Silver resistance in Pseudomonas stutzeri

Silver resistance was studied in a silver-resistant Pseudomonas stutzeri AG259 strain and compared to a silver-sensitive P. stutzeri JM303 strain. Silver resistance was not due to silver complexation to intracellular polyphosphate or the presence of low molecular weight metal-binding protein(s). Both the silver-resistant and silver-sensitive P. stutzeri strains produced H₂S, with the silver-resistant AG259 strain producing lower amounts of H₂S than the silver-sensitive JM303 strain. However, intracellular acid-labile sulfide levels were generally higher in the silver-resistant P. stutzeri AG259 strain. Silver resistance may be due to formation of silver-sulfide complexes in the silver-resistant P. stutzeri AG259 strain.     

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.     

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 3.8×10^-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 betweenP. stutzeri JM300 andP. stutzeri strain ZoBell. These results also further validate the utility ofP. stutzeri as a benchmark organism for modeling gene transfer by natural transformation in both soil and marine habitats.     

Muscodor albus, a potential biocontrol agent against plant-parasitic nematodes of economically important vegetable crops in Washington State, USA

The fungus, Muscodor albus, was tested for nematicidal and nematostatic potential against four plant-parasitic nematode species with three different feeding modes on economically important vegetable crops in the Pacific Northwest. Meloidogyne chitwoodi, Meloidogyne hapla, Paratrichodorus allius, and Pratylenchus penetrans were exposed for 72 h to volatiles generated by M. albus cultured on rye grain in sealed chambers at 24 °C in the laboratory. In addition, the nematodes were inoculated into soil fumigated with M. albus, and incubated for 7 days prior to the introduction of host plants under greenhouse conditions. The mean percent mortality of nematodes exposed to M. albus in the chamber was 82.7% for P. allius, 82.1% for P. penetrans, and 95% for M. chitwoodi; mortality in the nontreated controls was 5.8%, 7%, and 3.9%, respectively. Only 21.6% of M. hapla juveniles died in comparison to 8.9% in controls. However, 69.5% of the treated juveniles displayed reduced motility and lower response to physical stimulus by probing, in comparison to the control juveniles. This is evidence of nematostasis due to M. albus exposure. The greenhouse study showed that M. albus caused significant reduction to all nematode species in host roots and in rhizosphere soil. The percent mortality caused by M. albus applied at 0.5% and 1.0% w/w in comparison to the controls was as follows: 91% and 100% for P. allius in the soil; 100% for P. penetrans in bean roots and soil; 85% and 95% for M. chitwoodi in potato roots, and 56% and 100% in the soil; 100% for M. hapla both in pepper roots and soil. In this study, M. albus has shown both nematostatic and nematicidal properties.     

Immunochemical patterns of distribution of nitrous oxide reductase and nitrite reductase (cytochrome cd 1) among denitrifying pseudomonads

The novel multicopper enzyme nitrous oxide reductase from Pseudomonas perfectomarina was purified to homogeneity to study its properties and distribution in various pseudomonads and other selected denitrifying genera by immunochemical techniques. Quantitation of immunochemical crossreactivity by micro-complement fixation within the denitrifying pseudomonads of Palleroni's ribosomal ribonucleic acid group I corresponded to the taxonomic positions established by nucleic acid hybridization. The assignment of P. perfectomarina to the stutzeri-group (as strain ZoBell) was consolidated by immunochemical crossreactivity based on nitrous oxide reductase. Crossreactivity of nitrite reductase (cytochrome cd ₁) with a respective P. perfectomarina rabbit antiserum was limited to strain DSM 50227 of P. stutzeri; although it could not contribute information towards broader relationships within rRNA group I, it lent further prove to the unity of these two species.     

