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     

Desulfurization of light gas oil by a novel recombinant strain from Pseudomonas aeruginosa

The dsz desulfurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred into the chromosomes of Pseudomonas aeruginosa NCIMB 9571 by using a transposon vector. Resting cells of the recombinant strain, PAR41, desulfurized 63 mg sulfur l^–1 of light gas oil (LGO) containing 360 mg S l^–1. The desulfurization activity for LGO by the resting cells of strain PAR41 grown with n-tetradecane (50% v/v) was much higher (1018-fold) than in glucose-grown cells. P. aeruginosa NCIMB 9571 is able to take up water-insoluble compounds from an oil phase which is enhanced by n-alkane.     

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.     

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     

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.     

Functional characterization of genes involved in alkane oxidation by Pseudomonas aeruginosa

Most clinical isolates identified as Pseudomonas aeruginosa grow on long-chain n-alkanes, while environmental P. aeruginosa isolates often grow on medium- as well as long-chain n-alkanes. Heterologous expression showed that the two alkane hydroxylase homologs of P. aeruginosa PAO1 (AlkB1 and AlkB2) oxidize C₁₂-C₁₆ n-alkanes, while two rubredoxin (RubA1 and RubA2) and a rubredoxin reductase (RubB) homologs can replace their P. putida GPo1 counterparts in n-octane oxidation. The two long-chain alkane hydroxylase genes are present in all environmental and clinical isolates of P. aeruginosa strains tested in this study. This revised version was published online in August 2006 with corrections to the Cover Date.     

The stereoisomers of pyochelin, a siderophore of Pseudomonas aeruginosa

The Pseudomonas aeruginosa siderophore pyochelin is obtained from the bacterial culture medium as a mixture of two epimers. Chromatically isolated pure stereoisomers equilibrate readily in most solvents. Experiments will be reported which allow to isolate one of the isomers in pure form and which shed some additional light on the epimerization reaction.     

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     

Extracellular polysaccharides of a copper-sensitive and a copper-resistant Pseudomonas aeruginosa strain: synthesis,chemical nature and copper binding

Extracellular polysaccharides (EPS) of a copper-sensitive (Cu^s) and a copper-resistant (Cu^r) Pseudomonas aeruginosa strain were investigated in terms of their production, chemical nature and response towards copper exposure. The extent of EPS synthesis by the resistant strain (4.78 mg mg^–1 cell dry wt.) was considerably higher over its sensitive counterpart (2.78 mg mg^–1 dry wt.). FTIR-spectroscopy and gas chromatography revealed that both the polymers were acidic in nature, containing alginate as the major component along with various neutral- and amino-sugars. Acid content in the Cu^r EPS (480.54 mg g^–1) was greater than that in the Cu^s EPS (442.0 mg g^–1). Presence of Cu²⁺ in the growth medium caused a dramatic stimulation (approximately 4-fold) in EPS synthesis by the Cu^r strain, while in a similar condition, the Cu^s failed to exhibit such response. The polymer of the resistant strain showed elevated Cu²⁺ binding (320 mg g^–1 EPS) compared to that of the sensitive type (270 mg g^–1). The overall observations show the potential of the Cu^r EPS for its deployment in metal bioremediation.     

Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid

Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l^–1. Presence of the plasmid in P. aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host. Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P. aeruginosa transformant.     

