The production of phenazine antibiotics by the Pseudomonas aureofaciens strain with plasmid-controlled resistance to cobalt and nickel

Plasmid pBS501 responsible for the resistance of the wild-type Pseudomonas sp. BS501 (pBS501) to cobalt and nickel ions was conjugatively transferred to the rhizosphere Pseudomonas aureofaciens strain BS1393, which is able to synthesize phenazine antibiotics and to suppress a wide range of phytopathogenic microorganisms. The transconjugant P. aureofaciens BS1393 (pBS501) turned out to be resistant to cobalt and nickel with an MIC of 8 mM. When grown in a synthetic medium with 0.25 mM cobalt, the transconjugant accumulated 6 times more cobalt than the wild-type strain BS501 (pBS501) (1.2 and 0.2 microgram Co/mg protein). Electron microscopic studies showed that cobalt accumulates on the surface of transconjugant cells in the form of electron-opaque granules. In a culture medium with 2 mM cobalt or nickel, strain BS1393 produced phenazine-1-carboxylic acid in trace amounts. The transconjugant P. aureofaciens BS1393 (pBS501) produced this antibiotic in still smaller amounts. Unlike the parent strain BS1393, the transconjugant P. aureofaciens BS1393 (pBS501) was able to suppress in vitro the growth of the phytopathogenic fungus Gaeumannomyces graminis var. tritici 1818 in a medium containing 0.5 mM cobalt or nickel.     

Effect of Sodium Salicylate on the Population Dynamics of the Rhizobacterium Pseudomonas aureofaciens in the Wheat Rhizoplane and Adjacent Soil

The effect of sodium salicylate on the population dynamics of the rhizobacterium Pseudomonas aureofaciens BS1393 and its variant bearing the naphthalene biodegradation plasmid pBS216 was studied in the wheat rhizoplane and adjacent soil. Optimum salicylate concentration for the maintenance of the plasmid-bearing strain and for the normal growth of wheat was found to be 250 g/g soil. When the soil was supplemented with salicylate, the population of P. aureofaciens BS1393(pBS216) in the wheat rhizoplane and adjacent soil was, respectively, 4- and 20-fold higher than that of the parent strain lacking the plasmid.     

The <Emphasis Type="Italic">cnr</Emphasis>-like operon in strain <Emphasis Type="Italic">Comamonas</Emphasis> sp. encoding resistance to cobalt and nickel

Plasmid pBS501 was detected in the strain Comamonas sp. BS501. This plasmid specifies high level of induced resistance (5 mM) to cobalt/nickel both in the host strain and in related strains C. testosteroni B-1241 and C. acidovorans B-1251. Hybridization analysis revealed a homology of pBS501 restriction fragments with the only well-characterized operon cnrXYHCBAT that resides in plasmid pMOL28 from Cupriavidus metallidurans CH34. Essential differences in the structural organization of the cobalt/nickel resistance determinant were found between plasmid pBS501 and the cnr operon.     

Resistance of certain strains of Pseudomonas bacteria to toxic effect of heavy metal ions]

The effect of heavy metal cations (cobalt, nickel, and copper) and the anion detergent sodium dodecyl sulfate (SDS) on the barrier properties of the plasma membrane (PM) of the Co-sensitive stain Pseudomonas putida BS394 and the Co-resistant strains Pseudomonas sp. BS501 (wild-type strain) and Pseudomonas putida BS394 (pBS501) (transformed strain) was studied by high-frequency electro-orientational spectroscopy. The cations were found to rank, in order of decreasing damage inflicted on the PM, as copper > cobalt > nickel. The strains studied were found to rank, in order of increasing resistance of the PM to damage inflicted by copper and cobalt cations, as P. putida BS394 < P. putida BS394 (pBS501) < Pseudomonas sp. BS501. In order of increasing resistance to SDS, the strains ranked inversely. The strains did not differ in sensitivity to nickel cations. Investigation of the surface of intact and trypsin-treated cells by microelectrophoresis showed that the surface layers of the cell wall of wild-type and transformed cells contained increased amounts of proteins. The surface proteins of Co-resistant cells had molecular masses of 49, 40, and 32 kDa. Exposure of Co-resistant cells to trypsin considerably reduced their resistance to cobalt cations. It is assumed that the resistance of the PM of the wild-type and transformed pseudomonads to heavy metal cations is determined by plasmid pBS501 and is related to the synthesis of protective surface proteins of the cell wall.     

