Silver,copper, lead and zinc accumulation byPseudomonas stutzeri AG259 andStreptomyces albus : electron microscopy and energy dispersive X-ray studies

Metal accumulation by a silver-resistant Pseudomonas stutzeri AG259 strain and a Streptomyces albus strain was investigated in a mixed metal solution of silver, copper, lead and zinc. The location of silver, lead and copper on cells was determined by transmission electron microscopy coupled with an X-ray analysis system. In P. stutzeri cells silver was detected as dense deposits on the cells. Copper and lead were distributed over the cells. S. albus accumulated these metals only on part of cells with a higher concentration per cell than in P. stutzeri.     

Influence of nitrate starvation on nitrite accumulation during denitrification byPseudomonas stutzeri

Anaerobically denitrifyingPseudomonas stutzeri may transiently accumulate from 0% to 100% of nitrate as nitrite, depending on the nitrate availability during the preceding 24 h. The variations of transient nitrite level were related to the length of previous nitrate starvation. Cells harvested in a late anaerobic growth phase did not accumulate any nitrite during denitrification. Five hours of nitrate starvation caused about 40% (mol/mol) nitrite formation from the further added nitrate and 16 h nearly 80%. Concomitant with this, whereas the nitrate reduction capacity was not significantly affected, the initial nitrite reduction capacity was decreased. These results suggested that nitrate starvation caused a rapid loss of the originally produced nitrite-reducing capacity in the preculture. The lost capacity could be regenerated when nitrate or nitrite was resupplied to the cells. To investigate the nature of the restoration of nitrite-reducing capacity, chloramphenicol was used. The presence of chloramphenicol at 83 μg/ml entirely stopped this restoration, which was otherwise observed in all instances. This indicated that the recovery of nitrite-reducing activity required de novo protein synthesis, which was further confirmed by Western immunoblot assay of cd₁ nitrite reductase.     

Electroporation of pKK1 silver-resistance plasmid fromPseudomonas stutzeri AG259 intoPseudomonas putida CYM318

Plasmid pKK1 (49.4 Mda), which encodes for Ag⁺ resistance, was isolated fromPseudomonas stutzeri AG259 (pKK1) and introduced intoPseudomonas putida CYM318 by high-voltage electroporation. Upon acquiring pKK1,P. putida CYM318 became resistant to AgNO₃. This demonstrated that electroporation is a useful method to introduce a nonconjugative metal-resistance plasmid into a bacterial strain and to conduct further comparative research on Ag accumulation.     

Degradation of 2-chlorobenzoic and 2,5-dichlorobenzoic acids in pure culture byPseudomonas stutzeri

A strain ofPseudomonas stutzeri KS25 utilizing 2-chlorobenzoic and 2,5-dichlorobenzoic acids as the sole carbon and energy source was isolated from polychlorophenol-contaminated soil and sewage, using the method of enrichment cultures. This strain was also able to grow on 2-fluoro-, 2-iodo-, 2-bromo- and 2,5-dihydroxybenzoate, but did not utilize 3-, 4-chloro-, 2,4- and 2,6-dichlorobenzoates as the sole carbon and energy source, however, it cometabolized 3-chloro-, 2,4-and 2,6-dichlorobenzoates, but not 4-chlorobenzoate. The yield of released chlorine during utilization of 2-chloro- and 2,5-dichlorobenzoates amounted to 100 % of the theoretical. The concentration of 2-chloro- and 2,5-dichlorobenzoates, not substantially inhibiting the isolated microorganism, was within the range 0.25–0.5 and 2.5–3.0 g/L, respectively.     

Degradation of 2-chlorobenzoic and 2,5-dichlorobenzoic acids in soil columns byPseudomonas stutzeri

The heterocontinuous flow cultivation technique was used for the study of 2-chlorobenzoic and 2,5-dichlorobenzoic acid degradation in soil columns inoculated withPseudomonas stutzeri. 2-Chlorobenzoic and 2,5-dichlorobenzoic acids disappeared from the soil columns within 8 and 12 d, respectively. The presence of the haloaromatics increased the survival of strain KS25 in soil. Viable cell numbers in the soil columns flushed with 2-chlorobenzoic and 2,5-dichlorobenzoic acids were 1.3 and 2 times higher, respectively, than those without the chlorobenzoic acids after 30 d of incubation.     

