[Characterization of the Lipopolysaccharides of Pseudomonas chlororaphis].

Lipopolysaccharides (LPS) from two strains ot Pseudomonas chlororaphis subsp. aureofaciens,UCM B-111 and UCM B-306, were isolated and characterized. The LPS preparations exhibited low toxicity, high pyrogenicity and high antiviral activity. Mild acid hydrolysis was used to obtain the O-specific polysaccharides. Their structures were established by monosaccharide analysis and determination of absolute configurations, as well as by 1D and 2D NMR spectroscopy. The O-polysaccharides were shown to contain the linear tri- or tetrasaccharide repeating units. Both O-polysaccharides were structurally heterogeneous: P. chlororaphis subsp. aureofaciens UCM B-111--> 4)-αD-GalpNAc6Ac-(1 --> 3)-ß-D-QuipNAc-(1 --> 6)-αD-GlcpNAc-(l --> ßD-GlcpNAc-(l --> 3)] GalNAc -60%; degree of the non-stoichiometric 6-O-acetylation of GalNAc -60%; P. chlorophis subsp. aureofaciens UCM B-306 --> 3)-α-D-Rhap-(1 --> 4)-α-D-GalpNAcAN-(1 --> 3)-αD-QuipNAc4NAc-(1 -->, where GalNAcAN is 2-acetamido-2-deoxy-D-galacturonamide, the degree of non-stoichiometric amidation of the GalNAcA residue -60%.

[Antifungal and antiviral substances of Pseudomonas chlororaphis subsp. aureofaciens strains--components of gaupsin].

Phenazine-1-carboxylic, 2-hydroxy-phenazine-carboxylic acid and 2-hydroxy-phenazine active against phytopathogenic fungi were detected in fermentation broth of Pseudomonas chlororaphis subsp.aureofaciens strains UCM B-111 and UCM B-306--components of insectofungicide biopreparation gaupsin using chromato-mass-spectrometric methods; strain B-306 produced antifungal antibiotic pyrrolnitrin together with phenazines. Supernatants of fermentation broth of P chlororaphis subsp. aureofaciens B-111 and B-306 strains grown in King A medium and exopolymers preparations obtained from these supernatants using evaporation, dialysis and liophylisation were highly active against tobacco mosaic virus (TMV). At a dose of 10 mg/ml they reduced TMV infectivity by 76-96%, at concentrations 1 and 0.1 mg/ml the antiviral effect was decreased to 40-62 and 14-27%, respectively. Dialysis did not influence the antiviral activity of isolated preparations. The latter contained 2-7.6 % of carbohydrates including neutral monosaccharides: fucose, mannose, galactose and glucose.

[Sensitivity of Pseudomonas chlororaphis to antibiotics and chemical tools of plant protection].

Examination of sensitivity of 10 Pseudomonas chlororaphis strains belonging to different subspecies to 54 antibiotics has shown that all studied representatives of Pchlororaphis subsp. chlororaphis, P. chlororaphis subsp. aureofaciens and Pchlororaphis subsp. aurantiaca were sensitive to aminoglycoside antibiotics and fluoroquinolones derivatives. Only part of studied strains has shown sensitivity to some beta-lactam antibiotics, imipeneme and meropeneme. In contrast to representatives of two other subspecies both strains of P. chlororaphis subsp. chlororaphis proved to be sensitive to chlortetracycline and cefepime that allows to consider this difference as the characteristic useful for differentiation. All studied P. chlororaphis strains were resistant to chemical fungicides (Scor and Svitch) and the insect growth regulators (Match, Lufox, Engio, Actellik). Resistance of bacteria to these herbicides gives evidence that their combined use is possible.

Pseudomonas batumici sp. nov., the antibiotic-producing bacteria isolated from soil of the Caucasus Black Sea coast.

