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[Study of the integral toxicity of aqueous media, polluted by petroleum and petroleum products using bacterial tests].

A biotest kit was used to assess the integral toxicity level of aquatic medium contamination with petroleum and petroleum-based products. The integral toxicity dynamics was also monitored during biodegradation of petroleum and petroleum-based products by an association of petroleum-degrading strains including Acinetobacter sp., Mycobacterium flavescens, and Rhodococcus sp. The following bacterial tests were used: the bioluminescence (BL) test based on Photobacterium leiognathi; electro-orientation (EO), optoosmotic (OO), and growth test; as well as the reducing activity (RA) test based on the Agrobacterium radiobacter culture. No significant increase in the integral toxicity level of aquatic medium was observed when diesel fuel and kerosene contamination had been subjected to biodegradation. Although express biotests (EO, OO, RA, and BL) detected a pronounced increase in the integral toxicity of aquatic medium, long-term growth biotest revealed no statistically significant increase in the toxicity level.

Acinetobacter↗

[Formation of artificial nitrogen-fixing symbiosis with rape (Brassica napus var. napus) plants in nonsterile soil].

The treatment of rape plants grown in nonsterile soil with 2,4-dichlorophenoxyacetic acid (auxin-like growth-promoting substance) or their inoculation with the bacterial association Micrococcus sp. + Rhodococcus sp. and/or with the mixed nitrogen-fixing culture Azotobacter nigricans + Bacillus sp. led to the formation of paranodules on the rape roots. The introduced bacteria were detected both in the intercellular space and inside the cells of the paranodules and the rape roots. The nitrogen-fixing activity of the paranodulated plants was two times higher than that of the inoculated plants lacking paranodules and five times higher than that of the control (i.e., not inoculated) plants. The paranodulation led to a 40% increase in the crop yield of rape plants and provided for a statistically significant increase in the total nitrogen as well as protein nitrogen contents of the plants.

2,4-Dichlorophenoxyacetic Acid↗

Structure-specificity relationships for haloalkane dehalogenases.

A structural analysis of the substrate specificity of hydrolytic dehalogenases originating from three different bacterial isolates has been performed using the multiple computer-automated structure evaluation methodology. This methodology identifies structural fragments in substrate molecules that either activate or deactivate biological processes. The analysis presented in this contribution is based on newly measured dehalogenation data combined with data from the literature (91 substrates). The enzymes under study represent different specificity classes of haloalkane dehalogenases (haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, Rhodococcus erythropolis Y2, and Sphingomonas paucimobilis UT26). Three sets of structural rules have been identified to explain their substrate specificity and to predict activity for untested substrates. Predictions of activity and inactivity based on the structural rules from this analysis were provided for those compounds that were not yet tested experimentally. Predictions were also made for the compounds with available experimental data not used for the model construction (i.e., the external validation set). Correct predictions were obtained for 28 of 30 compounds in the validation set. Incorrect predictions were noted for two substrates outside the chemical domain of the set of compounds for which the structural rules were generated. A mechanistic interpretation of the structural rules generated provided a fundamental understanding of the structure-specificity relationships for the family of haloalkane dehalogenases.

Biodegradation, Environmental↗

[Effect of the media salinity on destruction of petroleum oils by nocardioform bacteria].

Oil degradation by cultures of Rhodococcus erythropolis and Dietzia maris was found to depend on the NaCl concentration in the medium. Optimal utilization of turbine oil by R. erythropolis and D. maris was observed at 0.5 and 2 to 5% NaCl concentration, respectively. Mineral oil and a mixture of paraffins (C14-C18) were utilized within a broader range of the medium salinity. As shown by fluorescent microscopy, D. maris colonies formed on the oil drop surface, whereas R. erythropolis cells penetrated the drops. The strains studied may populate various ecological niches in oil-containing ecosystems. They are promising for the development of microbial preparations for cleaning the environment from oil pollution.

Actinomycetales↗

[Antagonistic interactions between stress factors during the growth of microorganisms under conditions simulating the parameters of their natural ecotopes].

Two stress factors, hypoxia (microaerobic conditions) and a high salt concentration, if applied simultaneously to aerobic microorganisms, display an antagonistic mode of interaction. As a result, the NaCl level that is usually optimal for moderate halophiles (5-6%) becomes optimal for the growth of weak halophiles (Rhodococcus erythropolis and Shewanella sp. CN32); the halotolerant yeast Yarrowia lypolytica acquires halophilic properties (with a growth optimum at a NaCl concentration of 10%), and the growth rate of the extremely halophilic Halobacterium salinarum increases at supraoptimal salt concentrations (25-34%). This phenomenon is apparently due to multiple changes in metabolic reactions. In particular, high salt concentrations suppress respiration and the formation of enzymes (superoxide dismutase and catalase) that protect the cell from toxic oxygen species. Therefore, establishment of microaerobic conditions compensates for the loss of these protective mechanisms and enables cell growth at higher salt concentrations than under aerobic conditions. Of some importance can also be the increase in the intracellular concentrations of osmoprotectants caused by the suppression of their intracellular oxidation. The implications of this phenomenon for the ecophysiology of microorganisms (including oiloxidizing species) and for the classification of weak and moderate halophiles are discussed.