Germanium accumulation by bacteria

Germanium accumulation was investigated in 23 bacterial strains. Bacillus strains accumulated the most Ge. Increasing the pH of the incubation medium from 7 to 8.5, as well as substituting catechol for glucose resulted in increased Ge accumulation. The apparent K _s and V _max of Ge accumulation in Bacillus cereus NRC 3045 were found to be 4.0 g/l and 2.2 mg/g dry wt/h, respectively. When cells from three different Bacillus strains were incubated in the presence of 2,4-dinitrophenol or toluene, Ge accumulation was completely inhibited. At 6° C, two out of three Bacillus strains showed a large decrease in Ge accumulation. In addition, non-viable Bacillus cells killed by UV irradiation did not accumulate Ge. These results strongly suggest that Ge accumulation by some Bacillus strains may be an energy-dependent process.     

Silver accumulation and resistance in Pseudomonas stutzeri

Silver (Ag) resistance and accumulation were investigated in Ag-resistant Pseudomonas stutzeri strain AG259 and Ag-sensitive P. stutzeri strain JM303. Both strains exhibited a similar pattern of silver accumulation although to different final concentrations. Energy-dispersive X-ray analyses revealed the association of dense silver deposits with the Ag-resistant strain, but not the Ag-sensitive strain. Toluene permeabilization or incubation of cells at 2°C resulted in decreased Ag accumulation in both strains. This suggests that Ag accumulation may be energy dependent. A decrease in Ag accumulation was observed when cells were pretreated with 2,4-dinitrophenol (2,4-DNP). No decrease was observed using carbonyl cyanide m-chlorphenyl-hydrazone (CCCP). However, it was observed that both 2,4-DNP and CCCP complexed to Ag, making interpretation of accumulation results difficult. Washing of cells incubated in the presence of Ag with ethylenediaminetetraacetic acid (EDTA) or hydrochloric acid did not result in decreased Ag accumulation.     

Threonine metabolism byPseudomonas aeruginosa

83 strains ofPseudomonas aeruginosa were unable to utilizel-threonine as carbon-energy source, although this compound served as sole nitrogen source. Auxotrophs ofP. aeruginosa 9-D2 that requiredl-serine or glycine for growth could grow in the presence ofl-threonine. Extracts ofP. aeruginosa 9-D2 grown in the presence ofl-threonine contained threonine dehydrogenase and alpha-amino beta-ketobutyrate: CoA ligase activities; threonine aldolase was not detectable. Cells grown in the absence ofl-threonine produced no detectable threonine dehydrogenase.l-Leucine neither stimulated nor repressed threonine dehydrogenase levels. Glycine, and to a lesser extentl-serine, repressedl-threonine-mediated threonine dehydrogenase synthesis. A mutant of strain 9-D2 was isolated that could utilizel-threonine as sole carbon-energy source. This strain produced elevated levels of threonine dehydrogenase, but only slightly higher levels of alpha-amino beta-ketobutyrate: CoA ligase activities.     

Germanium and silver resistance, accumulation, and toxicity in microorganisms.

Germanium is an inert metal with no known biological function in prokaryotic or eukaryotic organisms. Its toxicity is low compared to that of silver. Germanium is accumulated in certain bacterial strains by either energy-independent passive binding or an energy-dependent mechanism. Little is known about the molecular aspects of silver resistance, toxicity, and accumulation in bacterial strains. This is surprising because silver has been used as an antimicrobial agent in the medical field for centuries. It is likely that silver ions are excluded (resulting in decreased silver accumulation) from certain bacterial strains or immobilized intracellularly to prevent toxic effects from being exerted. These mechanisms of silver resistance have not been fully elucidated. This review examines the toxicity and accumulation of germanium and silver in selected microbial species. In addition, resistance mechanisms to these biologically nonessential metals is discussed, with more emphasis placed on silver-resistant bacteria due to the knowledge available.     

Degradation ofn-alkane-1-sulfonates byPseudomonas

We have isolated two strains ofPseudomonas, AJ 1 and AJ 2, growing on the C₄–C₇ and the C₈–C₁₂ n-alkane-1-sulfonates, respectively, as the only source of their carbon and energy. The alkane sulfonates are dissimilated by these strains, ultimately to yield carbon dioxide, water and sulfate. In the primary oxidation reaction(s) the corresponding fatty acid and sulfate are formed. The fatty acid is subsequently degraded by Β-oxidation. For strain AJ 1, growing on the lower alkane sulfonates, we could exclude various pathways of degradation which might have been present in addition to the one mentioned above.     