Detection and characterization of natural transformation in the marine bacterium Pseudomonas stutzeri strain ZoBell

Soil isolates of Pseudomonas stutzeri have been shown previously to acquire genes by natural transformation. In this study a marine isolate, Pseudomonas stutzeri strain ZoBell, formerly Pseudomonas perfectomarina, was also shown to transform naturally. Transformation was detected by the Juni plate method and frequencies of transformation were determined by filter transformation procedures. Maximum frequencies of transformation were detected for three independent antibiotic resistance loci. Transformation frequencies were on the order of 4×10^-5 transformants per recipient, a frequency over 100 times that of spontancous antibiotic resistance. Transfer of antibiotic resistance was inhibited by DNase I digestion. Marine isolates achieved maximum competence 14 h after transfer of exponential cultures to filters on solid media, although lower levels of competence were detected immediately following filter immobilization. Like soil isolates, P. stutzeri strain ZoBell is capable of cell contact transformation, but unlike soil isolates where transformation frequencies are greater for cell contact transformation as compared to transformation with purified DNA, the maximum frequency of transformation achieved by cell contact in the marine strain was approximately 10-fold less than transformation frequencies with purified DNA. These studies establish the first marine model for the study of natural transformation.This paper is dedicated to John L. Ingraham, Professor Emeritus of Microbiology at the University of California, Davis. Professor Ingraham was the first person to recognize natural transformation in Pseudomonas stutzeri and has continued to contribute to our understanding of the process over the past eight years. This understanding of the genetics of P. stutzeri is only one of the many areas of microbiology to which Professor Ingraham has contributed in his exceptional career     

Ferrihydrite reduction by Geobacter species is stimulated by secondary bacteria

Geobacter species such as G. bremensis, G. pelophilus, and G. sulfurreducens are obligately anaerobic and grow in anoxic, non-reduced medium by fast reduction of soluble ferric citrate. In contrast, insoluble ferrihydrite was either only slowly or not reduced when supplied as electron acceptor in similar growth experiments. Ferrihydrite reduction was stimulated by addition of a reducing agent or by concomitant growth of secondary bacteria that were physiologically and phylogenetically as diverse as Escherichia coli, Lactococcus lactis, or Pseudomonas stutzeri. In control experiments with heat-inactivated Geobacter cells and viable secondary bacteria, no (E. coli, P. stutzeri) or only little (L. lactis) ferrihydrite was reduced. Redox indicator dyes showed that growing E. coli, P. stutzeri, or L. lactis cells lowered the redox potential of the medium in a similar way as a reducing agent did. The lowered redox potential was presumably the key factor that stimulated ferrihydrite reduction by all three Geobacter species. The observed differences in anoxic non-reduced medium with ferric citrate versus ferrihydrite as electron acceptor indicated that reduction of these electron acceptors involved different cellular components or different biochemical strategies. Furthermore, it appears that redox-sensitive components are involved, and/or that gene expression of components needed for ferrihydrite reduction is controlled by the redox state.Dedicated to Prof. Dr. Dr. h.c. mult. Hans Günter Schlegel on the occasion of his 80th birthday.     

Control of green mold and sour rot of stored lemon by biofumigation with Muscodor albus

Control of postharvest lemon diseases by biofumigation with the volatile-producing fungus Muscodor albus was investigated. In vitro exposure to M. albus volatile compounds for 3 days killed Penicillium digitatum and Geotrichum citri-aurantii, causes of green mold and sour rot of lemons, respectively. Lemons were wound-inoculated with P. digitatum and placed in closed 11-L plastic boxes with rye grain cultures of M. albus at ambient temperature. There was no contact between the fungus and the fruit. Biofumigation for 24–72 h controlled green mold significantly, even when treatment began 24 h after inoculation. Effectiveness was related to the amount of M. albus present. In tests conducted inside a 11.7-m³ degreening room with 5 ppm ethylene at 20 °C, green mold incidence on lemons was reduced on average from 89.8 to 26.2% after exposure to M. albus for 48 h. Ethylene accelerates color development in harvested citrus fruit. M. albus had no effect on color development. Biofumigation in small boxes immediately after inoculation controlled sour rot, but was ineffective if applied 24 h later. G. citri-aurantii may be less sensitive to the volatile compounds than P. digitatum or escapes exposure within the fruit rind. Biofumigation with M. albus could control decay effectively in storage rooms or shipping packages.     

Periplasmic location of nitrous oxide reductase and its apoform in denitrifying Pseudomonas stutzeri

Immunogold labelling techniques on ultrathin sections of low temperature embedded cells yielded evidence for the periplasmic location of the respiratory enzymes N₂O reductase and nitrite reductase (cytochrome cd ₁) in Pseudomonas stutzeri strain ZoBell. Cell fractionation by spheroplast preparation and two-dimensional electrophoresis showed the absence of a membrane association of these enzymes. Immunocytochemical localization of N₂O reductase in a mutant strain deficient in the chromophore of N₂O reductase showed the gold label at the cell periphery, indicating that the copper chromophore processing takes place after export of this protein's apoform.     