Rhizosphere strain of <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> capable of degrading naphthalene in the presence of cobalt/nickel

Combination of genetic systems of degradation of polyaromatic hydrocarbons, resistance to heavy metals, and promotion of plant growth/protection is one of the approaches to the creation of polyfunctional strains for phytoremediation of soils after co-contamination with organic pollutants and heavy metals. A plant-growth-promoting rhizosphere strain Pseudomonas chlororaphis PCL1391 (pBS216^*, pBS501) has been obtained, in which the nah operon of plasmid pBS216 provides naphthalene biodegradation and the cnr-like operon of plasmid pBS501 provides resistance to cobalt and nickel due to the extrusion of heavy metal cations from the cells. In the presence of 100 μM of nickel, the viability, growth rate, and naphthalene biodegradation efficiency of the resistant strain PCL1391 (pBS216^*, pBS501) were much higher as compared with the sensitive PCL1391 (pBS216). During the growth of the resistant strain, in contrast to the sensitive strain, nickel (100 μM) had no inhibiting effect on the activity of the key enzymes of naphthalene biodegradation. Original Russian Text ? T.V. Siunova, T.O. Anokhina, A.V. Mashukova, V.V. Kochetkov, A.M. Boronin, 2007, published in Mikrobiologiya, 2007, Vol. 76, No. 2, pp. 212–218.     

Rhizosphere bacteria Pseudomonas aureofaciens and Pseudomonas chlororaphis oxidizing naphthalene in the presence of arsenic

Rhizosphere strains of P. aureofaciens BS1393(pBS216, pKS1) and P. chlororaphis PCL1391(pBS216, pKS1), exhibiting the ability to stimulate the growth of plants and protect them from phytopathogens, have been obtained. In these strains, plasmid pBS216 ensures naphthalene degradation and plasmid pKS1 confers resistance to arsenic. In the presence of arsenic and naphthalene, the number of living cells and the growth rate of the arsenic-resistant strains were higher than those of the arsenic-sensitive strains BS1393(pBS216) and PCL1391(pBS216). During the cultivation of the resistant strains, arsenic had no inhibitory effect on the activity of the key enzymes of naphthalene biodegradation, except for catechol-2,3-dioxygenase. In a model system containing plant-microbial associations, strains BS1393(pBS216, pKS1) and PCL1391(pBS216, pKS1) degraded as much as 97% of added naphthalene in the presence of arsenic.     

Effect of Wild and Genetically Modified Rhizosphere Bacteria Pseudomonas aureofaciens on the Accumulation of Arsenic by Plants

Gene constructions rendering recombinant bacteria resistant to arsenic and increasing their ability to dissolve soil phosphates and/or arsenates were created by cloning the ars operon and the gene of citrate synthase from a chromosome of the strain Pseudomonas aeruginosa PA01. Genetically modified variants of the strain Pseudomonas aureofaciens BS1393 have been constructed that are resistant to high concentrations of arsenic and dissolve poorly soluble phosphates and/or arsenates. The recombinant strains P. aureofaciens BS1393(pUCP22::arsRBC) and P. aureofaciens BS1393(pUCP22::gltA) exerted positive effects on the survival of sorgo (Sorghum saccharatum L.) and its ability to accumulate arsenic.     

