V. V. Kochetkov

Growth promotion of blackcurrant softwood cuttings by recombinant strain Pseudomonas fluorescens BSP53a synthesizing an increased amount of indole-3-acetic acid

Two isogenic Pseudomonas ftuorescens strains differing in plant hormone production were used to inoculate softwood cuttings. Production of bacterial indole-3-acetic add (IAA) was shown to have a stimulatory effect on the root development of blackcurrant softwood cuttings, whereas for sour cherry it was inhibitory. The size of the population of the inoculated strain on the root surface of the cuttings correlated with the effect observed.

[Biodegradation of phenanthrene by Pseudomonas bacteria bearing rhizospheric plasmids in model plant-microbial associations].

The consumption of phenanthrene in soil by model plant–microbial associations including natural and transconjugant plasmid-bearing rhizospheric strains of Pseudomonas fluorescens and P. aureofaciens degrading polycyclic aromatic hydrocarbons was studied. It was shown that phytoremediation of soil polluted with phenanthrene in the rhizosphere of barley (Hordeum sativum L.) was inefficient in the absence of the degrading strains. Inoculation of barley seeds with both natural and transconjugant plasmid-bearing Pseudomonas strains able to degrade polycyclic aromatic hydrocarbons (PAH) protected plants from the phytotoxic action of phenanthrene and favored its degradation in soil. Rape (Brassica napus L.) was shown to be an appropriate sentinel plant, sensitive to phenanthrene, which can be used for testing the efficiency of phenanthrene degradation in soil. Biological testing with the use of sensitive rape plants can be applied for estimation of the efficiency of phyto/bioremediation of PAH-polluted soils.

Genomic analysis of Pseudomonas putida phage tf with localized single-strand DNA interruptions.

The complete sequence of the 46,267 bp genome of the lytic bacteriophage tf specific to Pseudomonas putida PpG1 has been determined. The phage genome has two sets of convergently transcribed genes and 186 bp long direct terminal repeats. The overall genomic architecture of the tf phage is similar to that of the previously described Pseudomonas aeruginosa phages PaP3, LUZ24 and phiMR299-2, and 39 out of the 72 products of predicted tf open reading frames have orthologs in these phages. Accordingly, tf was classified as belonging to the LUZ24-like bacteriophage group. However, taking into account very low homology levels between tf DNA and that of the other phages, tf should be considered as an evolutionary divergent member of the group. Two distinguishing features not reported for other members of the group were found in the tf genome. Firstly, a unique end structure – a blunt right end and a 4-nucleotide 3′-protruding left end – was observed. Secondly, 14 single-chain interruptions (nicks) were found in the top strand of the tf DNA. All nicks were mapped within a consensus sequence 5′-TACT/RTGMC-3′. Two nicks were analyzed in detail and were shown to be present in more than 90% of the phage population. Although localized nicks were previously found only in the DNA of T5-like and phiKMV-like phages, it seems increasingly likely that this enigmatic structural feature is common to various other bacteriophages.

Arsenic-contaminated soils genetically modifiedPseudomonas spp. and their arsemc-phytoremediation potential

Sorghum was inoculated withPseudomonas bacteria, including strains harboring an As-resistance plasmid, pBS3031, to enhance As-extraction by the plants.Pseudomonas strains (P.fluorescens 38a, P.putida 53a, and P.aureofaciens BS1393) were chosen because they are antagonistic to a wide range of phyto-pathogenic fungi and bacteria, and they can stimulate plant growth. The resistance of natural rhizospheric pseudomonads to sodium arsenite was assessed. Genetically modifiedPseudomonas strains resistant to As(III)/As(V) were obtained via conjugation or transformation. The effects of the strains on the growth of sorghum on sodium-arsenite-containing soils were assessed. The conclusions from this study are: (1) It is possible to increase the survivability of sorghum growing in sodium-arsenite-containing soil by using rhizosphere pseudomonads. (2) The presence of pBS3031 offers the strains a certain selective advantage in arsenite-contaminated soil. (3) The presence of pBS3031 impairs plant growth, due to the As-resistance mechanism determined by this plasmid: the transformation of the less toxic arsenate into the more toxic, plant-root-available arsenite by arsenate reductase and the active removal of arsenite from bacterial cells. (4) Such a mechanism makes it possible to develop a bacteria-assisted phytoremediation technology for the cleanup of As-contaminated soils and is the only possible way of removing the soil-sorbed arsenates from the environment.

[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 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.

[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 versus 0.2 μg 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. tritici1818 in a medium containing 0.5 mM cobalt or nickel.

The cnr-like operon in strain Comamonas 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.

DNA homology and adsorption specificity of Pseudomonas aeruginosa virulent bacteriophages

SummaryThe DNA homology and adsorption specificity of newly isolated virulent bacteriophages of P. aeruginosa have been studied. On the basis of this analysis all phages were divided into four groups: φk, φm, φmnP78-like and φmnF82-like bacteriophages. DNAs of φk as well as φm phages were shown to possess different restriction patterns although they have an extensive homology. Unlike other groups, φk phages were characterized by the presence of T4 DNA ligase-repaired, single-chain breaks.

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.

Ratio [13C]/[12C] as an index for express estimation of hydrocarbon-oxidizing potential of microbiota in soil polluted with crude oil

The hydrocarbon-oxidizing potential of soil microbiota and hydrocarbon-oxidizing microorganisms introduced into soil was studied based on the quantitative and isotopic characteristics of carbon in products formed in microbial degradation of oil hydrocarbons. Comparison of CO2 production rates in native soil and that polluted with crude oil showed the intensity of microbial mineralization of soil organic matter (SOM) in the presence of oil hydrocarbons to be higher as compared with non-polluted soil, that is, revealed a priming effect of oil. The amount of carbon of newly synthesized organic products (cell biomass and exometabolites) due to consumed petroleum was shown to significantly exceed that of SOM consumed for production of CO2. The result of microbial processes in oil-polluted soil was found to be a potent release of carbon dioxide to the atmosphere.