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ABSTRACT: The inactivation of the rpoS gene, which encodes the sigma S subunit of RNA polymerase of rhizospheric strain Pseudomonas chlororaphis 449 results in a drastic (5–8-fold) decrease in the synthesis of phenazine antibiotics, a decline in the acid and alkaline
phosphatase activities (pH 2.5 and 8.8, respectively), and the antagonistic activity of this strain against phytopathogenic
fungus Rhizoctonia solani. A mutation in the rpoS gene also causes a minor decrease of lipase and proteolytic activity in the supernatants of Pseudomonas chlororaphis 449 and has no substantial effect on the synthesis of three types of N-acyl homoserine lactones, the signal molecules of quarum sensing regulation, and the ability of bacteria to migrate over
the medium surface (swarming).
Molecular Genetics Microbiology and Virology 04/2012; 24(1):7-11. · 0.23 Impact Factor
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ABSTRACT: Strain Pseudomonas chlororaphis 449, an antagonist of a broad spectrum of phytopathogenic microorganisms isolated from the maize rhizosphere, was shown to
produce three phenazine antibiotics: phenazine-1-carboxylic acid (PCA), 2-hydroxylphenazine-1-carboxylic acid (2-OH-PCA),
and 2-hydroxylphenazine (2-OH-PHZ). Two Quorum Sensing (QS) systems of regulation were identified: Phz/R and CsaI/R. Genes
phzI and csaI were cloned and sequenced. Cells of strain 449 synthesize at least three types of AHL: N-butanoyl-L-homoserine lactone (C4-AHL), N-hexanoyl-L-homoserine lactone (C6-AHL), and N-(3-oxo-hexanoyl)-L-homoserine lactone (30C6-AHL). Transposon mutagenesis was used to generate mutants of strain 449 deficient in synthesis of phenazines, which carried
inactivated phzA and phzB genes of the phenazine operon and gene phzO. Mutations phzA
− and phzB
− caused a drastic reduction in the antagonistic activity of bacteria toward phytopathogenic fungi. Both mutants lost the ability
to protect cucumber and leguminous plants against phytopathogenic fungi Rhizoctonia solani and Sclerotinia sclerotiorum. These results suggest a significant role of phenazines in the antagonistic activity of P. chlororaphis 449.
Russian Journal of Genetics 04/2012; 44(12):1400-1408. · 0.43 Impact Factor
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ABSTRACT: The introduction into strain Pseudomonas chlororaphis 449 of plasmid pME6863 that contains the cloned gene for N-acyl-homoserine lactonase, AiiA, leads to the degradation of all three types of N-acylhomoserine lactones produced by this strain (N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, and N-3-oxo-hexanoyl-homoserine lactone). This causes a drastic reduction in the synthesis of phenazine pigment and decreases the
ability of cells to migrate on the surface of nutrient medium. However, the antagonistic activity of P. chlororaphis 449 toward phytopathogenic fungi Sclerotinia sclerotiorum and Rhizoctonia solani is not only decreased, but is even slightly increased; no essential changes in the exoprotease activity were observed. It
is assumed that one of the QS systems of P. chlororaphis 449 may exert the repression effect on the expression of genes, which determine the two latter cell activities.
Russian Journal of Genetics 04/2012; 45(1):30-34. · 0.43 Impact Factor
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ABSTRACT: 228 strains of soil and rhizosphere pseudomonads isolated in different geographic zones were screened, with the use of two
tester systems, for the capacity to produce N-acyl-homoserine lactones (AHLs), which are autoinducers involved in quorum-sensing (QS) regulation. AHL production was found
in 11.4% of the strains investigated. In five Pseudomonas chlororaphis strains shown to be active AHL producers and chosen for further study, PCR identified two QS systems that involved the phzI, phzR, csaI, and csaR genes; this finding suggests the conservative nature of these regulation systems in P. chloroaphis. Strain P. chlororaphis 449, chosen as a model object and studied in greater detail, produced three AHL species including N-butanoyl-homoserine lactone and N-hexanoyl-homoserine lactone. This strain produced three types of phenazine antibiotics, as well as siderophores and cyanide;
it also exhibited antagonistic properties toward a wide spectrum of phytopathogenic fungi. The phzI and csaI genes, coding for synthases of AHLs of two types, were cloned and sequenced; mutants with knocked-out phzI and csaI genes were obtained. With the use of transposon mutagenesis and the gene substitution method, mutations were obtained in
the global expression regulator genes gacS, coding for the GacA-GacS regulation system kinase, and rpoS, coding for the sigma S subunit of RNA polymerase. The effect of these mutations on the AHL synthesis and on the regulation
of various metabolic processes in P. chlororaphis was studied.