The production and utilization of nitric oxide by a new,denitrifying strain of Pseudomonas aeruginosa

When a new strain of Pseudomonas aeruginosa was grown aerobically and then transferred to anaerobic conditions, cells reduced NO ₃ ^– quantitatively to NO ₂ ^– in NO ₃ ^– -respiration. In the absence of nitrate, NO ₂ ^– was immediately reduced to NO or N₂O but not to N₂ indicating that NO ₂ ^– -reductase but not N₂O-reductase was active. The formation of the products NO or N₂O depended on the pH in the medium and the concentration of NO ₂ ^– present. When P. aeruginosa was grown anaerobically for at least three davs N₂O-reductase was also active. Such cells reduced NO to N₂ via N₂O. The new strain generated a H⁺-gradient and grew by reducing N₂O to N₂ but not by converting NO to N₂O. For comparison, Azospirillum brasilense Sp7 showed the same pattern of NO-reduction. In contrast, Paracoccus denitrificans formed 3.5 H⁺/NO during the reduction of NO to N₂O in oxidant pulse experiments but could not grow in the presence of NO. Thus the NO-reduction pattern in P. denitrificans on one side and P. aeruginosa and A. brasilense on the other was very different. The mechanistic implications of such differences are discussed.     

Modelling antibiotic- and anti-quorum sensing treatment of a spatially-structured Pseudomonas aeruginosa population

The bacterial cell to cell signalling system known as quorum sensing (QS) is essential for the regulation of virulence in many pathogens and offers a specific biochemical target for novel antibacterial therapies. Expanding on earlier work, in which consideration was given to the primary QS system (lasR system) in a homogeneous population of the common human pathogen Pseudomonas aeruginosa, we build a simple spatial model of an early-stage P. aeruginosa biofilm subject to treatment with topically applied anti-QS drugs (of two specific kinds) and conventional antibiotics. In the case of a slowly growing biofilm we show that both kinds of anti-quorum sensing drug are effective in reducing the level of the relevant signal molecule (3-oxo-C12-homoserine lactone; henceforth AHL), in each case obtaining an explicit bound on the steady-state AHL profile in terms of a prescribed surface drug concentration. Using numerical methods, we are also able to reproduce the hysteretic phenomena exhibited by the homogeneous model, in particular showing that for each kind of anti-QS drug there is a parameter regime in which a catastrophic collapse occurs in the steady-state AHL concentration as the surface drug concentration passes some critical value; an alternative way of interpreting this result is to say that, for a prescribed surface drug concentration, there is a critical biofilm depth such that treatment is successful until this depth is reached, but fails thereafter. In the thick-biofilm limit we show that the critical concentration of each drug increases exponentially with the biofilm thickness (or, conversely, that the critical depth increases logarithmically with surface drug concentration); this is dramatically different to the behaviour observed in the corresponding homogeneous model, where the critical concentrations grow linearly with bacterial carrying capacity, and thus highlights the relative difficulty of treating a large, spatially-structured population with diffusing antibacterials.     

Isolation and characterization of Pseudomonas aeruginosa mutants deficient in the utilization of the terpenoid citronellic acid

Eighteen non-sibling mutants of Pseudomonas aeruginosa PAO were isolated that were deficient in the utilization of the -methyl branched acid citronellic acid but not in the utilization of the unbranched n-octanoic acid (Cau mutants). These mutants are also deficient in the utilization of citronellol and citronellal. R68.45 plasmid-mediated transfer of chromosomal material has been used to map one of the mutations at about 52 min on the PAO chromosome and to show linkage of some, but not all, of the other mutations to this region. This system is of interest for bioremediation in oil spill areas since -methyl branches block normal -oxidation and cause recalcitrance of organic molecules present in petroleum products.     

Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp wastes

A protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa MN7. The strain was found to produce proteases when it was grown in media containing only shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. The use of 60 g/l SWP resulted in a high protease production. Elastase, the major protease produced by P. aeruginosa MN7, was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration, and ultrafiltration using a 10-kDa cut-off membrane, with a 5.2-fold increase in specific activity and 38.4% recovery. The molecular weight of the purified elastase was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for protease activity were 60 degrees C and 8.0, respectively. The activity of the enzyme was totally lost in the presence of ethylene glycol tetraacetic acid, suggesting that the purified enzyme is a metalloprotease. The purified enzyme was highly stable in the presence of organic solvents, retaining 100% of its initial activity after 60 days of incubation at 30 degrees C in the presence of dimethyl sulfoxide and methanol. The lasB gene, encoding the MN7 elastase, was isolated and its DNA sequence was determined.     