Bacteria↗

Identification of the source of faecal pollution in contaminated rivers.

Identifying the source of faecal pollution is important to enable appropriate management of faecal pollution of water. Four independent assays for faecal source discrimination have been developed and implemented in our lab. These assays detect fluorescent whitening agents, faecal sterols, Bifidobacterium adolescentis and Rhodococcus coprophilus. The combination of these indicators is able to identify human derived faecal pollution in rivers containing inputs from septic tanks, municipal oxidation ponds, farmed animals and feral animals.

Agriculture↗

[Effect of shale kerogen oxidation products on biodegradation of petroleum and petroleum products in soil and water].

Effect of oxidation products of shale kerogen (high-molecular-weight acids with 6-22 carbon atoms) on biodegradation of oil and oil products in soil and water was studied. High-molecular-weight acids (HMWA) not only affected the layer of oil and/or oil products and dispersed it into small particles, but also stimulated growth of Rhodococcus erythropolis VKM AS-1339D, degraders of oil and oil products. Addition of 0.001-0.003% HMWA to a medium to be purified from oil products increased the extent of bacterial biodegradation by a factor of 1.1-5.0.

Acids↗

The use of chemical and molecular microbial indicators for faecal source identification.

Identifying the source of faecal pollution is important to enable appropriate management of faecal pollution of water. We are developing and evaluating a combination of these microbial and chemical indicators better able to identify the source of faecal pollution. These assays make use of a combination of direct PCR, culturing, and colony hybridisation to identify source specific species of Bifidobacterium, Rhodococcus and Bacteroides. In conjunction with assays for (a) fluorescent whitening agents and (b) faecal sterols and stanols, these indicators were able to identify human derived faecal pollution in river water containing inputs from septic tanks, municipal oxidation ponds, farmed animals and feral animals. Differentiating amongst the animal sources was more difficult and will require development of molecular assays for organisms specific to each animal group.

Agriculture↗

[The effect of physicochemical environmental factors on the growth of gram-positive bacterial dissociants].

The growth of R-, S- and M (g)-dissociants of Streptococcus lactis, Bacillus coagulans, Rhodococcus rubropertinctus under the action of some physico-chemical factors: temperature, pH, ultraviolet (UV) rays, high concentration of NaCl and storage have been compared. R-variants gain selective advantage under the influence of UV-irradiation, high temperature and storage; S-variants--at decreasing of active pH of medium; M (g)-variants--at decreasing of growth temperature, high values of pH, increased NaCl concentration. The dissociation has been concluded to enlarge the limits of the species survival.

Bacillus↗

[The quantity and structure of the root-associated microbial complexes of two greenhouse rose cultivars].

The study of the root-associated microbial complexes of affected and healthy rose plants of two cultivars (Grand gala and Royal velvet) grown in a greenhouse showed that the biomass of eukaryotic microorganisms in the rhizoplane and rhizosphere of healthy rose plants and in the surrounding soil was considerably lower than in the same loci of affected plants. In contrast, the biomass of root-associated prokaryotic microorganisms was higher in the case of healthy than in the case of affected rose plants. The root-associated bacterial complexes of both affected and healthy rose plants were dominated by the genera Arthrobacter, Rhodococcus, and Myxobacterium and did not contain phytopathogenic bacteria. The root-associated fungal complex of healthy roses was dominated by fungi of the genus Trichoderma, whereas that of the affected rose plants was dominated by the species Aureobasidium microstictum. The affected cane cuttings and cankers occurring on affected canes were found to contain Coniothyrium fuckelii (the causal fungus of rose stem canker) and sclerotia of Botrytis cinerea (the causal fungus of gray rot). The micromycete complex of healthy rose plants was not so diverse as was the micromycete complex of affected rose plants.

Arthrobacter↗

[Intensification of microbial degradation of crude oil and oil products in the presence of perfluorodecalin].

