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Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.

Kinetics↗

Pseudomonas sp. strain KC represents a new genomovar within Pseudomonas stutzeri.

Pseudomonas sp. strain KC (= ATCC 55595 = DSM 7136) is a denitrifying aquifer isolate that produces and secretes pyridine-2,6-bis(thiocarboxylate) (PDTC), a chelating agent that fortuitously transforms carbon tetrachloride without producing chloroform. Although KC has been used successfully for full-scale bioremediation of carbon tetrachloride, its taxonomy has proven difficult to resolve, as it retains properties of both Pseudomonas stutzeri and Pseudomonas putida. In the present work, a polyphasic approach was used to conclude that strain KC represents a new genomovar (genomovar 9) within the species P. stutzeri.

Biodegradation, Environmental↗

Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene.

Unlike enteric bacteria, Pseudomonas spp. generally lack thymidine phosphorylase and thymidine kinase activities, thus preventing their utilization of exogenous thymine or thymidine and precluding specific radioactive labeling of their DNA in vivo. To overcome this limitation, a DNA fragment encoding thymidine kinase (EC 2.7.1.21) from Escherichia coli was cloned into pKT230, a small, broad-host-range plasmid derived from plasmid RSF1010. From transformed E. coli colonies, the recombinant plasmid bearing the thymidine kinase gene was conjugally transferred to Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes. Thymidine kinase activity was expressed in all of these species, and all gained the ability to incorporate exogenous [2-14C]thymidine into their DNA. Thymidine incorporation into P. stutzeri was enhanced 12-fold more in mutants lacking thymidylate synthetase activity. These mutants produced higher levels of thymidine kinase and were thymidine auxotrophs; thymineless death resulted from removal of thymidine from a growing culture.

DNA Replication↗

Biology of Pseudomonas stutzeri.

Pseudomonas stutzeri is a nonfluorescent denitrifying bacterium widely distributed in the environment, and it has also been isolated as an opportunistic pathogen from humans. Over the past 15 years, much progress has been made in elucidating the taxonomy of this diverse taxonomical group, demonstrating the clonality of its populations. The species has received much attention because of its particular metabolic properties: it has been proposed as a model organism for denitrification studies; many strains have natural transformation properties, making it relevant for study of the transfer of genes in the environment; several strains are able to fix dinitrogen; and others participate in the degradation of pollutants or interact with toxic metals. This review considers the history of the discovery, nomenclatural changes, and early studies, together with the relevant biological and ecological properties, of P. stutzeri.

Biology↗

A suspected hospital outbreak of pseudobacteremia due to Pseudomonas stutzeri.

Pseudomonas stutzeri was recovered from blood cultures of 24 patients from 1977 through 1979 at one Mayo Clinic-affiliated hospital. During the investigation it was determined that aqueous green soap--used throughout the hospital to prepare the skin for iv insertions--had probably become contaminated with P. stutzeri. The use of aqueous green soap was discontinued, but eight additional cases of pseudobacteremia occurred in 1980-1981 and one case occurred in 1982. With one exception, all of the patients appeared to have pseudobacteremia rather than true bacteremia; the outbreak ceased only after aqueous green soap was deleted as a standard stock item from the hospital formulary.

Adolescent↗

Sodium-dependent growth and respiration of a nonhalophilic bacterium, Pseudomonas stutzeri.

Pseudomonas stutzeri (van Niel strain) requires Na+ for growth. Its growth rate was a sigmoidal function of Na+ concentration, being maximal and constant from 2 to 50 mM-Na+, and half maximal at about 0-5 mM-Na+. The relationship between cell concentration and Na+ concentration was non-linear; cell concentration increased abruptly when Na+ was greater than 0-3 mM. Accumulation of Na+ in the organism during growth was not detected. In the presence of K+, respiration was enhanced specifically by Na+. The respiration rate of the organism growing in the culture was a linear function of the growth rate when limited by the Na+ concentration, whereas the maximum rate induced by excess Na+ was independent of the growth rate.

Oxygen Consumption↗

Purification and characterization of two forms of maltotetraose-forming amylase from Pseudomonas stutzeri.

