two-component systems, AraC regulators). In this review the Fur regulon (experimentally proven and/or predicted) of P. aeruginosa will be presented. An interesting feature revealed by this analysis of Fur-regulated genes is the overlap between the iron and the sulfur regulons as well with the quorum sensing system.     

Ureolytic nitrification at low pH by Nitrosospira spec.

An ureolytic ammonium-oxidizing chemolithotroph belonging to the genus Nitrosospira was shown to nitrify at pH 4.5 in a pH-stat with urea as a substrate. With ammonium as the sole substrate nitrification did not occur at pH values below 5.5. Nitrosomonas europaea ATCC 19718 and Nitrosospira briensis ATCC 25971 did not possess urease activity. The results indicate that in acid soils nitrification by ureolytic ammonium-oxidizing chemolithotrophs may not be restricted to microsites of neutral pH.     

Protection of catheter surfaces from adhesion of Pseudomonas aeruginosa by a combination of silver ions and lectins

A catheter surface was modified by coating a cellulose acetate polymer. Adhesion of Pseudomonas aeruginosa ATCC 27853 to the surface was investigated by exposing bacterial cultures to three treatments: polymer impregnated with silver ions (Ag⁺), polymer surfaces coated with lectins and a combination of Ag⁺ and a lectin coating. The effective concentration of Ag⁺ providing protection against bacterial biofilm development was 100g/ml and higher. Lectins alone at 10% also showed inhibition of bacterial attachment. However, the best result was achieved against bacterial adhesion and growth on surfaces using a combination of 100 g Ag⁺/ml and a lectin coating as a surface treatment. This surface treatment was also effective against both fresh culture and a two-week-old culture containing P. aeruginosa producing exopolymers. Our results suggest that Ag⁺impregnation combined with a lectin coating warrants further investigation as a potential means of protecting catheters.     

Preventive effect of several drugs againstPseudomonas aeruginosa infection and the toxicity of combined tumor necrosis factor with lipopolysaccharide: Relationship between lethality and the arachidonic cascade

The participation of tumor necrosis factor (TNF) and lipopolysaccharide (LPS) inPseudomonas aeruginosa (Pa) infection was examined. The lethal challenge of Pa or TNF and LPS injection could be prevented by pretreatment with anti-TNF antibody, polymyxin B, ONO 1078, or Shosaiko-to. The combined effects of TNF and LPS may be deeply related to the lethality of Pa infection. The activities of leukotriene(LT) C₄/D₄/E₄ or platelet activating factor (PAF) were also related to the lethality of Pa infection, probably due to the subsequently produced TNF which acts in combination with LPS. Activating the host defence mechanism with biological response modifiers like Chinese medicines was effective against Pa infection. One mechanism could involve an activity as an LT inhibitor or PAF antagonist. Following the administration of TNF and/or LPS, the serum levels of arachidonic cascade products underwent various changes. With a combination of TNF and LPS, there was a synergistic increment of prostaglandins, thromboxane, and LT. Following pretreatment with Shosaiko-to, suppression of LTs was dominant even with the combination of TNF and LPS, which might be related to the lethality of the infection or combined TNF with LPS.     

Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A

Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated -glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting -glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile.     

Mesosome formation is accompanied by hydrogen peroxide accumulation in bacteria during the rifampicin effect

Ultrastructural alteration and hydrogen peroxide localization were examined in Xanthomonas campestris pv. phaseoli during rifampicin effect using transmission electron microscopy. Bacterial cells were treated with rifampicin and then were examined by electron microscopy to observe the changes of ultrastructure or hydrogen peroxide accumulation in living cells that took place before lysis. Intriguingly, rifampicin treatment led to presence of an additional location of hydrogen peroxide accumulation within the cells. There was an association between the frequency and size of the additional location of hydrogen peroxide accumulation and the concentration of rifampicin. Furthermore, an additional ultrastructure, mesosomes, was also present in cells during rifampicin effect. The frequency and size of mesosome increased with the increasing concentration of rifampicin. Result of multiple linear regression showed that the size of mesosome plays as a key factor in the quantity of excess hydrogen peroxide accumulation in cells during rifampicin effect. Linear correlation was confirmed between quantity of excess hydrogen peroxide accumulation and the size of mesosome in cells during rifampicin effect. This finding intensely indicated that mesosomes are just the additional location of hydrogen peroxide accumulation in cells under cellular injury caused by rifampicin treatment. The mesosome formation is always accompanied by excess hydrogen peroxide accumulation in X. campestris pv. phaseoli during rifampicin effect.     