Microbiology 04/2012; 75(4):398-400. · 3.06 Impact Factor
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ABSTRACT: Gene vfr of Pseudomonas chlororaphis 449 previously described only in Pseudomonas aeruginosa was identified, cloned, and sequenced; its localization in the chromosome was determined. Amino acid sequence of the protein
encoded by gene vfr in P. chlororaphis 449 was shown to have a 83% identity with the Vfr protein of P. aeruginosa PAO1 and a 63% identity with the CRP protein of Escherichia coli. Amino acid residues that ensure the most important structural properties of the CRP protein, i.e., its binding to cAMP,
RNA polymerase, and DNA, were identical or highly conserved in Vfr proteins of P. aeruginosa and P. chlororaphis 449. The cloned vfr gene of P. chlororaphis 449 was complemented partially the mutation at gene crp in cells of E. coli AM306 enhancing ten times synthesis of β-galactosidase dependent on the CRP protein. Unlike P. aeruginosa, the Vfr protein in cells of P. chlororaphis 449 does not participate in the regulation of synthesis of N-acyl-homoserine lactones.
Russian Journal of Genetics 04/2012; 45(9):1055-1061. · 0.43 Impact Factor
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ABSTRACT: The participation of global regulators GrrS (sensor kinase GacA/GacS-like regulatory system) and sigma S subunit of RNA polymerase
in the control of phosphatase synthesis in a soil bacterium Serratia plymuthica was shown. In cells of null mutants for genes grrS and rpoS synthesis of acid and alkaline phosphatases was markedly decreased.
Russian Journal of Genetics 11/2007; 43(12):1428-1430. · 0.43 Impact Factor
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ABSTRACT: To study the regulation of expression of the Serratia plymuthica gene chiA encoding a 58-kDa endochitinase, its 586-bp-long upstream regulatory region was cloned, sequenced and fused to a promoterless lac operon in phage lambdaRS45 to obtain a single-copy transcriptional fusion (P F1chiA )-lac in lysogens of Escherichia coli wild-type strains or their mutants deficient in various global regulators of transcription. The level of P F1chiA -lac expression increased about 20- and 90-fold, respectively, in E. coli K12 Deltahns and double Deltahns stpA mutants deficient in H-NS, and in both H-NS and StpA DNA-binding histone-like proteins, as compared to levels in the wild-type strain. In a Deltalrp mutant deficient in the leucine-responsive transcriptional regulator Lrp, the level of P F1chiA -lac expression increased only up to threefold, whereas even smaller differences relative to the wild-type strain were observed in rpoS and Deltacrp mutants deficient in the sigmaS subunit of RNA polymerase and catabolite-repression protein (CRP), respectively. Deletion of the inverted-repeat sequences and curved DNA regions located in the upstream region of chiA essentially did not influence strain IC1270's chiA promoter activity in E. coli .
Journal of Basic Microbiology 02/2005; 45(6):426-37. · 1.27 Impact Factor
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ABSTRACT: It was earlier shown that expression of the microcin C51 operon in Escherichia coli cells is activated upon decelerated growth of cells during their transition to the stationary growth phase and depends on the sigmaS subunit of RNA polymerase. Using a single-copy construct containing the cloned promoter region of the microcin C51 operon and a promoterless lac operon (P(mcc)-lac), it was shown that the promoter of the microcin operon was also induced by stress caused by the transition of cells at the exponential growth phase into the medium without glucose as a sole carbon source. Activation of P(mcc)-lac expression upon severe glucose starvation occurred in rpoS+ and rpoS- strains. In cells carrying the rpoD800 mutation that renders the sigma70 subunit of RNA polymerase temperature-sensitive, an activation of P(mcc)-lac expression was observed at nonpermissive temperature, in contrast to its complete inhibition in E. coli cells at the phase of delayed growth. Other stressors-nitrogen starvation, high temperatures, osmotic shock, tetracycline and chloramphenicol-did not activate P(mcc)-lac expression in cells at the exponential growth phase.