Pseudomonas stutzeri MO-19 produced two active forms of extracellular maltotetraose-forming amylase. Both forms, G4-1 and G4-2, were purified to electrophoretic homogeneity. The molecular masses of G4(-1) and G4(-2) were 57 kd and 46 kd by SDS-polyacrylamide gel electrophoresis, respectively. An identical N-terminal sequence up to 20 amino acid residues and similar amino acid compositions were obtained from both forms, but different C-terminal amino acids, leucine from G4(-1) and alanine from G4(-2), were released by carboxypeptidase Y. By in vitro incubation with a culture supernatant containing protease activity, G4(-1) was converted into G4(-2) without any loss of the amylase activity. It was concluded that G4(-2) was a product derived by the limited proteolysis of G4(-1), and that the proteolysis occurred in the C-terminal region of G4-1. G4-2 was more thermostable than G4(-1), and had a 20-fold higher Michaelis constant value for glycogen, which was 50 mg/ml against 2.3 mg/ml of G4(-1). G4(-1) adsorbed onto raw starch granules while G4(-2) did not.

Amino Acid Sequence↗

Comparison of dentrification by Paracoccus denitrificans, Pseudomonas stutzeri and Pseudomonas aeruginosa.

The course of denitrification of nitrate, nitrite and both compounds together by static cultures of Paracoccus denitrificans, Pseudomonas stutzeri and Pseudomonas aeruginosa was studied. These strains represent three different types of denitrification: 1. reduction of nitrate to gaseous nitrogen without accumulation of nitrite (P. denitrificans); 2. partial accumulation of nitrite in growing cultures during reduction of nitrate to gaseous nitrogen (P. aeruginosa) and 3. two-phase denitrification that includes reduction of nitrates at the very beginning of the process, and then, after depletion of the former, the reduction of nitrates to gaseous nitrogen (P. stutzeri). These observations differ from the results reported in the literature and possible reasons are discussed.

Nitrates↗

Metal binding by pyridine-2,6-bis(monothiocarboxylic acid), a biochelator produced by Pseudomonas stutzeri and Pseudomonas putida.

Pyridine-2,6-bis(monothiocarboxylic acid) (pdtc), a natural metal chelator produced by Pseudomonas stutzeri and Pseudomonas putida that promotes the degradation of carbon tetrachloride, was synthesized and studied by potentiometric and spectrophotometric techniques. The first two stepwise protonation constants (pK) for successive proton addition to pdtc were found to be 5.48 and 2.58. The third stepwise protonation constant was estimated to be 1.3. The stability (affinity) constants for iron(III), nickel(II), and cobalt(III) were determined by potentiometric or spectrophotometric titration. The results show that pdtc has strong affinity for Fe(III) and comparable affinities for various other metals. The stability constants (log K) are 33.93 for Co(pdtc)2(1-); 33.36 for Fe(pdtc)2(1-); and 33.28 for Ni(pdtc)2(2-). These protonation constants and high affinity constants show that over a physiological pH range the ferric pdtc complex has one of the highest effective stability constants for iron binding among known bacterial chelators.

Chelating Agents↗

Genotype versus phenotype in the circumscription of bacterial species: the case of Pseudomonas stutzeri and Pseudomonas chloritidismutans.

The phenotypic characteristic of strain AW-1(T) of Pseudomonas chloritidismutans that is most relevant from the taxonomic point of view appears to be the capacity of growth under anaerobic conditions using chlorate as electron acceptor. This property is not restricted to this species only within the genus Pseudomonas, since it is also present in strains of genomovars 1 or 5, and 3 of Pseudomonas stutzeri. P. chloritidismutans has been described as a non-denitrifying species, but the isolation of variants that are able to grow anaerobically in the presence of nitrate is possible after subcultivation under selective conditions. The subdivision of P. stutzeri into a number of species on the basis of these characteristics does not help to clarify the phylogenetic relationships among the members of an otherwise coherent group of strains, and the considerations presented in this communication support the reclassification of the new species name P. chloritidismutans, which in our opinion, should be considered as a Junior name of P. stutzeri. A multilocus sequence analysis, together with a phenotypic analysis of the anaerobic oxidative metabolism, gives new insights into the phylogeny and evolution of the species.

