[Isolation and characteristics of plasmids from Aeromonas]. / Vydelenie i kharakteristika plazmid bakterii Aeromonas.

Fourteen out of fifty-two examined strains of Aeromonas contain plasmids. One of them determines the resistance to tetracycline, streptomycin, and chloramphenicol. The pAs30 plasmid, 53 t. n. p. in size, is capable of conjugative transfer to bacteria of different genera, which permits us to consider it a broad host range plasmid. Using 12 restriction endonucleases, the physical map of the plasmid was plotted, on which the genes responsible for transporting functions and streptomycin resistance were tentatively located.

[Characterization of the fluorescent pigment pyoverdine Pm, produced by Pseudomonas putida bacteria]. / Kharakteristika fluorestsiruiushchego pigmenta pioverdina Pm, produtsiruemogo bakteriiami Pseudomonas putida.

It has been shown that under iron-limited conditions Pseudomonas putida M produces large amounts of the fluorescent pigment, pyoverdine Pm, which is a siderophore and exhibits antibacterial activity. The absorption spectrum for the pyoverdine Pm has two main peaks, at 230 nm and 400 nm, respectively. It was demonstrated that pyoverdine Pm molecule besides dihydroxyquinoline moiety has a peptide chain contain five amino acids: threonine, serine, lysine, hydroxyaspartic acid and N delta-hydroxyornithine in a molar ratio 3:2:1:1:1.

[Controlled-expression vector for Pseudomonas bacteria involving regulatory elements of trpIBA genes of Pseudomonas putida]. / Vektor reguliruemoi ékspressii dlia bakterii Pseudomonas, vkliuchaiushchii reguliatornye élementy TrpIBA genov Pseudomonas putida.

A bireplicon controlled-expression vector pPS10 was developed based, on trpIBA genes of Pseudomonas putida. It is a low-copy-number vector in Pseudomonas bacteria, and a high-copy-number vector in Escherichia coli. The vector is 10.4 kilobase pairs (kb), determines resistance to kanamycin, carries a replicon of cryptic Pseudomonas pMK1 plasmid; a pBR322 replicon; the par locus of pMT2 plasmid; and the trpI gene of P. putida, which encodes the activator protein and the promoter Pba of trpBA genes. Expression of the promoter is induced by the TrpI protein activator and the precursor of tryptophan, indole-3-glycerolphosphate (InGP). InGP is an unstable compound, and its accumulation in bacterial cells is ensured by using trpE mutants grown in the presence of anthranilate; no InGP is produced among trpE mutants on the media supplemented with tryptophan. As shown in the pheA gene of E. coli, the expression of genetic material cloned under control of the Pba promoter into the pPS10 vector, may be enhanced more than 70-fold in cells of Pseudomonas under conditions of InGP accumulation.

[Genetic control of the synthesis of enzymes of the common site for the aromatic pathway in Pseudomonas bacteria]. / Geneticheskii kontrol' sinteza fermentov obshchego uchastka aromaticheskogo puti u bakterii Pseudomonas.

We have shown recently that the first stage of the common site for the aromatic pathway in Pseudomonas aeruginosa, P. putida, P. fluorescens, P. acidovorans, and P. testosteroni is controlled by repression of 3-deoxy-D-arabinoheptulosenate-7-phosphate-synthase synthesis with phenylalanine and tyrosine. This work is devoted to the study of genetic control of the remaining six stages of the common site for aromatic pathway in representatives of Pseudomonas genus using auxotrophic and regulatory mutants it was shown that genes aroB, aroD, aroE, aroL, aroA, and aroC operate constitutively in P. putida, P. mendocina, P. marginata, and P. acidovorans.

[The role of pyrimidines in the biosynthesis of the fluorescing pigment pyoverdin Pm in Pseudomonas putida M]. / Rol' pirimidinov v biosinteze fliuorestsiruiushchego pigmenta pioverdina Pm u bakterii Pseudomonas putida M.

Dihydroorotate was shown to be a predecessor of deoxyquinoline nucleus of a fluorescing enzyme pyoverdin Pm in Pseudomonas putida M. The data was obtained in experiments with a set of Pyr- mutants with different steps of pyrimidine synthesis blocked. A scheme for deoxyquinoline nucleus of the enzyme including dihydroorotate participation is proposed.