Four novel strains of saprophytic bacteria were isolated from the soil samples collected in the moist subtropics region (the Black Sea coast of the Caucasus) and studied using methods of polyphasic taxonomic analysis. Microorganisms were Gram-negative, oxidase positive, aerobic, rod-shaped motile bacteria that produced antibiotic named batumin with high and selective activity against staphylococci; its total formula was C30H48N2O7 Phylogenetic analysis of 16S rRNA gene sequences (1376 bp, accession number in Genbank--JF306642) indicated that the isolates belonged to the gamma-Proteobacteria formed a separate branch within the genus Pseudomonas and had 98% 16S rRNA gene sequence similarity with Pseudomonas gingeri. The latter essentially differed from the studied strains in its phenotypic characteristics. The predominant cellular fatty acids of isolates were similar and included Cl6:0, C16:1, C18:1, and up to 22.9% of deltaC17:0; their DNA G+C content was 64.0 mol%. An analysis of taxonomic data indicated that the studied isolates represented a novel species, for which the name Pseudomonas batumici sp. nov. is proposed with the type strain UCM B-321 (Ukrainian Collection of Microorganisms).

[The Black Sea bacteria--producers of hydrolytic enzymes].

Investigation of 15 different glycoside activities of 64 strains isolated from water and invertebra of the Black Sea has shown that 64% of the studied strains displayed the capacity to synthesize enzymes with alpha-L-ramnosidase activity which varied from 0.01 to 0.20 un/ml depending on the strain. The greatest number of the enzyme producers was found in representatives of Alteromonas macleodii. Other investigated glycosidase activities: alpha-amylase, beta-N-acetyl-D-glucosaminidase, beta-D-glucuronide, alpha-N-acetyl-D-galactosaminidase, beta-N-acetyl-D-galactosaminidase, beta-D-galactosidase, alpha-D-galactosidase, beta-D-glucosidase, KM-cellulase activities though have been found, but mainly with inconsiderable indices, alpha-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, beta-D-xylosidase and alpha-D-xylosidase activities were found in neither of the studied strains. Strains with rather high proteolytic activity were found among marine species of bacteria. It has been established that 18 strains (28%) of 64 marine isolates were characterized by rather high level of total proteolytic activity (from 0.1 to 05 un/ml), 43.75% of them displayed inconsiderable (up to 0.1 un/ml) or only trace (up to 0.01 un/ml), 18.75% did not display any hydrolytic activity in respect of casein. Investigation of substrate specificity to a number of fibrillar and globular proteins of 9 studied strains, which displayed considerable general (caseinolytic) activity has shown that 8 of them displayed fibrinolytic activity from 0.15 to 2.175 un/ml. All 9 strains were characterized by gelatin activity. Collagenase and keratinase activity was also revealed. Neither of 9 studied strains displayed elastase activity.

[Use of Pseudomonas bacteria strains for determining water-assimilable carbon].

The ability of 12 Pseudomonas putida and Pseudomonas fluorescens strains for growth in the tap water has been studied. All of them grew well in pasteurized Kyiv tap water at 500 colony-forming units--CFU/ml inoculum reaching the maximal titre 1.7 x 10(4)-4.6 x 10(6) CFU/ml after 240 hours of incubation. The most intensive growth was observed in strain P. putida UCM B-143, capable to assimilation of 84 different sources of carbon nutrition. The linear dependence between strain 143 growth and acetate carbon concentration in the interval of 10 to 60 microg/l has been demonstrated in deionized water with addition of necessary mineral salts; its yield was 4.5 x 10(6) CFU/microg of assimilable acetate carbon (AOC). Determination of AOC of water using standard methods, standard strain P. fluorescens UCM B-385 (P17, ATCC 49642) and selected strain P. putida B-143 gave the similar values of AOC quantities in the Kyiv tap water (536 and 600 microg of AOC in 11, accordingly). Viability of strain 143 grown in the artesian water decreased more quickly than that of the standard strain which is possibly connected with origin of both cultures: strain 143 was isolated from wheat rhizosphere while strain P17--from the drinking water.

[Analysis of relation between structure and antibiotic activity of Batumin].