A cytochrome cd 1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus

Respiratory nitrite reductase (NIR) has been purified from the soluble extract of denitrifying cells of Alcaligenes eutrophus strain H16 to apparent electrophoretic homogeneity. The enzyme was induced under anoxic conditions in the presence of nitrite. Purified NIR showed typical features of a cytochrome cd ₁-type nitrite reductase. It appeared to be a dimer of 60 kDa subunits, its activity was only weakly inhibited by the copper chelator diethyldithiocarbamate, and spectral analysis revealed absorption maxima which were characteristic for the presence of heme c and heme d ₁. The isoelectric point of 8.6 was considerably higher than the pI determined for cd ₁ nitrite reductases from pseudomonads. Eighteen amino acids at the N-terminus of the A. eutrophus NIR, obtained by protein sequencing, showed no significant homology to the N-terminal region of nitrite reductases from Pseudomonas stutzeri and Pseudomonas aeruginosa.     

Formate dependent nitrate and nitrite reduction to ammonia by Citrobacter freundii and competition with denitrifying bacteria

Citrobacter freundii, Paracoccus denitrificans and Pseudomonas stutzeri were grown either singly or in mixed culture in anaerobic nitrate or nitrite limited chemostats with formate and/or succinate as electron donors and carbon sources. C. freundii reduced nitrate or nitrite stoichiometrically to ammonia. Maximum molar growth yields for nitrate (nitrite) were 15.3 (9.9) g/mol for C. freundii on formate with succinate as carbon source, 15.3 (9.5) g/mol for Ps. stutzeri on succinate and 32.3 (20.4) g/mol for Pa. denitrificans on succinate. The almost identical growth yields indicate that the ATP output of the anaerobic processes in the nitrate (nitrite) ammonifying organism and Ps. stutzeri are nearly the same. In mixed cultures with either Ps. stutzeri or Pa. denitrificans, C. freundii was the best competitor for nitrate. These results show that in anaerobic environments C. freundii may compete successfully with denitrifying organisms.     

Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001

A strain YZN-001 was isolated from swine manure effluent and was identified as Pseudomonas stutzeri. It can utilise not only nitrate and nitrite, but also ammonium. The strain had the capability to fully remove as much as 275.08 mg L⁻¹ NO₃⁻–N and 171.40 mg L⁻¹ NO₂⁻–N under aerobic conditions. Furthermore, At 30 °C, the utilization of ammonium is approximately 95% by 18 h with a similar level removed by 72 h and 2 weeks at 10 and 4 °C, respectively. Triplicate sets of tightly sealed serum bottles were used to test the heterotrophic nitrifying ability of P. stutzeri YZN-001. The results showing that 39% of removed NH₄⁺–N was completely oxidised to nitrogen gas by 18 h. Indicating that the strain has heterotrophic nitrification and aerobic denitrification abilities, with the notable ability to remove ammonium at low temperatures, demonstrating a potential using the strain for future application in waste water treatment.     

Effect of chlorobenzoates on the degradation of polychlorinated biphenyls (PCB) by Pseudomonas stutzeri

Chlorobenzoic acids (CBA) are frequently dead-end products of partial aerobic biodegradation of polychlorinated biphenyls (PCB). When CBA produced from PCB accumulate in the growth medium, they can inhibit the bacterial growth and consequently, slow down PCB biodegradation. In this study, the effects of seven mono- and dichlorinated CBA on growth of Pseudomonas stutzeri on different substrates and on the PCB degradation by this strain in a liquid mineral medium were tested. 3-CBA was the strongest growth inhibitor for P. stutzeri growing on glucose, benzoate and biphenyl. It was found to inhibit heavily the elimination of some di- and trichlorinated biphenyls. In contrast, its influence on the elimination of more chlorinated congeners was much less significant.The authors are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Technical University, 812 37 Bratislava, Slovakia.     