Phenanthrene degradation by bacteria of the genera <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Burkholderia</Emphasis> in model soil systems

Degradation of phenanthrene by strains Pseudomona, Moscow, KMK, 2004simova, I.A. and Chernov, I.s putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) were studied in model soil systems. The differences in accumulation and uptake rate of phenanthrene intermediates between the strains under study have been shown. Accumulation of 1-hydroxy-2-naphthoic acid in soil in the course of phenanthrene degradation by strain BS3702 (pBS1143) in a model system has been revealed. The efficiency of phenanthrene biodegradation was assessed using the mathematical model proposed previously for assessment of naphthalene degradation efficiency. The efficiency of degradation of both phenanthrene and the intermediate products of its degradation in phenanthrene-contaminated soil is expected to increase with the joint use of strains P. Putida BS3701 (pBS1141, pBS1142) and Burkholderia sp. BS3702 (pBS1143).     

Effect of rhizosphere bacteria <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> on the resistance of micropropagated plants to phytopathogens

Effects of colonization of micropropagated potato (Solanum tuberosum L.) and strawberry (Fragaria L.) plants by the rhizosphere bacterium Pseudomonas aureofaciens strain BS1393 (VKM B-2188 D) on plant growth and resistance to bacterial and fungal phytopathogens were studied. Pseudomonad colonization improved the physiological characteristics of plants and enhanced their adaptation to in vivo conditions. The presence of P. aureofaciens cells in various plant tissues (leaves, stems, and roots) in vitro was demonstrated on the background of plant cocolonization by two associative strains—P. aureofaciens strain BS1393 (VKM-B-2188 D) and Methylovorus mays (VKM-B-2221). The colonized plants displayed an increased resistance to the phytopathogens Erwinia carotovora, Sclerotinia sclerotiorum, and Phytophthora infestans. These results demonstrate that pseudomonades are promising for practical application in the microbial protection of plants against phytopathogens.     

Rhizosphere strain of Pseudomonas chlororaphis capable of degrading naphthalene in the presence of cobalt/nickel

Combination of genetic systems of degradation of polyaromatic hydrocarbons, resistance to heavy metals, and promotion of plant growth/protection is one of the approaches to the creation of polyfunctional strains for phytoremediation of soils after combined contamination with organic pollutants and heavy metals. A plant-growth-promoting rhizosphere strain Pseudomonas chlororaphis PCL1391 (pBS216*, pBS501) has been obtained, in which the nah operon of plasmid pBS216 provides naphthalene biodegradation and the cnr-like operon of plasmid pBS501 provides resistance to cobalt and nickel due to the withdrawal of heavy metal cations from the cells. In the presence of 100 microM of nickel, the viability, growth rate, and naphthalene biodegradation efficiency of the resistant strain PCL1391 (pBS216*, pBS501) were much higher as compared with the sensitive PCL1391 (pBS216). During the growth of the resistant strain, in contrast to the sensitive strain, nickel (100 microM) had no inhibiting effect on the activity of the key enzymes of naphthalene biodegradation.     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10^?5–10^?4 per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

Effect of sodium salicylate on the population dynamics of a rhizospheric strain of Pseudomonas aureofaciens in soil and on wheat roots

The effect of sodium salicylate on the population dynamics of the rhizobacterium Pseudomonas aureofaciens BS1393 and its variant bearing the naphthalene biodegradation plasmid pBS216 was studied in the wheat rhizoplane and adjacent soil. Optimum salicylate concentration for the maintenance of the plasmid-bearing strain and for the normal growth of wheat was found to be 250 micrograms/g soil. When the soil was supplemented with salicylate, the population of P. aureofaciens BS1393(pBS216) in the wheat rhizoplane and adjacent soil was, respectively, 4- and 20-fold higher than that of the parent strain lacking the plasmid.     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Isolation of a Tn501 insertion mutant lacking porin protein P of Pseudomonas aeruginosa

