Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t _1/2 values of 7.0 and 1.5 min at 55°C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instabiliby due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M _r value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.     

Staphylolytic enzyme from Pseudomonas aeruginosa: characterization and immunocytochemical localization

Staphylolytic enzyme, a specific peptidase produced by Pseudomonas aeruginosa, has been characterized by using immunochemical procedures. Lytic activity was detected in the extracellular medium of Pseudomonas cultures at the beginning of the stationary growth phase. No activity was detected in bacterial cells. However, lytic protein antigen was present in periplasmic and cytoplasmic fractions, suggesting that staphylolytic enzyme is synthesized as an inactive precursor which becomes active during translocation to the extracellular broth. Results obtained in immunolocalization experiments indicate the presence of the precursor in the outer part of cells. The export pathway of staphylolytic enzyme through the periplasmic space is proposed.Abbreviations DCE dialyzed crude extract - CFU colonies forming units - LU lytic unit     

Regulation of amidase formation in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity

The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation between amidase and urease formation was observed. The results suggest that amidase formation in strain PAO is subject to nitrogen control and that glutamine or some compound derived from it mediates the nitrogen repression of amidase.     

Characterization of monoclonal antibodies against altered (T103I) amidase from Pseudomonas aeruginosa

Monoclonal antibodies (MAbs) against mutant (T103I) amidase from Pseudomonas aeruginosa were raised by hybridoma technology. To select MAbs suitable for immunoaffinity chromatography, hybridoma clones secreting polyol-responsive MAbs (PR-MAbs) were screened that bind antigen tightly but release under mild and nondenaturing elution conditions. It was found that about 10% of enzyme-linked immunosorbent assay (ELISA)-positive hybridoma produce these MAbs as their ag-ab complex can be disrupted by propylene glycol in the presence of a suitable salt. Two of these hybridoma clones (F6G7 and E2A6) secreting PR-MAbs against mutant amidase were selected for optimization of experimental conditions for elution of amidase by using ELISA elution assay. These hybridoma cell lines secreted MAbs of IgM class that were purified in a single step by gel filtration chromatography, which revealed a single protein band on native polyacrylamide gel electrophoresis (PAGE). Specificity studies of this MAb revealed that it recognized specifically a common epitope on mutant and wild-type amidases as determined by direct ELISA. This MAb exhibited a higher affinity for denatured forms of wild-type and mutant amidases than for native forms as revealed by affinity constants (K), suggesting that it recognizes a cryptic epitope on an amidase molecule. Furthermore, MAb E2A6 inhibited about 60% of wild-type amidase activity, whereas it activated about 60% of mutant amidase (T103I) activity. The data presented in this work suggest that this MAb acts as a very useful probe to detect conformational changes in native and denatured amidases as well as to differentiate wild-type and mutant (T103I) amidases.     

A tracheal culture model of respiratory tract infection withPseudomonas Aeruginosa

Summary The pathogenesis ofPseudomonas aeruginosa for the respiratory tract has been examined using hamster tracheal organ cultures. Tracheal rings prepared from male Syrian hamsters, strain LSH/LAK, were infected withP. aeruginosa for 4 h and processed at 4-h intervals for 24 h for examination by light- and electron microscopy. Tissue destruction was observed within 8 h after infection with 10⁸ colony-forming units (cfu)/ml and within 12 h after infection with 10⁴ or 10⁶ cfu/ml. Ciliated cells that contained abnormal subcellular organelles were expelled from the epithelium. By 20 h the epithelial borders were composed primarily of nonciliated cells. Transmission- and scanning electron microscopy revealed details of the cellular destruction and attachment ofP. aeruginosa to the ciliated epithelium.Pseudomonas aeruginosa causes a rapid destruction of the epithelium of hamster trachea in cultures. Hamster tracheal organ cultures have been shown to be useful in studying the pathogenesis ofP. aeruginosa for the respiratory tract. This work was supported by Grants G-430B and G-431B from the Cystic Fibrosis Foundation.     