Natural transformation of Pseudomonas stutzeri by plasmids that contain cloned fragments of chromosomal deoxyribonucleic acid

Natural transformation of plasmids by Pseudomonas stutzeri was found to depend on their bearing inserts of chromosomal DNA. A set of plasmids derived from the nonconjugative broad host range plasmid pSa151 was constructed by integrating various chromosomal DNA fragments into the single EcoR1 site of pSa151. Selection for the kanamycin resistance determined by pSa151 demonstrated that the derivative plasmids were taken into the cells by natural transformation and stably maintained; each could be reisolated unchanged. Thirty-two different derivative plasmids, 14 to 31 kbase pairs in size, all transformed. The frequency of transformation increased with the size of the chromosomal insert over a twenty fold range. These results suggest that the mechanism of transformation of plasmids by the Gram-negative P. stutzeri is similar to those proposed to operate in Gram-positive bacteria.Dedicated to Prof. Dr. H.-G. Schlegel on the occasion of his 60 th birthday     

Expression of the Alcaligenes eutrophus poly(β-hydroxybutyric acid)-synthetic pathway in Pseudomonas sp.

The 12.5-kb EcoRI restriction fragment PP1 of Alcaligenes eutrophus strain H16, which encodes for -ketothiolase, NADP-dependent acetoacetyl-CoA reductase and poly(-hydroxybutyric acid)-synthase was mobilized to six different species of the genus Pseudomonas belonging to the rRNA homology group I. Pseudomonas aeruginosa, P. fluorescens, P. putida, P. oleovorans, P. stutzeri and P. syringae, which are unable to synthesize and accumulate poly(-hydroxybutyric acid), PHB, were employed as recipients. Whereas the A. eutrophus PHB-synthetic enzymes were only marginally expressed in P. stutzeri, they were readily expressed in the other species. For example, the specific activity of PHB-synthase was 1.8 U/g protein in transconjugants of P. stutzeri but was between 21 and 77 U/mg protein in transconjugants of the other species. All recombinant strains harboring plasmid pVK101::PP1 except those of P. stutzeri accumulated PHB; the PHB content of the cells grown on gluconate under nitrogen limitation varied between 8 and 24.3% of the cellular dry mass.Abbreviations PHB poly(-hydroxybutyric acid) - PHA poly(hydroxyalkanoic acid)     

Natural genetic transformation of Pseudomonas stutzeri by sand-adsorbed DNA

In a soil/sediment model system we have shown recently that a gram-positive bacterium with natural competence (Bacillus subtilis) can take up transforming DNA adsorbed to sand minerals. Here we examined whether also a naturally transformable soil bacterium of the gramnegative pseudomonad (Pseudomonas stutzeri) can be transformed by mineral-associated DNA. for these studies the transformation protocol of this species was further improved and characterized. The peak of competence during growth of P. stutzeri was determined to occur at the beginning of the stationary phase. The competence state was conserved during shock freezing and thawing of cells in 10% glycerol. Kinetic experiments showed that transformant formation after addition of DNA to competent cells proceeded for more than 2 h with DNA adsorption to cells being the rate limiting step. By means of the defined protocol P. stutzeri was shown to be transformed by sand-adsorbed DNA. Transformation by adsorbed or dissolved DNA occurred between 16° and 44°C. Efficiency and DNaseI-sensitivity of transformation by DNA adsorbed to sand or in liquid were comparable. It is concluded that uptake of particle-bound DNA by P. stutzeri in soil is possible. This finding adds evidence to the view that transformation occurs in natural environments where DNA is assumed to be significantly associated with mineral/particulate material and thereby is protected against enzymatic degradation.     

Nitrate respiration in Pseudomonas stutzeri A15 and its involvement in rice and wheat root colonization

Unlike most bacteria, the nitrogen-fixing rice-associated Pseudomonas stutzeri A15 disposes of three different nitrate reductases that enable conversion of nitrate to nitrite through three physiologically distinct processes, called nitrate assimilation, nitrate respiration and nitrate dissimilation. To study the role of nitrate respiration in rhizosphere fitness, a Pseudomonas stutzeri narG mutant was constructed and characterized by assessing its growth characteristics and whole-cell nitrate reductase activity in different oxygen tensions. Unexpectedly, the Pseudomonas stutzeri A15 narG mutant appeared to be a better root colonizer, outcompeting the wild type strain in a wheat and rice hydroponic system.     