Antimicrobial activity of six derivatives and products of chemical modification of antistaphylococcal antibiotic Batumin and one natural analog of this antibiotic formed by the strain-producer has been investigated. Not a single substance, which could exceed Batumin or be equal to it as to activity and selectivity in respect of staphylococci was detected among the studied compounds. It was shown that a unique antimicrobial effect of Batumin was determined by several peculiarities of its molecule: presence of double bonds, group--CONH2, as well as carboxyl and hydroxyl groups. The substitution or modification of each of these groups decreased activity against staphylococci and qualitatively changed antimicrobial spectrum of the antibiotic.

[Antimicrobial spectrum of antibiotic batumin].

Antibacterial activity of antibiotic batumin, isolated from Pseudomonas genus has been studied using CLSI standard methods. Batumin was highly active against all studied strains belonging to 10 species of Staphylococcus genus (minimal inhibitory concentration (MIC)--0.25-0.5 microg/ml); it has shown a moderate activity against enterobacteria of genera Salmonella, Bordetella, Escherichia, Klebsiella (MIC 8-64 microg/ml) and practically did not inhibit strains ofmicrococci, streptococci, sporeforming bacteria including Clostridium sporogenes (MIC 256 microg/ml or above). Strains of yeasts and microscopic fungi Candida tropicalis, C. utilis, C. albicans, Saccharomyces cerevisiae u Aspergillus niger were resistant to batumin. Antibiotic activity depended on test-culture cells concentration and medium pH and was greatest at pH 5.5 characteristic of human skin.

[Pseudomonas rathonis--a new species of "Pseudomonas aeruginosa-complex"].

Pseudomonas strains (n = 8), isolated from soil and attributed to a new species of Pseudomonas rathonis have been studied by the methods of polyphasic taxonomic analysis. The data of 16 S rRNA sequence, sequence of gyr B-gene, DNA-DNA molecular hybridization, data of fatty acid composition and phenotypical analysis proved, that the studied bacteria belonged to "Pseudomonas aeruginosa-complex". The set of characteristics to differentiate Pseudomonas rathonis from other 11 species of this complex has been proposed.

[Shewanella genus bacteria isolated from the Black sea water and molluscs].

Six (6) strains of bacteria isolated from the Black Sea water and mollusks Mytilaster lineatus have been assigned to Shewanella genus on the basis of phenotypical and molecular-genetic study. Partial sequence of 16S rRNA gene revealed 100% similarity of three of these strains to S. baltica, although they had some phenotypical differences. Three more strains were genetically related to S. colwelliana and S. affinis (99-100% of 16S rRNA sequences similarity), but they differed from these species as well as from each other in some phenotypical properties, so the question about their taxonomic status still remains open. In aerobic conditions the Black Sea strains of S. baltica adsorbed significant quantities of Fe III from the media. They reduced it to Fe (II) in anaerobic conditions. Strains isolated from mollusks were not able to reduce Fe (III).

[Taxonomic analysis of Pseudomonas strains with uncertain taxonomic status]. / Taksonomicheskii analiz shtammov bakterii roda Pseudomonas s neopredelennoi vidovoi prinadlezhnost'iu.

Pseudomonas strains isolated from soil and rhizosphere and designated as "Pseudomonas rathonis", Pseudomonas sp. B and Pseudomonas sp. C have been characterized by 113 phenotypic properties and studied by methods of molecular-genetic analysis. The 5'-terminal hypervariable 16S rRNA regions, containing from 208 to 344 nucleotides have been amplified and sequenced. The comparative analysis of sequence results gave evidence about phylogenetic relatedness of studied bacteria (98-100% of 16S rRNA sequences identity) to some saprophytic and plant-pathogenic Pseudomonas species. The latter substantially differed from studied strains in phenotypic characteristics. The DNA-DNA hybridization results have shown the low (0-36%) level of chromosomic DNA homology of "Pseudomonas rathonis", Pseudomonas sp. B, Pseudomonas sp. C strains with the type strains of P. fluorescens, P. putida, P. mendocina, P. pseudoalcaligenes. Obtained data give evidence that these strains belong to some novel Pseudomonas species.

[Identification of the Pseudomonas genus bacteria by computer analysis]. / Identifikatsiia bakterii roda Pseudomonas metodami komp'iuternogo analiza.