Chlorates↗

Comparative responses of Pseudomonas stutzeri and Pseudomonas aeruginosa to antibacterial agents.

The sensitivity of six strains of Pseudomonas stutzeri (NCIMB 568, 10783, 11358, 11359, JM 302, JM 375) to cationic antiseptics, mercury compounds, the parabens, phenolics, EDTA and various antibiotics was compared with Pseudomonas aeruginosa NCIMB 8626. All Ps. stutzeri strains were highly sensitive to chlorhexidine diacetate, organomercurials and triclosan, but rather less so to quarternary ammonium compounds (QACs). They were also sensitive to other biocidal agents and more sensitive to many antibiotics than the strain of Ps. aeruginosa. There was little correlation between uptake of chlorhexidine diacetate or cetylpyridinium chloride by dense suspensions of organisms, leakage of intracellular constituents and loss of cell viability.

Anti-Bacterial Agents↗

Exchange of chromosomal markers by natural transformation between the soil isolate, Pseudomonas stutzeri JM300, and the marine isolate, Pseudomonas stutzeri strain ZoBell.

Both the soil isolate, Pseudomonas stutzeri JM300, and the marine isolate, Pseudomonas stutzeri strain ZoBell, have been shown previously to be naturally transformable. This study reports the detection of genetic exchange by natural transformation between these two isolates. Transformation frequency was determined by filter transformation procedures. Three independent antibiotic resistance loci were used as chromosomal markers to monitor this exchange event: resistance to rifampicin, streptomycin, and nalidixic acid. The maximum frequencies of transformation were on the order of 3.1 to 3.8 x 10(-6) transformants per recipient; frequencies over an order of magnitude greater than those for spontaneous antibiotic resistance, although they are lower than those observed for soil:soil or marine:marine strain crosses. This exchange was inhibited by DNase I. Transformation was observed between soil and marine strains, both by filter transformation using purified DNA solutions and when transforming DNA was added in the form of viable donor cells. The results from this study support the close genetic relationship between P. stutzeri JM300 and P. stutzeri strain ZoBell. These results also further validate the utility of P. stutzeri as a benchmark organism for modeling gene transfer by natural transformation in both soil and marine habitats.

Deoxyribonuclease I↗

Development and application of a dapB-based in vivo expression technology system to study colonization of rice by the endophytic nitrogen-fixing bacterium Pseudomonas stutzeri A15.

Pseudomonas stutzeri A15 is a nitrogen-fixing bacterium isolated from paddy rice. Strain A15 is able to colonize and infect rice roots. This strain may provide rice plants with fixed nitrogen and hence promote plant growth. In this article, we describe the use of dapB-based in vivo expression technology to identify P. stutzeri A15 genes that are specifically induced during colonization and infection (cii). We focused on the identification of P. stutzeri A15 genes that are switched on during rice root colonization and are switched off during free-living growth on synthetic medium. Several transcriptional fusions induced in the rice rhizosphere were isolated. Some of the corresponding genes are involved in the stress response, chemotaxis, metabolism, and global regulation, while others encode putative proteins with unknown functions or without significant homology to known proteins.

Bacterial Proteins↗

Interaction between NifL and NifA in the nitrogen-fixing Pseudomonas stutzeri A1501.

Pseudomonas stutzeri strain A1501 isolated from rice fixes nitrogen under microaerobic conditions in the free-living state. This paper describes the properties of nifL and nifA mutants as well as the physical interaction between NifL and NifA proteins. A nifL mutant strain that carried a mutation non-polar on nifA expression retained nitrogenase activity. Complementation with a plasmid containing only nifL led to a decrease in nitrogenase activity in both the wild-type and the nifL mutant, suggesting that NifL acts as an antiactivator of NifA activity. Using the yeast two-hybrid system and purified protein domains of NifA and NifL, an interaction was shown between the C-terminal domain of NifL and the central domain of NifA, suggesting that NifL antiactivator activity is mediated by direct protein interaction with NifA.

Bacterial Proteins↗

Genetic characterization and evolutionary implications of a chromosomally encoded naphthalene-degradation upper pathway from Pseudomonas stutzeri AN10.