Anaerobic expression of nitric oxide reductase from denitrifying Pseudomonas stutzeri

NO reductase synthesis was investigated immunochemically and by activity assays in cells of Pseudomonas stutzeri ZoBell grown in continuous culture at discrete aeration levels, or in O₂-limited batch cultures supplemented with N oxides as respiratory substrate. Under aerobic conditions, NO reductase was not expressed in P. stutzeri. Oxygen limitation in combination with the presence of nitrate or nitrite derepressed NO reductase synthesis. On transition from aerobic to anaerobic conditions in continuous culture, NO reductase was synthesized below 3% air saturation and reached maximum expression under anaerobic conditions. By use of mutant strains defective in nitrate respiration or nitrite respiration, the inducing effect of individual N oxides on NO reductase synthesis could be discriminated. Nitrite caused definite, concentration-dependent induction, while nitrate promoted moderate enzyme synthesis or amplified effects of nitrite. Exogenous nitric oxide (NO) in concentrations 25 M induced trace amounts of NO reductase; in higher concentrations it arrested cell growth. Nitrite reductase or NO reductase were not detected immunochemically under these conditions. NO generated as an intermediate appeared not to induce NO reductase significantly. Antiserum raised against the P. stutzeri NO reductase showed crossreaction with cell extracts from P. stutzeri JM300, but not with several other denitrifying pseudomonads or Paracoccus denitrificans.     

Defects in cytochrome cd 1-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomonas stutzeri

Mutants with defective respiratory nitrite utilization (Nir^- phenotype) were obtained by transposon Tn5 insertion into genomic DNA of the ZoBell strain of Pseudomonas stutzeri. Three representative mutants were characterized with respect to their activities of nitrite and nitric oxide reduction, cytochrome cd ₁ content, and pattern of soluble c-type cytochromes. Mutant strain MK201 over-produced cytochrome c ₅₅₂ about fourfold by comparison with the wild type, but possessed an in vitro functional cytochrome cd ₁. Mutant strain MK202 lacked cytochrome cd ₁ and, simultaneously, had low amounts of cytochrome c ₅₅₂ and the split -peak c-type cytochrome. Strain MK203 synthesized nitrite reductase defective in the heme d ₁ prosthetic group. Irrespective of these biochemically distinct Nir^- phenotypes, all mutants preserved the nitric oxidereducing capability of the wild type. The mutant characteristics demonstrate that cytochrome cd ₁ is essential for nitrite respiration of P. stutzeri and establish the presence of a nitric oxide-reducing system distinct from cytochrome cd ₁. They also indicate the functional or regulatory interdependence of c-type cytochromes.     

A lower specificity PhaC2 synthase from Pseudomonas stutzeri catalyses the production of copolyesters consisting of short-chain-length and medium-chain-length 3-hydroxyalkanoates

A polyhydroxyalkanoate (PHA) synthase gene phaC2 _Ps from Pseudomonas stutzeri strain 1317 was introduced into a PHA synthase gene phbC _Re negative mutant, Ralstonia eutropha PHB⁻4. It conferred on the host strain the ability to synthesize PHA, the monomer compositions of which varied widely when grown on different carbon sources. During cultivation on gluconate, the presence of phaC2 _Ps in R. eutropha PHB⁻4 led to the accumulation of polyhydroxybutyrate (PHB) homopolymer in an amount of 40.9 wt% in dry cells. With fatty acids, the recombinant successfully produced PHA copolyesters containing both short-chain-length and medium-chain-length 3-hydroxyalkanoate (3HA) of 4–12 carbon atoms in length. When cultivated on a mixture of gluconate and fatty acid, the monomer composition of accumulated PHA was greatly affected and the monomer content was easily regulated by the addition of fatty acids in the cultivation medium. After the (R)-3-hydroxydecanol-ACP:CoA transacylase gene phaG _Pp from Pseudomonas putida was introduced into phaC2 _Ps-containing R. eutropha PHB⁻4, poly(3HB-co-3HA) copolyester with a very high 3-hydroxybutyrate (3HB) fraction (97.3 mol%) was produced from gluconate and the monomer compositions of PHA synthesized from fatty acids were also altered. This study clearly demonstrated that PhaC2_Ps cloned from P. stutzeri 1317 has extraordinarily low substrate specificity in vivo, though it has only 54% identity in comparison to a previously described low-substrate-specificity PHA synthase PhaC1_Ps from Pseudomonas sp. 61–3. This study also indicated that the monomer composition and content of the synthesized PHA can be effectively modulated by controlling the addition of carbon sources or by modifying metabolic pathways in the hosts.