A computer program for the simplified phenotypic identification of Pseudomonas has been developed. The information concerning 66 species included in up-to-date Pseudomonas genus characterized by 113 tests was accumulated in a database. The identification key is represented in interactive mode on a website http://www.imv.kiev.ua/PsmIK/default.htm. The program was used for the identification of 46 Pseudomonas strains isolated from rhizosphere. For 23 more strains unidentified by conventional technique, the level of similarity was 67-74%. This fact allows suggesting that they might be representatives of new Pseudomonas species.

[Auxotrophy and utilization of oxidized and reduced mineral sulfur forms by Brevundimonas diminuta strains]. / Auksotrofiia i usvoenie oksilennykh i vosstanovlennykh mineral'nykh soedinenii sery shtammami Brevundimonas diminuta.

Connection between auxotrophy of Brevundimonas diminuta strains for sulphur-containing amino acids and presence of oxidized or reduced sulphur forms in the medium has been shown. When grown in synthetic medium supplied with sulphate or sulphite B. diminuta strains required cystine, biotin, pantothenate and vitamin B12 for growth and utilized very limited spectrum--from 8 to 12--carbon sources. In the presence of 5 g/l of thiosulphate in the medium instead of mentioned sulphur-containing salts strains of this species did not require cystine and assimilated the ammonium nitrogen. The intensity of their growth in these conditions was lower and carbon nutrition spectra more limited than in custine-containing medium. Tetrathionate, elemental sulphur, sulphide were less efficient than thiosulphate, but they also provided growth of B. diminuta strains in the media supplied with ammonium salts, necessary vitamins and such universal C-sources as aspartate, glutamate, proline. Neither oxidized nor reduced mineral sulphur forms were able to substitute sulphur-containing amino acids for Comamonas terrigena strain requiring methionine.

[Isolation of highly active strain producing the antistaphylococcal antibiotic batumin]. / Poluchenie vysokoaktivnogo shtamma--produtsenta antistafilokokkovogo antibiotika batumina.

The use of chemical and UV-induced mutageneses allowed us to increase the biosynthetic activity of the strain capable of producing new antistaphylococcal antibiotic, batumin. The strain of Pseudomonas batumici N17 producing 87-100 mg batumin per liter culture liquid was selected. Its activity was 3.5-5 times higher than the activity of the most potent natural strain. P. batumici N17 was shown to be stable in relation to the synthesis of batumin.

[The insecto-fungicidal preparation, gaupsin, isolated from Pseudomonas aureofaciens strains]. / Insektofungitsidnyi preparat gaupsin na osnove shtammov Pseudomonas aureofaciens.

The integrated insectofungicidal preparation Gaupsin was developed on the basis of the Pseudomonas aureofaciens strains UKM V-111, which is active against bacterial and fungal phytopathogens, and UKM V-306, active against codling moth larvae. Gaupsin is an effective means for protection of orchards against moths and fungi. A method for production of Gaupsin in the liquid form with a titer of not less than 1 x 10(10) cells/ml under aeration conditions was elaborated. After spraying, the preparation remained on apple leaves for seven days. The efficiency of Gaupsin against codling moth was 88-94%. The effect of a fungal attack decreased 10 to 25-fold.

[The use of batumin-containing disks for the rapid identification of staphylococci]. / Ispol'zovanie diskov s batuminom dlia ékspress-identifikatsii stafilokokkov.

The study of 467 microbial strains obtained from collections and from clinical sources revealed that microorganisms of the genus Staphylococcus were highly sensitive to batumin, a new antibiotic obtained from bacteria of the genus Pseudomonas. 378 strains of 15 Staphylococcus species proved to be highly sensitive to the diagnostic preparation "Diastaph", developed on the basis of batumin (antibiotic-impregnated discs); After 18-hour incubation the diameter of the growth inhibition zones on agar-containing culture media was 18-38 mm. Strains belonging to the genera Micrococcus, Dermacoccus, Kocuria and Kytococcus, as well as the tested representatives of other taxa (Planococcus, Streptococcus, Corynebacterium, Acinetobacter, Pseudomonas, Neisseria, the representatives of all tested genera of the family Enterobacteriaceae, fungi of the genus Candida) were insensitive to the diagnosticum. "Diastaph" permits not only the rapid identification of staphylococci pure cultures, but also the determination of their presence in association with other microbial species directly in pathological material, which makes it possible to recommend this diagnostic preparation for use in medical, veterinary and sanitary microbiology.