[Cloning the trpBA-genes from Pseudomonas mendocina and Pseudomonas marginata]. / Klonirovanie tryBA-genov Pseudomonas mendocina i Pseudomonas marginata.

The trpBA genes of Pseudomonas mendocina and P. marginata were cloned in Escherichia coli using pBR322 as a vector. Transcription of the cloned genes took place from their own promoters, in the following order: trpB-->trpA. The latter genes were not linked to the trpF gene and could not be induced by indole glycerol phosphate. Thus, the trpBA cluster of P. mendocina and P. marginata are distinguished from that of P. putida, P. aeruginosa (trpIBA) and P. acidovorans (trpFBA).

[Role of phenylalanine in the biosynthesis of fluorescent pigment in Pseudomonas putida bacteria]. / Rol' fenilalanina v biosinteze fluorestsiruiushchego pigmenta u bakterii Pseudomonas putida.

The contemporary data of the participation of phenylalanine in the biosynthesis of fluorescent pigment pyoverdine PM of Pseudomonas putida strain M are presented. Using aro1phu1 mutant of this strain, it has been shown that one of the precursors of the dihydroxyquinoline moiety of the pyoverdine PM is phenylalanine in the D- or L-form. These results were confirmed in experiments with 14C-phenylalanine incorporation. Pyoverdine PM that was synthesized by aro1phu1 mutant from exogenous phenylalanine is identical with the pigment from wild type cells.

[Temperate SM phage from Pseudomonas aeruginosa as a vector for cloning genetic information]. / Umerennyi fag SM Pseudomonas aeruginosa kak vektor dlia klonirovaniia geneticheskoi informatsii.

The recombinant bacteriophages with the genomes containing the DNA fragments of bacteria Erwinia chrysanthemi, including the pectatelyase gene, were constructed on the base of Pseudomonas aeruginosa temperate bacteriophage SM. The gene transferred into Pseudomonas aeruginosa PAO1 cells by transfection is expressed in the new bacterial host. The restriction maps of the recombinant bacteriophages are constructed and the position of an insert is defined. Bacteriophage SM was found to be capable of reproducing in Pseudomonas aeruginosa PAO1 cells when its DNA was shortened to 88% or increased to 111% of the normal genome length. Except for bacteriophage SM, the recombinant bacteriophage SM-2 with an unique restriction endonuclease site for XbaI can also be used as a vector for cloning. Bacteriophage SM capacity in cloning of heterological DNA at HindIII sites is not less than 8 Md, capacity of bacteriophage SM-2 is not less than 5 and 8 Md at XbaI and HindIII sites respectively.

[Regulation of the activity and synthesis of 3-deoxy-D-arabinoheptuloso-7-phosphate synthase in the obligate methylotroph Methylobacillus flagellatum KT]. / Reguliatsiia aktivnosti i sinteza 3-dezoksi-D-arabinogeptulozo-7-fosfatsintazy u obligatnogo metilotrofa Methylobacillus fagellatum KT.

The activity of the first enzyme of aromatic path 3-deoxy-D-arabino-heptuloso-7-phosphate-synthase (DAHP-synthase) is regulated by retro-inhibition and is a subject of repression. Analysis of partially purified preparations of the enzyme has revealed three isoenzymes: DAHP-synthase-Tyr, DAHP-synthase-Trp and DAHP-synthase-Phe, each of them being regulated by a corresponding amino acid. DAHP-synthase-Phe is a dominant isoenzyme presenting 70% of the enzyme activity, 30% inhibition of which is possible by 7.0 mkM of phenylalanine. DAHP-synthase-Tyr and DAHP-synthase-Trp are minor isoenzymes (sharing 15% of enzyme activity each) and are controlled by tyrosine and tryptophane correspondingly. 50% of inhibition of activity is possible by adding 0.7 and 0.8 mkM of corresponding amino acid. Regulation of the enzyme synthesis was studied in the Trp-, Phe- and Tyr- mutants. The enzyme activity was registered under the conditions of limiting and surplus of each aromatic amino acid. The synthesis of DAHP-synthase in M. flagellatum KT is repressed by tryptophane and tyrosine decreasing the synthesis 18.8 and 15.6 fold.