Uptake of volatilen-alkanes byPseudomonas PG-I

Using a bacterial speciesPseudomonas PG-1, evidence has been obtained which indicates that uptake ofn-pentane ton-octane by microbial cells takes place primarily from the gas phase either directly orvia the aqueous phase. Specific growth rate increased along with the increase in substrate concentration but above the alkane concentration of 0.3% by volume, specific growth rate decreased indicating substrate inhibition of growth. In the case of less volatile alkanes,n-nonane andn-decane, substrate transfer is predominantly through substrate solubilization system elaborated by the cells. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited growth on these two alkanes but had negligible effect on growth onn-pentane ton-octane.     

Genetics of alkane oxidation byPseudomonas oleovorans

Many Pseudomonads are able to use linear alkanes as sole carbon and energy source. The genetics and enzymology of alkane metabolism have been investigated in depth forPseudomonas oleovorans, which is able to oxidize C5-C12 n-alkanes by virtue of two gene regions, localized on the OCT-plasmid. The so-calledalk-genes have been cloned in pLAFR1, and were subsequent analyzed using minicell expression experiments, DNA sequencing and deletion analysis. This has led to the identification and characterization of thealkBFGHJKL andalkST genes which encode all proteins necessary to convert alkanes to the corresponding acyl-CoA derivatives. These then enter the -oxidation-cycle, and can be utilized as carbon- and energy sources. Medium (C6-C12)- or long-chain (C13-C20) n-alkanes can be utilized by many strains, some of which have been partially characterized. The alkane-oxidizing enzymes used by some of these strains (e.g. twoP. aeruginosa strains, aP. denitrificans strain and a marinePseudomonas sp.) appear to be closely related to those encoded by the OCT-plasmid.     

Production of xylonic acid byPseudomonas fragi

Summary Eleven microbial strains were tested for their ability to produce xylonic acid from xylose. The production of xylonic acid by one of the strains,Pseudomonas fragi ATCC 4973, was further studied in laboratory fermenter scale. The yield of xylonic acid was 92 % of original sugar. Xylonic acid production seemed to be growth associated and it was found to be very sensitive to the decrease of pH.     

Thetrans-eliminative breakdown of Na-polygalacturonate byPseudomonas fluorescens

Three strains ofPseudomonas fluorescens were isolated, which produced an inducible enzyme capable of converting Na-polygalacturonate into several reaction products which strongly absorbed ultraviolet light with a maximum at 232 mµ. Paper and column chromatography revealed that these products constituted a homologous series of oligo-uronides. The fastest moving compound appeared to be an unsaturated mono-uronic acid, 4-deoxy-l-threo-5-hexoseulose uronic acid, whereas indirect evidence showed the others to consist of the unsaturated mono-uronic acid linked by glycosidic bonding to 1, 2, 3, or 4 galacturonic acid moieties. Therefore, the enzyme concerned appears to be an endo-polygalacturonic acidtrans-eliminase. Enzyme extracts prepared from the isolates did not show a hydrolytic activity.The effects of some factors (pH, substrate and enzyme concentrations, inhibitory substances) on enzyme kinetics were investigated.Study of the literature on bacterial pectolytic enzymes suggests that many of these have been wrongly described as depolymerases, polygalacturonases, macerating enzymes, etc., and are better classified astrans-eliminases.     

A nonhemolytic phospholipase C produced byPseudomonas cepacia

Pseudomonas cepacia Pc224c, a nonhemolytic strain originally isolated from the sputum of a cystic fibrosis patient, produced an extracellular, heat-labile phospholipase C activity, which was measured quantitatively on the synthetic substratep-nitrophenylphophorylcholine. Cell-free supernatants from cultures grown to late log phase in MOPS-minimal salts-Tryptose medium contained specific activity at least 38 times greater than that from cultures grown in Tryptose minimal medium, Tryptic Soy broth, or peptone medium. Production was inhibited by the presence of inorganic phosphate in the medium and enhanced by aeration of the culture. The PLC ofP. cepacia is nonhemolytic and does not exhibit lecithinase activity on egg-yolk agar.     

Catabolism of naphthalene and salicylate byPseudomonas fluorescens

Analysis of spent naphthalene growth media ofPseudomonas fluorescens by GC-MS revealed the presence of salicylate. Gentisate 1,2-dioxygenase and pyrocatechol 1,2-dioxygenase were induced by growth on naphthalene, whereas only pyrocatechol 1,2-dioxygenase was induced during growth on salicylate. These results suggest the existence of alternative degradative routes of salicylate,via gentisate and pyrocatechol, which are involved in the catabolism of naphthalene.     

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 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.