Phenotypic diversity of Pseudoalteromonas citrea from different marine habitats and emendation of the description.

Four strains of marine, aerobic, agar-decomposing bacteria with one polar flagellum and with DNA G + C contents of 38.9-40.2 mol% were isolated from the Far-Eastern mussels Crenomytilus grayanus and Patinopecten yessoensis. These four strains were identified as Pseudoalteromonas; however, they were phenotypically different from species described previously according to carbon compound utilization tests and the BIOLOG identification system. High agar-decomposing activity was found in two strains, in one of which agarase, alpha-galactosidase, pustulanase and laminarinase had been detected. The level of DNA homology of three of the strains was 70-100%. The fourth isolate was genetically less related to the others (67% DNA relatedness) and phenotypically was more distant from other members of this group; however, all four strains were assigned to a single species genotypically. DNA from the strains isolated from mussels showed 40-45% genetic relatedness with the DNA of Alteromonas atlantica, 8-36% with DNA of Pseudoalteromonas haloplanktis subsp. haloplanktis, Pseudoalteromonas haloplanktis subsp. tetraodonis, Pseudoalteromonas undina, Pseudoalteromonas nigrifaciens and Pseudoalteromonasas carrageenovora, 53% with Pseudoalteromonas elyakovii, 32-48% with marine P. nigrifaciens from mussels and 14-16% with Alteromonas macleodii. The DNA-DNA hybridization data revealed that the levels of relatedness between the strains isolated and the type strains of Pseudoalteromonas citrea and Pseudoalteromonas fuliginea described recently were significant (95-85%). These results were confirmed by serological data employing polyclonal antibodies to cell surface antigens. The strains isolated from mussels were identified as P. citrea. The hybridization data showed that the name P. fuliginea Romanenko et al. 1994 should be recognized as a junior subjective synonym of P. citrea Gauthier 1977. A notable phenotypic diversity of P. citrea which might be a reflection of their ecological habitats is discussed.

Fluviols, bicyclic nitrogen-rich antibiotics produced by Pseudomonas fluorescens.

Fifteen fluorescent pseudomonads, isolated from the rhizosphere of agricultural plants, were similar in both their phenotypic properties and the chemical nature of produced pigments, to the previously described Pseudomonas fluorescens var. pseudoiodinum. DNA-DNA hybridisation data showed their genetic similarity (but not identity) to different biovars of P. fluorescens. A family of antibiotics-fluviols belonging to pyrazolo-[4,3-e]as-triazine derivatives was isolated from studied strains; isolation, properties, antimicrobial and antitumour activity of fluviols are described.

[The effect of the antibiotic AL-87 on the amino acid yield from Staphylococcus aureus 209P cells]. / Vliianie antibiotika AL-87 na vykhod aminokislot iz kletok Staphylococcus aureus 209P.

Antibiotic AL-87 has been studied for its effect on the composition of intracellular free amino acids and of amino acids in culture fluid of Staphylococcus aureus 209 P. It is established that the content of amino acids in the culture fluid of S. aureus 209 P is doubled due to antibiotics, while the content of intracellular free amino acids considerably decreases. Spectrum of free amino acids of S. aureus 209 P is presented by 17 basic amino acids. When there is a sub-bacteriostatic concentration of the antibiotic in the medium all free amino acids tend to leave the cells, the content of aspartic acid, serine, threonine and leucine in the medium being increased. Data obtained when studying the effect of antibiotic AL-87 on the composition of free amino acids of Staphylococcus agree well with the previously obtained results from the study of the fatty acid composition of cells. In the light of these data it may be supposed that an increase of the membrane permeability and as a result of it an outlet of amino acids into the medium is one of constituents of the mechanism of antibiotic AL-87 action on Staphylococcus cells.