Summary In order to demonstrate a role for anion-specific protein P channels in phosphate transport in Pseudomonas aeruginosa PAO, we wished to isolate a transposon insertion mutant deficient in protein P. A number of transposon delivery systems were tested which yielded, for the most part, whole plasmid inserts. Plasmid pMT1000 (Tsuda et al. 1984), a temperature-sensitive R68 plasmid carrying the transposon Tn501, was successfully employed in the isolation of a Tn501 insertion mutant lacking protein P under normally inducing conditions. To identify the mutant deficient in protein P, a protein P-specific polyclonal antiserum was used. This mutant, strain H576, was deficient in high-affinity phosphate transport exhibiting a Km for uptake (3.60±0.64 M) almost ten times greater than that of the wild type strain (Km=0.39 M). There was, however, no change in the V_max for high-affinity phosphate transport as a result of the loss of protein P in this mutant. The protein P-deficiency of the mutant correlated with a growth defect in a phosphate-limited medium resulting in an 18%–35% decrease in growth when compared with the wild type.     

Inhibitor effects on the accumulation and efflux of nickel ions in plasmid pMOL28-harboring strains of Alcaligenes eutrophus

The accumulation and efflux of ⁶³Ni²⁺ ions were studied using two strains of the strictly respiratory bacterium Alcaligenes eutrophus, the wild type strain N9A and its transconjugant N9A-M243 which harbors plasmid pMOL28.1 encoding constitutive resistance to nickel. When 1 M ⁶³Ni²⁺ is added to respiring cells, strain N9A accumulates high, but M243 only negligibly small amounts of nickel. When the cells were preincubated for about 20 h under anoxic conditions and were then exposed to 1 M ⁶³Ni²⁺ anaerobically, both strains accumulated approximately the same amounts of nickel. Aeration of these preloaded cells resulted in rapid efflux by strain M243 but renewed uptake of nickel by N9A. ⁶³Ni²⁺ uptake and efflux are highly sensitive to protonophores such as CCCP, FCCP and TCS but insensitive to DCCD (each at 20 M concentrations). Measurements on the effects of the inhibitors on biosynthetic processes requiring ATP either from substrate phosphorylation or from electron transport phosphorylation made sure that at the concentrations used the inhibitor effects were specific. Thus the results suggest that in the nickelresistant strain M243 under normal aerobic conditions two constitutive energy-dependent nickel transport systems can function concomitantly, a chromosomally-determined specific uptake system and a plasmid-mediated specific nickel efflux system.     

Use of glucose and carbon isotope fractionation by microbial cells immobilized on solid-phase surface

By the example of glucose uptake by the soil bacteria Pseudomonas aureofaciens BS1393(pBS216) and Rhodococcus sp. 3–30 immobilized on a solid-phase surface (quartz sand), their growth parameters were determined: growth rate (doubling time), total CO₂ production, CO₂ production per cell, lag period with respect to substrate uptake, respiratory quotient. The growth of P. aureofaciens and Rhodococcus sp. on glucose revealed (1) differences of the lag period with respect to substrate (lag time of ～4 h for P. aureofaciens and ～26 h for Rhodococcus sp.); (2) differences between the maximal rates of CO₂ production (～50 μg C-CO₂ g^?1 sand h^?1 for P. aureofaciens and ～8.5 μg C-CO₂ g^?1 sand h^?1 for Rhodococcus sp.); (3) differences in CO₂ production per cell (～1.94 × 10^?9 μM CO₂/CFU for P. aureofaciens and more than ～3.4 × 10^?9 μM CO₂/CFU for Rhodococcus sp.). The kinetics of the metabolic CO₂ isotopic composition was shown to be determined by the difference in the carbon isotopic characteristics of products in the cell. Upon introduction of glucose into the medium (the preparatory stage of the metabolism), the uptake of intracellular ¹³C-depleted products (lipids) is noted; at the stage of the maximal cell growth rate, introduced glucose is mainly metabolized; and at the final stage, upon exhaustion of substrate, the “stored” products—the lipid fraction—get involved in the metabolism. At the maximal rate of glucose uptake, the CO₂ carbon isotopic fractionation coefficient relative to organic products of microbial biosynthesis was determined to be α = 1.009 ± 0.002.     