Alginate acetylation influences initial surface colonization by mucoid Pseudomonas aeruginosa

Mucoid strains of Pseudomonas aeruginosa overproduce the exopolysaccharide alginate, which is substituted with O-acetyl groups. Under non-growing conditions in phosphate buffer, a mucoid clinical strain formed microcolonies on steel surfaces, while an acetylation-defective mutant was unable to form cell clusters. Enzymatic degradation of alginate by alginate lyase prevented microcolony formation of the mucoid parent strain. In a continuous-culture flow-cell system, using gluconate minimal medium, the mucoid strain with acetylated alginate formed microcolonies and grew into heterogenous biofilms, whereas the acetylation-defective mutant produced a thinner and more homogeneous biofilm. A lowered viscosity of extracellular material from the acetylation-defective mutant indicated a weakening of exopolymer interactions by loss of acetyl groups. These results suggest that acetyl substituents are necessary for the function of high-molecular-mass alginate to mediate cell aggregation into microcolonies in the early stages of biofilm development by mucoid P. aeruginosa, thereby determining the architecture of the mature biofilm.     

Isolation and characterization of TMPD-oxidase mutants of Pseudomonas aeruginosa

Mutants showing negative oxidase-reaction have been isolated from Pseudomonas aeruginosa following mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine. These mutants were compared to the wild type cells with respect to their respiratory activities and cytochrome contents. They exhibit lower respiration rates and contain much less cytochrome c's which are responsible for their weak or negative oxidase-reaction in these mutants. This is supported in part from an initial linear relationship observed between the measured oxidase activities and the lower cytochrome c contents in these mutants. Further evidence comes from analyzing oxidase-negative cells of P. syringae, in which low cytochrome c contents similar to these oxidase mutants account for negative oxidase activities. Cytochrome o was the sole oxidase found among these mutants as well as in the wild type cell, suggesting that cytochrome c+o complex is responsible for the tetramethyl-p-phenylenediamine-oxidase activity in these mutants as the case in the wild-type cells. From the spectral characteristics it seems that all mutants contain about the same amount and type of terminal oxidase as that of the wild-type cells. The mutation occurred which altered the oxidase activities in these mutants appears to affect cytochrome c gene(s), but not the terminal oxidase gene(s).Abbreviations TMPD Tetramethyl-p-phenylenediamine - MD minimal Davis     

Biodegradation of 8-anilino-1-naphthalenesulfonic acid by Pseudomonas aeruginosa

Pseudomonas aeruginosa, isolated from soil near tannery effluent was able to degrade 8-anilino-1-naphthalenesulfonic acid (ANSA), a sulfonated aromatic amine. The organism degraded this amine up to a concentration of 1,200 mg l⁻¹ using glucose and ammonium nitrate as carbon and nitrogen sources respectively. The degradation started when the organism reached its late exponential growth phase. Salicylic acid and β-ketoadipic acid were identified as intermediate compounds using HPLC and GC–MS and provide evidence for ortho pathway reactions. Further proof for the pathway is obtained from the dioxygenase activity of the strain growing exponentially in medium with ANSA and glucose.     

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     

Glutathione exhibits antibacterial activity and increases tetracycline efficacy against Pseudomonas aeruginosa

Glutathione (GSH) plays important roles in pulmonary diseases, and inhaled GSH therapy has been used to treat cystic fibrosis (CF) patients in clinical trials. The results in this report revealed that GSH altered the sensitivity of Pseudomonas aeruginosa to different antibiotics through pathways unrelated to the oxidative stress as generally perceived. In addition, GSH and its oxidized form inhibited the growth of P. aeruginosa. Supported by the National Natural Science Foundation of China (Grant Nos. 30870097 and 30611120520)