[Characteristics of the R-plasmid pM3 (IncP-9) of a broad circle of hosts]. / Kharakteristika R-plazmidy pM3 (IncP-9) shirokogo kruga khoziaev.

A new broad host range plasmid pM3 (IncP-9) was found in a facultative methylotrophic bacteria Pseudomonas putida and described. The pM3 plasmid is characterized by thermo-instability in Enterobacteriaceae family of bacteria at 36 degrees C or higher temperatures. It is also unable to be inherited as an autonomous element in the obligate methylotrophic bacteria Methylobacillus M75. The peculiarities of plasmid inheritance make possible to use it as a tool for genetic research, for instance, to construct the donor strains in Methylobacillus M75 able to mobilize the chromosomal genes for conjugational transfer in isogenic systems of crosses.

[Genetic control of tryptophan hypersynthesis in regulatory mutants of Pseudomonas putida]. / Geneticheskii kontrol' sverkhsinteza triptofana u reguliatornykh mutantov Pseudomonas putida.

The 6-fluorotryptophan resistant MR1 mutant was obtained from Pseudomonas putida M30 (Tyr- Phe-) strain. The mutant was able to excrete tryptophan (60 micrograms/ml) and has derepressed aroF gene encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase. The mutation isolated was identified as aroR with the help of cloning early aroF gene of P. putida. On the next step of selection, regulatory mutant MR2 was obtained producing 240 micrograms/ml of tryptophan. The MR2 has derepressed unlinked trpE and trpDC genes and represents a mutant of the trpR type. Expression of the trpE gene of P. putida MR2 weakened in the presence of tryptophan excess in the medium, which points to attenuation of this gene. From the prototrophic variant of P. putida MR2 the MRP3 mutant producing 850 micrograms/ml of tryptophan was obtained. This mutant was characterized by twofold increase in the activity of the anthranilate synthase encoded by the trpE gene. The assay of the activity of tryptophanyl-tRNA synthase in P. putida MRP3 demonstrated that the mutant has TrpS+ phenotype.

[Regulation of the synthesis of 3-deoxy-D-arabinoheptuloso-7-phosphate-synthase in Pseudomonas bacteria]. / Reguliatsiia sinteza 3-deoksi-D-arabinogeptulozo-7-fosfat-sintazy u bakterii Pseudomonas.

Regulation of synthesis of 3-deoxy-D-arabinoheptulose-7-phosphate-synthase (DAHP-synthase) was analysed in 12 strains of Pseudomonas belonging to 10 species. Repression of synthesis of DAHP-synthase was registered in half of strains under study, this being controlled by tyrosin and phenilalanine in P. aeruginosa PAO1 and PAT 2152, P. fluorescens BKMB 896, P. acidovorans BKMB 1251 and P. testosteroni BKMB 1241 and by only phenilalanine in P. maltophilia BKMB 30. In the rest of cases (in P. putida AC29, P. stutzeri BKMB 148, P. mendocina BKMB 1299, P. marginata BKMB 1298, P. vesicularis BKMB 974 and P. fluorescens BKMB 35) the enzyme was synthesized constitutively.

[Cloning genes for biosynthesis of Pseudomonas putida tryptophan in Escherichia coli cells]. / Klonirovanie genov biosinteza triptofana Pseudomonas putida v kletkakh Escherichia coli.

The trpE, trpC and trpIBA genes of Pseudomonas putida were cloned by complementation of the corresponding auxotrophic mutations of Escherichia coli using pBR322 as a vector. With the exception of trpE, transcription of all genes in new host takes place under control of their own promoters. Expression of the trpD gene linked to trpC was not registered in E. coli. Repressible trpC enzyme was synthesized constitutively in E. coli. Characteristic regulation of P. putida trpBA genes via induction by indolglycerol phosphate is retained in E. coli. The activator gene trpI and tryptophan synthase genes were closely linked in the trpIBA sequence.

[Regulation of phenylalanine biosynthesis in the obligate methylotroph Methylobacillus M75]. / Reguliatsiia biosinteza fenilalanina u obligatnogo metilotrofa Methylobacillus M75.

