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.     

Enzymatic detoxification of cyanide: clues from Pseudomonas aeruginosa Rhodanese

Cyanide is a dreaded chemical because of its toxic properties. Although cyanide acts as a general metabolic inhibitor, it is synthesized, excreted and metabolized by hundreds of organisms, including bacteria, algae, fungi, plants, and insects, as a mean to avoid predation or competition. Several cyanide compounds are also produced by industrial activities, resulting in serious environmental pollution. Bioremediation has been exploited as a possible alternative to chemical detoxification of cyanide compounds, and various microbial systems allowing cyanide degradation have been described. Enzymatic pathways involving hydrolytic, oxidative, reductive, and substitution/transfer reactions are implicated in detoxification of cyanide by bacteria and fungi. Amongst enzymes involved in transfer reactions, rhodanese catalyzes sulfane sulfur transfer from thiosulfate to cyanide, leading to the formation of the less toxic thiocyanate. Mitochondrial rhodanese has been associated with protection of aerobic respiration from cyanide poisoning. Here, the biochemical and physiological properties of microbial sulfurtransferases are reviewed in the light of the importance of rhodanese in cyanide detoxification by the cyanogenic bacterium Pseudomonas aeruginosa. Critical issues limiting the application of a rhodanese-based cellular system to cyanide bioremediation are also discussed.     

Release of rhodanese from Pseudomonas aeruginosa by cold shock and its localization within the cell.

Whole cells of Pseudomonas aeruginosa possess rhodanese activity. The enzyme can be released by rapidly resuspending the cells in cold Tris--HCl buffer. Approximately 95% of the rhodanese activity is released by cold shock. Release of the enzyme can be inhibited either by preincubating the cells with Mg2+ or by incorporating Mg2+ into the shocking buffer. The effect of Mg2+ can be reversed by washing the cells twice with buffer prior to cold shock. While rhodanese can be released from P. aeruginosa by cold shock, lactic dehydrogenase, a cytoplasmic enzyme, remains within the cell. Diazo-7-amino-1,3-napthalenedisulfonic acid, a compound which does not penetrate the cytoplasmic membrane, completely inactivated rhodanese and alkaline phosphatase, a periplasmic enzyme, whereas lactic dehydrogenase retained its full activity. These data suggest that rhodanese in P. aeruginosa, like alkaline phosphatase, is located distal to the cytoplasmic membrane in the periplasmic space. Electron micrographs also show that portions of the lipopolysaccharide outer membrane are shed from the cell during cold shock, while cells preincubated with Mg2+ did not release segments of their outer membrane.     

Hydrogen cyanide, a secondary metabolite of Pseudomonas aeruginosa.

Seventy-four of 110 strains of Pseudomonas aeruginosa tested produced detectable amounts of HCN from growth in 2% peptone or nutrient agar. Of the 25 species of12 bacterial and fungal genera tested, other than P. aeruginosa, only P. fluorescens and P. polycolor gave positive HCN tests. Cyanide is produced after cessation of active growth. Iron was stimulatory to cyanogenesis in concentration above 1 muM, while copper, zinc, cobalt, and manganese at concentrations of 20 muM had no effect. Cyanogenesis id dependent on the temperature of incubation within ranges which allow complete growth. Inorganic phosphate in concentrations between 90 and 300 mM allows growth but inhibits HCN production. Growth of cells anaerobically, using nitrate as the electron acceptor, results in low cyanide yields, which can be partially reversed by subsequent aerobic incubation. These results indicate that HCN is a secondary metabolite of P. aeruginosa.     

Glycine metabolism by Pseudomonas aeruginosa: hydrogen cyanide biosynthesis.

Hydrogen cyanide (HCN) production by Pseudomonas aeruginosa in a synthetic medium is stimulated by the presence of glycine. Methionine enhances this stimulation but will not substitute for glycine as a stimulator of cyanogenesis. Threonine and phenylalanine are effective substitutes for glycine in the stimulation of HCN production. Glycine, threonine, and serine are good radioisotope precursors of HCN, but methionine and phenylalanine are not. Cell extracts of P. aeruginosa convert [14C]threonine to [14C]glycine. H14CN is produced with low dilution of label from either [1-14C]glycine or [2-14C]glycine, indicating a randomization of label either in the primary or secondary metabolism of glycine. When whole cells were fed [1,2-14C]glycine, cyanide and bicarbonate were the only radioactive extracellular products observed.     

Hydrogen cyanide production by Pseudomonas aeruginosa at reduced oxygen levels

Hydrogen cyanide production by Pseudomonas aeruginosa growing in a synthetic medium required aerobosis but operated efficiently at low dissolved oxygen concentration. Half maximum levels of cyanogenesis occurred at 0.015 microM oxygen; maximum cyanogenesis occurred over a wide range, 0.1-180 microM, of oxygen concentrations. These cells lost the ability to produce cyanide upon aerobic incubation in the absence of both the carbon energy source (L-glutamate) and the metabolic precursor of hydrogen cyanide (glycine). This loss of cyanogenesis was dependent on oxygen concentration; 1.0 microM oxygen produced no detectable loss, whereas 180 microM oxygen caused a rapid decline in cyanogenic ability. The endogenous cyanide production rate of cells in the presence of carbon energy source was not significantly influenced by oxygen concentration. During the batch culture cycle, the acquisition of the ability to produce HCN was preceded by oxygen reduction to growth-limiting levels. Cells which had lost the ability to produce hydrogen cyanide by oxygen treatment required protein synthesis before they could again become cyanogenic.     

Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa

Bacterial biofilms at times undergo regulated and coordinated dispersal events where sessile biofilm cells convert to free-swimming, planktonic bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, we previously observed that dispersal occurs concurrently with three interrelated processes within mature biofilms: (i) production of oxidative or nitrosative stress-inducing molecules inside biofilm structures, (ii) bacteriophage induction, and (iii) cell lysis. Here we examine whether specific reactive oxygen or nitrogen intermediates play a role in cell dispersal from P. aeruginosa biofilms. We demonstrate the involvement of anaerobic respiration processes in P. aeruginosa biofilm dispersal and show that nitric oxide (NO), used widely as a signaling molecule in biological systems, causes dispersal of P. aeruginosa biofilm bacteria. Dispersal was induced with low, sublethal concentrations (25 to 500 nM) of the NO donor sodium nitroprusside (SNP). Moreover, a P. aeruginosa mutant lacking the only enzyme capable of generating metabolic NO through anaerobic respiration (nitrite reductase, DeltanirS) did not disperse, whereas a NO reductase mutant (DeltanorCB) exhibited greatly enhanced dispersal. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. We observed that exposure to SNP (500 nM) greatly enhanced the efficacy of antimicrobial compounds (tobramycin, hydrogen peroxide, and sodium dodecyl sulfate) in the removal of established P. aeruginosa biofilms from a glass surface. Combined exposure to both NO and antimicrobial agents may therefore offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.     

Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning

In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans. Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P. aeruginosa that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (hcnC) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant (egl-9) resistant to P. aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of P. aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium.     

Quantification of Pseudomonas aeruginosa hydrogen cyanide production by a polarographic approach

Pseudomonas aeruginosa is an opportunistic pathogen responsible for numerous infections acquired in hospital especially in persons whose immune systems are weakened, such as with patient suffering from AIDS or cystic fibrosis. This bacterium produces a great diversity of virulence factors among them hydrogen cyanide (HCN) which is one of the most potent and toxic. A precise quantification of HCN or CN(-) ion is essential to understand the involvement of this toxin in the pathogenesis of P. aeruginosa. In the present study, we present a new technique based on a polarographic approach to measure the production kinetics of HCN/CN(-) by P. aeruginosa strains, in several media commonly used in microbiology labs. The method was validated using mutants (hcnB- and hcnC-) which are unable to produce detectable HCN/CN(-). The kinetics of HCN/CN(-) production by P. aeruginosa in Luria Bertani (LB) medium showed a parabolic shape with a peak observed at 4, 5 and 8h for strains PA14, PAO1 and MPAO1, respectively. When bacteria were grown in ordinary nutrient broth (ONB) 2.5% medium, a less adapted medium for bacterial growth, the general profile of the kinetics was conserved but peak production was delayed (10 and 12h for PAO1 and MPAO1, respectively). When the bacteria were cultured in minimum medium MMC, bacterial growth was particularly slow and HCN/CN(-) production was markedly reduced. Taken together, this new polarographic method appears as a useful technique to detect and quantify HCN/CN(-) in routine media where the bacteria can express and regulate high amounts of toxins. With this method, we demonstrate that HCN/CN(-) production by P. aeruginosa is maximal at the end of the exponential growth phase and depends on the richness of the growth medium used.     

Peptidase activity and toxicity of strains of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a microorganism of the terrestrial and aquatic environment which may cause pulmonary, urinary and corneal infections. The pathogenesis of Pseudomonas-induced diseases seems to be linked to the production of extracellular substances such as exotoxins, hemolysins, leukocidins and hydrolytic enzymes, including various peptidases. Anyway there is not a clear view on the role other proteases have in the mechanism of pathogenesis, and even if their activity may always be associated to the toxicity of the microorganism. We have therefore determined the activity of a number of eso- and endopeptidases in 17 strains of P. aeruginosa isolated from patients with urinary pathologies. Four of these peptidase activities, namely elastase, neutral protease, aminopeptidase I and leucine aminopeptidase show a positive correlation with three parameters selected as indices of toxicity, i.e. the mucoid appearance, the gentamicin resistance and the adhesivity of colonies.     