Kinetic studies on surfactant production byPseudomonas aeruginosa 44T1

Summary Biosurfactant accumulation occurred in the exponential and stationary phases. Production started when the nitrogen level was very low. Surfactant was produced with a diauxic pattern. Rhamnolipid concentration increased as nitrogen levels increased. Maximum product yield (Y _p/x) 2.9 was detected when C/N ratio was 6.6 and specific rate of product formation (p _q) was calculated. The examination of these kinetics parameters such as product yield and specific rate of product formation should be taken into account to develop a high efficient production process.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30–60 and 1,4-butanediol-diglycidyl ether: 16–36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a M _r of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and process-compatible alternative to other types of chromatography.     

铜绿假单胞菌异质性耐药的研究进展

异质性耐药是指细菌中的同源亚群对某种抗生素表现出不同的敏感性,被认为是细菌由敏感进化成完全耐药的中间阶段.常规的临床检验无法有效检测出异质性耐药,这对临床治疗用药造成了巨大的威胁,引起患者的反复感染和用药失败.铜绿假单胞菌作为医院内感染的主要条件致病菌之一,其耐药机制已被广泛研究,而异质性耐药研究则相对较少.本文主要就...     

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.     

Mutants of Pseudomonas aeruginosa unable to inactivate allantoinase and NADP-dependent glutamate dehydrogenase

Both allantoinase and NADP-GDH in Pseudomonas aeruginosa were inactivated when cells reached the stationary phase of growth. Mutants unable to inactivate these enzymes were isolated. Results with recombinants showed that the mutation is not located in the structural genes of these enzymes but in an independent gene involved in the inactivation.Abbreviations NADP-GDH NADP-dependent glutamate dehydrogenase - Ani^- mutant allantoinase non-inactivating mutant - GOGAT glutamate synthase     

Induction and selection of formaldehyde-based resistance inPseudomonas aeruginosa

Summary A formaldehyde resistant (R) phenotype ofPseudomonas aeruginosa was isolated from a formaldehydesensitive (S) parent by sequential treatment with 1,3,5-tris-(ethyl)hexahydro-s-triazine (ET). The resistance of the (R) strain to treatment with ET was approximately 3-fold higher than the parental (S) strain. Two modes of resistance to ET, and simultaneous resistance to formaldehyde, are demonstrated: (1) transient or induced resistance is expressed during shor-term exposure to ET, and this resistance is gradually lost during subsequent growth in the absence of ET, and (2) resistance that results from a stable phenotypic change in the (S) strain following sequential treatment with ET ((R) strain phenotype). The observed activities of three forms of the formaldehyde oxidizing enzyme, formaldehyde dehydrogenase, are strongly correlated with the relative response of the (S) and (R) strains to treatment with ET. The observed resistance of the (R) strain appears to be due to high levels of an NAD⁺-linked, glutathione-dependent form of formaldehyde dehydrogenase as well as a dye-linked formaldehyde dehydrogenase. The transient or induced response of the (R) strain involves an increase in activity of the dye-linked formaldehyde dehydrogenase. The induced response of the (S) strain and an ATCC strain ofP. aeruginosa, however, is correlated with the two forms of the NAD⁺-linked enzyme (glutathione-dependent (EC 1.2.1.1) and independent (EC 1.2.1.46)) with no contribution from the dye-linked enzyme.     

R-bodies inPseudomonas aeruginosa strain 44T1

For the first time R-bodies are described in a new strain 44T1 ofPseudomonas aeruginosa. Its size was measured as being 0.22 to 0.37 m of width per 0.27 to 0.41 m of length and 5 to 9 spiral turns about 16 nm. These structures are similar to previously observed in bacteria and are related with physiological state of bacteria in minimal conditions of growth.     