The effect of transposons on the expression of the naphthalene biodegradation genes in Pseudomonas putida BS202(NPL-1) and derivative strains

NPL-1 and its derivative plasmid pBS106, which control the degradation of naphthalene and salicylate, were found to contain class II transposons of the Tn3 family. These transposons are involved in intraplasmid rearrangements, such as deletions and inversions, and can influence the expression of the catabolic and regulatory genes borne by biodegradation plasmids. The formation of a strong NahR-independent constitutive promoter by the inversion of a DNA fragment may be responsible for changing the character of naphthalene dioxygenase synthesis from inducible (in the case of plasmid NPL-1) to constitutive (in the case of plasmid NPL-41). The stability of plasmids NPL-1 and NPL-41 in Pseudomonas putida strains grown on different substrates depends on the expression of the nah and tnp genes.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 79–86.Original Russian Text Copyright © 2005 by Sokolov, Kosheleva, Filonov, Boronin.     

A Novel Alginate Lyase with High Activity on Acetylated Alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03

Summary To exploit alginate lyase which could degrade bacterial alginates, degenerate PCR and long range-inverse PCR (LR-IPCR) were used to isolate alginate lyase genes from soil bacteria. Gene algL, an alginate lyase-encoding gene from Pseudomonas sp. QD03 was cloned, and it was composed of a 1122 bp open reading frame (ORF) encoding 373 amino acid residues with the calculated molecular mass of 42.2 kDa. The deduced protein had a potential N-terminal signal peptide of 20 amino acid residues that was consistent with its proposed periplasmic location. Gene algL was expressed in pET24a (+)/E. coli BL21 (DE3) system. The recombinant AlgL was purified to electrophoretic homogeneity using affinity chromatography. The molecular weight of AlgL was estimated to be 42.8 kDa by SDS-PAGE. AlgL exhibited maximal activity at pH 7.5 and 37 °C. Na⁺, K⁺, Ca²⁺ and Ba²⁺ significantly enhanced the activity of AlgL. AlgL could degrade alginate and mannuronate blocks, but hardly degrade guluronate blocks. In particular, AlgL could degrade acetylated alginate of Pseudomonas aeruginosa FRD1 (approximately 0.54 mol of O-acetyl group per mol of alginate). It might be possible to use alginate lyase AlgL as an adjuvant therapeutic medicine for the treatment of disease associated with P. aeruginosa infection.     

Pro-oxidative synergic bactericidal effect of NO: kinetics and inhibition by nitroxides

NO plays diverse roles in physiological and pathological processes, occasionally resulting in opposing effects, particularly in cells subjected to oxidative stress. NO mostly protects eukaryotes against oxidative injury, but was demonstrated to kill prokaryotes synergistically with H₂O₂. This could be a promising therapeutic avenue. However, recent conflicting findings were reported describing dramatic protective activity of NO. The previous studies of NO effects on prokaryotes applied a transient oxidative stress while arbitrarily checking the residual bacterial viability after 30 or 60 min and ignoring the process kinetics. If NO-induced synergy and the oxidative stress are time-dependent, the elucidation of the cell killing kinetics is essential, particularly for survival curves exhibiting a “shoulder” sometimes reflecting sublethal damage as in the linear-quadratic survival models. We studied the kinetics of NO synergic effects on H₂O₂-induced killing of microbial pathogens. A synergic pro-oxidative activity toward gram-negative and gram-positive cells is demonstrated even at sub-μM/min flux of NO. For certain strains, the synergic effect progressively increased with the duration of cell exposure, and the linear-quadratic survival model best fit the observed survival data. In contrast to the failure of SOD to affect the bactericidal process, nitroxide SOD mimics abrogated the pro-oxidative synergy of NO/H₂O₂. These cell-permeative antioxidants, which hardly react with diamagnetic species and react neither with NO nor with H₂O₂, can detoxify redox-active transition metals and catalytically remove intracellular superoxide and nitrogen-derived reactive species such as ^•NO₂ or peroxynitrite. The possible mechanism underlying the bactericidal NO synergy under oxidative stress and the potential therapeutic gain are discussed.