The possibility of using perfluorinated organic compounds for growing microorganisms and degrading xenobiotics has been demonstrated for the first time with perfluorodecalin (PFD), a gas-transporting component of the blood substitute Perftoran. In particular, this is promising for intensifying microbial degradation of oil and oil products and production of biodegrader biomass in synthetic mineral media. Addition of PFD to a mineral medium with crude oil and masut increased 4.5-10.2 times maximum concentrations and growth rates of all bacterial strains under study (Pseudomonas, Rhodococcus, and Bacillus genera). The degree of oil product consumption was increased 8.7-12.7 times.

Bacillus↗

In vitro activity of teicoplanin and vancomycin against gram-positive bacteria from human clinical and veterinary sources.

The minimum inhibitory concentration (MIC) of teicoplanin and vancomycin was determined by the agar dilution method for 186 Gram-positive bacteria from human clinical and veterinary sources. Teicoplanin MIC values were less than or equal to 4 micrograms/ml for 94% of staphylococci (group A, n = 52) and less than or equal to 2 micrograms/ml for all streptococci, enterococci, aerococci and pediococci (group B, n = 75). Seventy-eight percent of Gram-positive rods, Rhodococcus and Leuconostoc spp. (group C, n = 59) were inhibited by 4 micrograms/ml. Teicoplanin resistance (MIC greater than or equal to 16 micrograms/ml) was demonstrated for all Nocardia strains and for some strains of Lactobacillus, E. rhusiopathiae, Leuconostoc, and S. haemolyticus. Cross-resistance between teicoplanin and vancomycin was observed for all Nocardia strains and for some strains of Lactobacillus, E. rhusiopathiae, and Leuconostoc. Three methicillin-resistant S. haemolyticus strains were either resistant or intermediately susceptible to teicoplanin and susceptible to vancomycin. Eight strains (motile enterococci four, E. rhusiopathiae three and Leuconostoc sp. one) were susceptible to teicoplanin and resistant to vancomycin. Teicoplanin disc diffusion on Danish Blood Agar with NeoSensitabs (Rosco), PDM AB Biodisc and locally prepared discs revealed a wide range of zone diameters in groups B and C. The relation between MIC values and zone diameters for teicoplanin was analysed by the error-rate bounded method. Zone size interpretive criteria as suggested by the manufacturers (greater than or equal to 15 mm) produced 2.7% (95% confidence limits 0.9-6.2%) and 1.6% (95% confidence limits 0.3-4.6%) very major errors for NeoSensitabs and PDM-disc, respectively. Using a zone size breakpoint for susceptibility of greater than or equal to 25 mm for NeoSensitabs and greater than or equal to 20 mm for PDM-disc, the proportions of very major errors were 0.5% (95% confidence limits 0.0-3.0%) at the expense of 5.9% (95% confidence limits 3.0-10.3%) indeterminate strains that belonged to E. rhusiopathiae, Leuconostoc, Lactobacillus and S. haemolyticus. However, using these zone size breakpoints five major errors (beta-haemolytic streptococci, group B three, S. aureus one, Leuconostoc sp. one) were observed for NeoSensitabs and two major errors (beta-haemolytic streptococcus, group B one, Leuconostoc sp. one) were observed for PDM-disc. Susceptibility testing against teicoplanin among these taxa should therefore include a determination of MIC.

Animals↗

[Lysozyme of the mollusk Unio pictorum and the sensitivity of alkanotrophic rhodococci to its effect].

A preparation of lysozyme from a freshwater bivalve, Unio pictorum, has been isolated by sorption to chitin, and its physicochemical properties have been studied. An assessment of the sensitivity of 48 strains of rhodococci, belonging to the species Rhodococcus rubber, R. luteus, and R. erythropolis (Specialized Collection of Alkanotrophic Microorganisms of the Institute of Ecology and Genetics of Microorganisms, Ural Division of the Russian Academy of Sciences), which were isolated from diverse natural waters, to lysozyme of the mollusk Unio pictorum demonstrated that the three species differ in their sensitivity to its effects. The high resistance of rhodococci to lysozyme is indicative of their considerable permanence in hydrobiocenoses (and, therefore, ability to maintain self-purification of microbiocenoses from hydrocarbons).

Alkanes↗

[The microbial transformation of phenanthrene and anthracene].

The transformation of phenanthrene and anthracene by Rhodococcus rhodnii 135, Pseudomonas fluorescens 26K, and Arthrobacter sp. K3 is studied. Twenty-one intermediates of phenanthrene and anthracene transformation are identified by HPLC, mass spectrometry, and NMR spectroscopy. P. fluorescens 26K and Arthrobacter sp. K3 are found to produce a wide range of intermediates, whereas R. rhodnii 135 oxidizes phenanthrene, resulting in the formation of a sole product, 3-hydroxyphenanthrene. Putative transformation pathways of phenanthrene and anthracene are proposed for the three bacterial strains studied. These strains can be used to obtain valuable compounds (such as hydroxylated polycyclic aromatic hydrocarbons) that are difficult to produce by chemical synthesis.