N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

Production of extracellular emulsifying agent byPseudomonas aeruginosa UG1

Summary Twenty-three bacterial strains were isolated from oil-contaminated soil samples. Of these, 20 displayed some ability to effect oil dispersion and they were screened quantitatively for the ability to emulsify 0.5% (v/v) reference oil. One strain, identified asPseudomonas aeruginosa UG1, produced extracellular material that emulsified reference oil, hexadecane and 2-methylnaphthalene at concentrations as high as 6% (v/v) in nutrient broth. Emulsification activity increased during a 10 day incubation period at 30°C. The activity was not influenced by pH over the range 5 to 9. The emulsifying agent was precipitated by cold ethanol. The highest emulsifying activity was detected in the extracellular fraction precipitated between 30 and 50% (v/v) ethanol. A linear relationship was observed between emulsifier concentration (mg/ml) and emulsifying activity. Genetic analysis showed that thePseudomonas aeruginosa UG1 strain did not carry extrachromosomal plasmids, suggesting that the gene(s) coding for emulsifying activity was carried on the chromosome.     

The effect of fifteen biocides on formaldehyde-resistant strains ofPseudomonas aeruginosa

Summary Evaluation of formaldehyde and fifteen biocides in formaldehyde sensitive (S) and resistant (R) strains ofPseudomonas aeruginosa revealed a pattern of response that allowed a comparison of the mode of action of these biocides. The response of these strains to the various biocides, as well as the induction of transient resistance or cross-resistance in the (S) strain, allowed a grouping of biocides based on this pattern of response. Group 1 biocides acted in a manner indistinguishable from formaldehyde for both the (S) and (R) strains. Group 2 biocides were not effective against either the (S) or (R) strains at concentrations calculated to release equimolar concentrations of formaldehyde. However, treatment of the (S) strain with formaldehyde or Group 2 biocides resulted in the development of cross-resistance. Group 3 biocides were equally effective against the (S) and (R) strain, but the (S) strain survivors of treatment with Group 3 biocides were resistant to formaldehyde. Group 4 biocides (controls) had no presumed connection to formaldehyde mode of action. These four groupings, based on pattern of response, also resulted in groupings of biocides based on chemical structure.     

Selection and preliminary characterization of a Pseudomonas aeruginosa strain mineralizing selected isomers in a branchedchain dodecylbenzenesulphonate mixture

A bacterium able to grow at the expense of some isomers in a commercial surfactant preparation consisting of branched-chain dodecylbenzenesulphonate was isolated (W51), and it was identified as a Pseudomonas aeruginosa strain. A faster growing derivative was selected (W51D) after enrichment in batch culture under microaerobic conditions, using the surfactant as the sole source of carbon and energy. Strain W51D is the first microorganism reported to degrade at least 70% of a branched-chain alkylbenzenesulphonate mixture and to be resistant to high concentrations of this surfactant. The ability to degrade the surfactant was shown to be transferred by conjugation to other P. aeruginosa strains and to an Escherichia coli strain.G. Soberón-Chávez and J. Campos are with the Instituto de Biotecnologia, Universidad Nacional Autónoma de México, Apdo Postal 510-3, Cuernavaca, Mor. 62250, México.A. Hädour and L. Ramos are with Estación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas, Protesor Albareda 1, Granada 18008, España. J. Ortigoza is with Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Apdo. Postal 42-186. México D.F. 11340. México.     

Isolation and characterization of mutant Pseudomonas aeruginosa strains unable to assimilate nitrate

Single-site mutants of Pseudomonas aeruginosa that lack the ability aerobically to assimilate nitrate and nitrite as sole sources of nitrogen have been isolated. Twentyone of these have been subdivided into four groups by transductional analysis. Mutants in only one group, designated nis, lost assimilatory nitrite reductase activity. Mutants in the other three transductional groups, designated ntmA, ntmB, ntmC, display a pleiotropic phenotype: utilization of a number of nitrogen-containing compounds including nitrite as sole nitrogen sources is impaired. Assimilatory nitrite reductase was shown to be the major route by which hydroxylamine is reduced in aerobically-grown cells.In memoriam of Professor R. Y. Stanier     