Involvement of chromosomally-encoded genes in malathion utilization by Pseudomonas aeruginosa AA112

Malathion is an organophosphate insecticide that has been widely used for both domestic and commercial agricultural purposes. However, malathion has the potential to produce toxic effects in mammalian systems. In this study, Pseudomonas aeruginosa AA 112 which was isolated from soil using enrichment technique could utilize the malathion as a sole carbon source and a source of energy. Pseudomonas aeruginosa AA112 was able to grow in MSMPY medium containing 42.75 mg/ml malathion. However, the optimum concentration of malathion which supported the maximum bacterial growth was found to be 22. 8 mg/ml. Malathion was used as an initial source of energy and carbon when it was found without additional carbon sources (in MSM medium) while it was utilized as second source of energy and carbon in a nutrient-supplemented medium (in MSMPY medium). Moreover, lead acetate test indicated that malathion was first attacked at a sulphur site 1-2 hours after the start of incubation. TLC and IR analysis indicated that malathion was completely degraded into diethyl succinate, hydrogen sulphide and phosphates. Therefore a malathion degradation pathway was proporsed. The degradation of malathion is attributed to the genes located on the chromosome and at least three proteins of high molecular size might be involved in malathion utilization. Bacteria able to use malathion as a food source or metabolize its residues in the environment to inactive, less toxic, and harmless compounds, could be used in bioremediation of an environmental pollution caused by the pesticide.     

Phosphate transport in Pseudomonas aeruginosa. Involvement of a periplasmic phosphate-binding protein

A binding protein for inorganic phosphate was purified to apparent homogeneity from the shock fluids of phosphate-limited Pseudomonas aeruginosa. The purified protein bound one molecule of phosphate per molecule of binding protein with an average Kd of 0.34 microM. Arsenate, pyrophosphate and polyphosphates up to 15 units long could inhibit the binding of phosphate to the binding protein, although organic phosphates, such as glucose 6-phosphate, glycerol 3-phosphate and adenosine 5'-monophosphate could not. Mutants lacking the phosphate-binding protein were isolated and shown to be deficient in phosphate transport compared with wild-type cells. Two kinetically distinct systems for phosphate uptake could be observed in wild-type cells, with apparent Km values of 0.46 +/- 0.10 microM (high affinity) and 12.0 +/- 1.6 microM (low affinity). In contrast, only a single low-affinity transport system was observable in mutants lacking the binding protein (Km apparent = 19.3 +/- 1.4 microM Pi), suggesting the involvement of the binding protein in the inducible high-affinity phosphate-uptake system of P. aeruginosa.     

Involvement of DNA helicases in chromate resistance by Pseudomonas aeruginosa PAO1

Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted within the PAO1 genome showed that mutant strains GGP-64 and AJ-22 were affected in open reading frames PA0967 and PA5345, which correspond to the ruvB and recG genes, respectively. These genes encode helicases RuvB and RecG involved in DNA replication, recombination and repair. The chromate resistance phenotype in mutants GGP-64 and AJ-22 was restored by cosmids bearing wild type ruvB or recG genes, respectively. Also, both mutant strains showed an increased susceptibility to the toxic oxyanions tellurite and selenite as well as to mitomycin C, but not to arsenite, paraquat and hydrogen peroxide. It was concluded that P. aeruginosa RuvB and RecG helicases are involved in repairing DNA damage caused by chromate or its derivatives.     

Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa

According to the widely accepted view, neutrophil elastase (NE), a neutrophil-specific serine protease, is a major contributor to Pseudomonas aeruginosa infection-associated host tissue inflammation and damage, which in severe cases can lead to death. Herein, we provide for the first time compelling evidence that the host rather employs NE to protect itself against P. aeruginosa infection. Using a clinically relevant model of pneumonia, targeted deficiency in NE increased the susceptibility of mice to P. aeruginosa. We found that NE was required for maximal intracellular killing of P. aeruginosa by neutrophils. In investigating the mechanism of NE-mediated killing of P. aeruginosa, we found that NE degraded the major outer membrane protein F, a protein with important functions, including porin activity, maintenance of structural integrity, and sensing of host immune system activation. Consistent with this, the use of an isogenic mutant deficient in outer membrane protein F negated the role of NE in host defense against P. aeruginosa infection.     

Iron reductases from Pseudomonas aeruginosa

Cell-free extracts of Pseudomonas aeruginosa contain enzyme activities which reduce Fe(III) to Fe(II) when iron is provided in certain chelates, but not when the iron is uncomplexed. Iron reductase activities for two substrates, ferripyochelin and ferric citrate, appear to be separate enzymes because of differences in heat stabilities, in locations in fractions of cell-free extracts, in reductant specificity, and in apparent sizes during gel filtration chromatography. Ferric citrate iron reductase is an extremely labile activity found in the cytoplasmic fraction, and ferripyochelin iron reductase is a more stable activity found in the periplasmic as well as cytoplasmic fraction of extracts. A small amount of activity detectable in the membrane fraction seemed to be loosely associated with the membranes. Although both enzymes have highest activity reduced nicotinamide adenine dinucleotide, reduced glutathione also worked with ferripyochelin iron reductase. In addition, oxygen caused an irreversible loss of a percentage of the ferripyochelin iron reductase following sparge of reaction mixtures, whereas the reductase for ferric citrate was not appreciably affected by oxygen.