Mutation in <Emphasis Type="Italic">cyaA</Emphasis> in <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> decreases cucumber root colonization

Strains of Enterobacter cloacae show promise as biological control agents for Pythium ultimum-induced damping-off on cucumber and other crops. Enterobacter cloacae M59 is a mini-Tn5 Km transposon mutant of strain 501R3. Populations of M59 were significantly lower on cucumber roots and decreased much more rapidly than those of strain 501R3 with increasing distance from the soil line. Strain M59 was decreased or deficient in growth and chemotaxis on most individual compounds detected in cucumber root exudate and on a synthetic cucumber root exudate medium. Strain M59 was also slightly less acid resistant than strain 501R3. Molecular characterization of strain M59 demonstrated that mini-Tn5 Km was inserted in cyaA, which encodes adenylate cyclase. Adenylate cyclase catalyzes the formation of cAMP and cAMP levels in cell lysates from strain M59 were approximately 2% those of strain 501R3. Addition of exogenous, nonphysiological concentrations of cAMP to strain M59 restored growth (1 mM) and chemotaxis (5 mM) on synthetic cucumber root exudate and increased cucumber seedling colonization (5 mM) by this strain without serving as a source of reduced carbon, nitrogen, or phosphorous. These results demonstrate a role for cyaA in colonization of cucumber roots by Enterobacter cloacae.     

Transformation of low-molecular linear caprolactam oligomers by the caprolactam-degrading bacterium <Emphasis Type="Italic">Pseudomonas putida</Emphasis> BS394(pBS268)

A biosensor based on the most active caprolactam-degrading strain Pseudomonas putida BS394(pBS268) was used in the study of aerobic degradation of linear caprolactam oligomers by bacterial cells. The changes in the respiratory activity of the strain depend quantitatively on caprolactam dimer concentration, making it possible to develop biosensors for detection of caprolactam oligomers in aqueous media. Based on mass spectrometry data, the scheme of transformation of linear caprolactam oligomers by the degrader strain P. putida BS394(pBS268) was proposed for the first time. It was found that oxidative transamination to respective dicarbonic acids may be one of the mechanisms of transformation of linear caprolactam oligomers. According to the scheme proposed, the ability of the caprolactam-degrading strain to transform linear oligomers results from the broad substrate specificities of two enzymes of the caprolactam degradation pathway: 2-oxoglutarate-6-aminohexanoate transaminase and 6-oxohexanoate dehydrogenase. Transformation of linear oligomers is genetically controlled by the CAP biodegradation plasmid pBS268.     

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd 1-free background (NirS−) of Pseudomonas stutzeri

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of Achromobacter cycloclastes nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd ₁ nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d ₁, indicating that both heterologous hosts synthesized a cytochrome cd ₁ without the d ₁-group.Abbreviations Cu-NIR Cu-containing nitrite reductase - DDC diethyldithiocarbamate - EPR electron paramagnetic resonance - IPTG isopropyl--D-galactoside - SDS sodium dodecyl sulfate - LB medium Luria-Bertani medium     

Nickel requirement for carbon monoxide dehydrogenase formation in Clostridium pasteurianum

Formation of carbon monoxide dehydrogenase in growing Clostridium pasteurianum was found to be dependent on trace nickel present as contaminant in the growth medium. The evidence is: i) Synthesis of the enzyme was increased, when NiCl₂ (0.1 M) was added to the medium; ii) Synthesis of the enzyme was almost completely inhibited when the cells were grown in the presence of nitrilotriacetate (0.1 mM) or of other chelating agents, which inhibited the uptake of trace nickel from the medium; iii) Inhibition of enzyme synthesis by the chelators could be specifically overcome by supplementing the medium with nickel (1M).