Regulation of phenylalanine biosynthesis has been studied in the bacterium Methylobacillus M75 on the level of enzymes 3-deoxy-D-arabinoheptulose-7-phosphate-synthase, chorismatmutase, prephenatdehyrataze. The DAHP-synthase is shown to be synthesized constitutively and its activity is inhibited by all aromatic aminoacids and antranilate. The synthesis of chorismatmutase and prephenatdehydratase is repressed by tyrosine, the activity of the latter enzyme, besides that, is inhibited by phenylalanine, the effect of which is decreased in the presence of tyrosine.

[Cloning of genes for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase from methylotrophic Pseudomonas in Escherichia coli]. / Klonirovanie genov 3-dezoksi-D-arabinogeptulozonat-7-fosfat-sintazy metilotrofnykh Pseudomonas v kletkakh Escherichia coli.

The genes of facultative methylotrophic bacteria Pseudomonas sp. M encoding tyrosine-sensitive and tryptophan-sensitive isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase were cloned separately on the pBR322 plasmid by complementation in Escherichia coli. Transcription of both genes in the new host was performed via the control of genes' own promoters. Cloned genes which are repressed by tyrosine and phenylalanine in their native host were expressed constitutively in E. coli.

[Interactions of the phytopathogenic bacteria Erwinia with phage P1 CRL100 CML]. / Osobennosti vzaimodeistviia fitopatogennykh bakterii Errwinia s fagom P1 CRL100 CML.

The bacteriophage P1 Cm clr100 lysogenises the bacteria E. chrysanthemi, E. aroideae, E. atroseptica being localized in the cytoplasm, replicating but causing no cell lysis. The prophage induction results in transformation of the lysogenic bacteria E. chrysanthemi into nonviable filamentous cells. However, a portion of cells gets rid of the prophage and gives rise to normal heritage inheritors permitting to use the bacteriophage as an efficient vehicle for introducing the transposons into the chromosome of E. chrysanthemi. The transposon Tn9 has been found to insert into the different chromosomal sites causing no inactivation of the genes, while the transposition of Tn5 from the bacteriophage P1::Tn5Cmclr100 induces the different mutations with the frequency up to 3%. The bacteriophage P1Cmclr100 may also serve a tool for construction of the homology regions in the chromosome of E. chrysanthemi and Flac+ plasmid for further construction of Hfr-type donors.

[The use of Pseudomonas sp. M plasmid pM3 for the transposon mutagenesis of Erwinia]. / Ispol'zovanie plazmidy pM3 Pseudomonas sp. M dlia transpozonnogo mutageneza bakterii Erwinia.

The transposon containing derivatives pMTF9 (Tn9), pMTF10 (Tn10) and pMTF59 (Tn5, Tn9) of the Pseudomonas sp. M conjugative plasmid pM3 demonstrating temperature-dependent instability in Erwinia cells incubated at 37 degrees C have been isolated. The obtained plasmids have been shown to be usable for transposon-mediated mutability in the bacterial cells of Erwinia generum incubated at 37 degrees C.

[Secretion of pectate lyase and protein composition of the outer membrane of Erwinia chrysanthemi ENA49 cells]. / Sekretsiia pektatliazy i sostav belkov naruzhnoi membrany kletok Erwinia chrysanthemi ENA49.

The combined changes (specters of isoenzymes of intracellular and extracellular pectatelyase, protein composition of periplasm and outer membrane) in the cells of E. chrysanthemi ENA49 from the periodical culture growing on the inducer containing medium have been studied. The beginning of active pectatelyase synthesis was established to be accompanied by the temporal intracellular accumulation of the enzyme. The cellular capability of pectatelyase secretion to the outer medium correlates with the presence of the definite protein in the outer membrane. The different pathways for secretion of pectatelyase isoenzymes PLb, PLc, PLd and PLe are suggested.

[recA-genes of Erwinia chrysanthemi ENA49]. / recA-gen Erwinia chrysanthemi ENA49.

The recA gene of Erwinia chrysanthemi ENA49 has been cloned in vivo in Escherichia coli K12, recA13 cells using the plasmid pULB113. On the basis or DNA repair and recombination deficiencies complementation, of restoration of the inducible "SOS"-response functions the functional identity of the cloned gene with the recA gene was concluded. The recA gene was localized in the 18th min region of the chromosomal genetical map of Erwinia chrysanthemi ENA49 between the genes proA and pheA.