Anthracenes↗

The isolation of microorganisms capable of phenol degradation.

The results of a study on the composition of microflora settling the pilot biofilter bed that purifies the exhausting gases from a cable factory's coil-wire varnishing division are presented in this study. The ability of isolated bacterial strains to biodegrade phenol was also evaluated using culture media of various compositions. Phenol was introduced into the medium at the following concentrations: 0.25, 0.5, 0.75 and 1 g x dm(-3). In addition, air in desiccators, where micro-organisms grew, was saturated with phenol. The isolated microorganisms were graded by the phenol decomposition rate using gas chromatography. The beds of biofilters utilized in industry appeared to be the source of microorganisms capable of degrading phenol. The most active were: Rhodococcus rhodochrous, Gordonia sputi, Pseudomonas putida. Their mixture showed higher degradation activity than the particular isolates. Isolated and identified bacteria metabolized phenol at high rate (about 14 to 42 g x m(-3) x h(-3)).

Air Pollutants↗

[Catalysis of the biodegradation of unusable medicines by alkanotrophic rhodococci].

Experiments on the biodegradation of unusable medicines containing a phenolic hydroxy group by actinobacteria of the genus Rhodococcus were performed. Six species and sixty-four strains were tested. It was found that rhodococci could degrade paracetamol, and some R. ruber strains showed high levels of its degradation. An efficient method for the identification and quantification of paracetamol and the products of its conversion (p-aminophenol, pyrocatechol, and hydroquinone) immediately in the culture liquid was developed. Conditions for the complete biodegradation of paracetamol dosage forms (pills) were optimized. The experimental results can be applied for the development of biotechnological methods for degrading medicines: faked, rejected, or those that are expired.

Acetaminophen↗

Activator proteins for glycosphingolipid hydrolysis by endoglycoceramidases. Elucidation of biological functions of cell-surface glycosphingolipids in situ by endoglycoceramidases made possible using these activator proteins.

Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). Recently, by extensive purification, it was separated from cell-lytic factor (hemolysin) and found to consist of three molecular species each with its own specificity (EGCases I, II, and III) (Ito, M., and Yamagata, T. (1989) J. Biol. Chem. 264, 9510-9519). A detergent was required for EGCases to express full activity, possibly due to their hydrophobic nature, and thus EGCases cannot be used for research on live cells. This paper presents findings on activator proteins in the culture supernatant of Rhodococcus sp. M-777 regarding the stimulation of EGCase activity in the absence of detergents. The activator protein, exhaustively purified and designated as activator II in this study, showed a single protein band on sodium dodecyl sulfate-, native-, and isoelectrofocussing-polyacrylamide slab gel electrophoresis after being stained with Coomassie Brilliant Blue. Its molecular weight and pI were 69,200 and 4.0, respectively. The activator protein enhanced the hydrolysis of glycosphingolipids in vitro and on the cell-surface by EGCase II in the absence of detergents in a concentration-dependent manner. Interestingly, activator II stimulated the activity of EGCase II much more than that of EGCase I on using asialo-GM1 as the substrate. This activator protein was found nonspecific to substrates susceptible to hydrolysis with EGCase II. Besides activator II, strain M-777 produced a second minor molecular species of activator protein designated as activator I which appeared specific for stimulating the activity of EGCase I in contrast to activator II. Following the addition of activator II, EGCase II hydrolyzed cell-surface glycosphingolipids quite efficiently at neutral pH at which hydrolysis hardly occurred at all in its absence. When using activator II in place of Triton X-100 for stimulating EGCase II activity, it was also noted to cause no damage to intact cells. It is thus possible by activator proteins to elucidate the biological functions of endogenous glycosphingolipids in situ by EGCases.

Amino Acid Sequence↗

Revision of the validity of CAMP tests for Listeria identification. Proposal of an alternative method for the determination of haemolytic activity by Listeria strains.

The validity of CAMP tests with Staphylococcus aureus and Rhodococcus (Corynebacterium) equi as defined for Listeria identification was revised. This characterization method appeared to be unreliable for two reasons: first, a positive CAMP test with R. equi is not specific for Listeria ivanovii as Listeria monocytogenes (and Listeria seeligeri) give also a clear positive reaction; second, doubtful reactions could be observed with S. aureus when assaying haemolytic and non-haemolytic Listeria strains (possibility of false negative and false positive results; subjectivity of the interpretation). The use of a Microplate technique previously described instead of CAMP tests is proposed for the reliable demonstration of the haemolytic character of Listeria in the routine identification of these organisms.

Bacterial Typing Techniques↗