Characterization of a rhodanese from the cyanogenic bacterium Pseudomonas aeruginosa

Pseudomonas aeruginosa, the rRNA group I type species of genus Pseudomonas, is a Gram-negative, aerobic bacterium responsible for serious infection in humans. P. aeruginosa pathogenicity has been associated with the production of several virulence factors, including cyanide. Here, the biochemical characterization of recombinant P. aeruginosa rhodanese (Pa RhdA), catalyzing the sulfur transfer from thiosulfate to a thiophilic acceptor, e.g., cyanide, is reported. Sequence homology analysis of Pa RhdA predicts the sulfur-transfer reaction to occur through persulfuration of the conserved catalytic Cys230 residue. Accordingly, the titration of active Pa RhdA with cyanide indicates the presence of one extra sulfur bound to the Cys230 Sgamma atom per active enzyme molecule. Values of K(m) for thiosulfate binding to Pa RhdA are 1.0 and 7.4mM at pH 7.3 and 8.6, respectively, and 25 degrees C. However, the value of K(m) for cyanide binding to Pa RhdA (=14 mM, at 25 degrees C) and the value of V(max) (=750 micromol min(-1)mg(-1), at 25 degrees C) for the Pa RhdA-catalyzed sulfur-transfer reaction are essentially pH- and substrate-independent. Therefore, the thiosulfate-dependent Pa RhdA persulfuration is favored at pH 7.3 (i.e., the cytosolic pH of the bacterial cell) rather than pH 8.6 (i.e., the standard pH for rhodanese activity assay). Within this pH range, conformational change(s) occur at the Pa RhdA active site during the catalytic cycle. As a whole, rhodanese may participate in multiple detoxification mechanisms protecting P. aeruginosa from endogenous and environmental cyanide.     

Pyoverdine-mediated iron transport Fate of iron and ligand inPseudomonas aeruginosa

Summary Incubated in the presence of [⁵⁵Fe]ferri[¹⁴C]pyoverdine, iron-poorPseudomonas aeruginosa accumulated more⁵⁵Fe than¹⁴C over a 60-min period. Distribution studies showed (a) more¹⁴C than⁵⁵Fe in the soluble fraction during the first 20 min, (b) approximately 60% of the⁵⁵Fe associated with the membranes at 60 min, and (c) approximately 85% of the¹⁴C in the soluble fraction at 60 min. Cells osmotically shocked after incubating with [⁵⁵Fe]ferri[¹⁴C]pyoverdine for 60 min released⁵⁵Fe but not¹⁴C, suggesting separation of metal and ligand in the periplasmic space. Whereas the mechanism of dissociation of iron and ligand is not known, the decrease in transport observed in the presence of dipyridyl suggests involvement of reduction in this process. Transport of iron was energized by the proton motive force instead of by intracellular levels of ATP. The hydrogen ion gradient was the major driving force of transport. Cyanide-poisoned cells accumulated more¹⁴C than⁵⁵Fe over 60 min. Here, iron accumulated in the soluble fraction instead of on the membranes.     

Na+(Li+)/Branched-chain amino acid cotransport inPseudomonas aeruginosa

Summary A transport system for branched-chain amino acids (designated as LIV-II system) inPseudomonas aeruginosa requires Na⁺ for its operation. Coupling cation for this system was identified by measuring cation movement during substrate entry using cation-selective electrodes. Uptakes of Na⁺ and Li^– were induced by the imposition of an inwardly-directed concentration gradient of leucine, isoleucine, or valine. No uptake of H^– was found, however, under the same conditions. In addition, effects of Na⁺ and Li⁺ on the kinetic property of the system were examined. At chloride salt concentration of 2.5mm, values of apparentK _m andV _max for leucine uptake were larger in the presence of Na⁺ than Li⁺. These results indicate that the LIV-II transport system is a Na⁺(Li⁺)/substrate cotransport system, although effects of Na⁺ and Li⁺ on kinetics of the system are different.