Pseudomonas stutzeri strain AN10 is a naphthalene-degrading strain whose dissimilatory genes are chromosomally encoded. We sequenced a total of 11514bp including the entire naphthalene-degradation upper pathway (nah) of P. stutzeri AN10. Nine open reading frames, nahAaAbAcAdBFCED, encoding the enzymes for the degradation of naphthalene to salicylate, were identified. The nah genes of P. stutzeri AN10 have been compared with genes encoding isofunctional proteins from other Pseudomonas naphthalene-degradation upper pathways. The implications of the sequence homologies to the evolution of aromatic catabolic pathways are discussed. Our findings indicate that this entire catabolic module of P. stutzeri AN10 was recruited from other microorganisms and a short period of time has elapsed after its incorporation within the P. stutzeri AN10 genome. Comparisons also suggest the coexistence of two entire nah upper pathways in a host strain, and further recombination between them. These events could accelerate the evolution of modern catabolic pathways.

Amino Acid Sequence↗

Organization and regulation of meta cleavage pathway genes for toluene and o-xylene derivative degradation in Pseudomonas stutzeri OX1.

Pseudomonas stutzeri OX1 meta pathway genes for toluene and o-xylene catabolism were analyzed, and loci encoding phenol hydroxylase, catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde dehydrogenase, and 2-hydroxymuconate semialdehyde hydrolase were mapped. Phenol hydroxylase converted a broad range of substrates, as it was also able to transform the nongrowth substrates 2,4-dimethylphenol and 2,5-dimethylphenol into 3,5-dimethylcatechol and 3,6-dimethylcatechol, respectively, which, however, were not cleaved by catechol 2,3-dioxygenase. The identified gene cluster displayed a gene order similar to that of the Pseudomonas sp. strain CF600 dmp operon for phenol catabolism and was found to be coregulated by the tou operon activator TouR. A hypothesis about the evolution of the toluene and o-xylene catabolic pathway in P. stutzeri OX1 is discussed.

Biodegradation, Environmental↗

Evolution of catabolic pathways and metabolic versatility in Pseudomonas stutzeri OX1.

Pseudomonas stutzeri OX1 is able to degrade toluene and ortho-xylene via the direct oxygenation of the aromatic ring. The genetic studies carried out suggest that the genes coding for the monooxygenase involved in the early steps of this catabolic route have been acquired by gene transfer. P stutzeri OX1 is also potentially able to utilize meta- and para-xylene as growth substrates. These two isomers are metabolized through a different pathway (TOL pathway). Both catabolic routes can be activated or inactivated by means of genomic rearrangements. The relevance of such recombination mechanisms in the evolution and the adaptability of P. stutzeri is discussed.

Biodegradation, Environmental↗

Tetrachloroethylene, trichloroethylene, and chlorinated phenols induce toluene-o-xylene monooxygenase activity in Pseudomonas stutzeri OX1.

Pseudomonas stutzeri OX1 naphthalene-oxidation activity is induced 3.0-fold by tetrachloroethylene (PCE) and 3.1-fold by trichloroethylene (TCE) at 100 microM. With the mutant P. stutzeri M1, which does not express toluene-o-xylene monooxygenase (ToMO, product of the tou operon), no naphthalene-oxidation activity induction by PCE and TCE was found; hence, PCE and TCE induce ToMO of P. stutzeri OX1. The chlorinated phenols 2-, 3-, and 4-chlorophenol induced ToMO expression 0.58-, 0.23- and 0.37-fold, respectively, compared to the direct inducer of the pathway, o-cresol. Using P. putida PaW340 (pPP4062, pFP3028), which has the tou promoter fused to the reporter catechol-2,3-dioxygenase, and the regulator gene touR, it was determined that the tou promoter was induced directly 5.7-, 7.1-, and 5.1-fold for 2-, 3-, and 4-chlorophenol, respectively (compared to an 8.8-fold induction with o-cresol). In addition, it was found that TCE and PCE do not directly induce the tou pathway and that components other than the tou structural and regulatory genes are necessary for induction. Gas chromatography results also showed that 100 microM TCE induced its own degradation (8-9%) in 16 h in P. stutzeri OX1, and all of the stoichiometric chloride from the degraded TCE was detected in solution.

Biodegradation, Environmental↗