Genetika 02/2005; 41(1):48-52. · 0.44 Impact Factor
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ABSTRACT: It was earlier shown that expression of the microcin C51 operon in Escherichia coli cells is activated upon decelerated growth of cells during their transition to the stationary growth phase and depends on the S subunit of RNA polymerase. Using a single-copy construct containing the cloned promoter region of the microcin C51 operon and a promoterless lac operon (Pmcc-lac), it was shown that the promoter of the microcin operon was also induced by stress caused by the transition of cells at the exponential growth phase into the medium without glucose as a sole carbon source. Activation of Pmcc-lac expression upon severe glucose starvation occurred in rpoS
+ and rpoS
– strains. In cells carrying the rpoD800 mutation that renders the 70 subunit of RNA polymerase temperature-sensitive, an activation of Pmcc-lac expression was observed at nonpermissive temperature, in contrast to its complete inhibition in E. coli cells at the phase of delayed growth. Other stressors—nitrogen starvation, high temperatures, osmotic shock, tetracycline and chloramphenicol—did not activate Pmcc-lac expression in cells at the exponential growth phase.
Russian Journal of Genetics 12/2004; 41(1):40-43. · 0.43 Impact Factor
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ABSTRACT: A search for phytase genes in 9 Bacillus strains from the collection of IMGAN was implemented. The growth optimum of strains IX-22, IX-12B, K17-2, K18, IMG I, IMG II, M4 and M8 was 50-60 degrees C; the optimal growth temperature for Bacillus sp. 790 was 45-47 degrees C. According to the sequence data of 16S RNA genes, Bacillus sp. 790 belongs to the B. subtilis/amyloliquefaciens group. The other 8 strains were identified as B. licheniformis. Selection of Bacillus strains, potentially containing the phytase genes, was performed via PCR with primers designed on the basis of the conserved sequence regions of the phyA gene from B. amyloliquefaciens FZB45 with chromosomal DNA being used as the template. The nucleotide sequences of all PCR fragments showed a high level of homology to the known Bacillus phytase genes. The gene libraries of B. licheniformis M8 and B. amyloliquefaciens 790 in E. coli were constructed and phytase-containing clones were selected from them. Twenty-four Pseudomonas strains of different species, 5 Xanthomonas maltophilia strains and 1 Xanthomonas malvacearum (all from the mentioned collection) were tested for phytase activity. Such activity was found in 13 Pseudomonas strains and in 6 Xanthomonas strains. The accumulation of phytase in Pseudomonas was shown to take place at later (over 2 days') growth stages. The optimum pH for phytase from 3 Pseudomonas strains were established. The enzymes were found to be most active at pH 5.5.
Molekuliarnaia genetika, mikrobiologiia i virusologiia 02/2004;
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I A Shaginian,
I A Khmel,
Iu M Romanova,
M A Veselova,
M Iu Chernukha,
L S Chernin,
S V Sidorenko, V A Lipasova,
V P Kovtun,
G V Alekseeva,
N V Alekseeva,
T V Stepanova,
A L Gintsburg
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ABSTRACT: Described in the paper are characteristics of B. cepacia clinical strains isolated from patients at Moscow hospitals. The strains were investigated for the presence of proteolytic, chitinolytic, hemolytic and lipase activities as well as for presence of components of the "Quorum sensing" gene activity regulatory system by using biological test-systems and in the polymerase chain reaction with primers to genes cepI and cepR.
Molekuliarnaia genetika, mikrobiologiia i virusologiia 02/2003;
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ABSTRACT: Signaling molecules assigned to N-acyl-homoserine-lactones (AHL) serve as autoinducers for the genes controlling the quorum sensing regulatory system. In many gram-negative bacteria, AHL are the key factors responsible for density-dependent regulation of exoenzyme and secondary metabolite production; they also participate in interaction between bacteria and higher organisms. The soil and rhisosphere bacteria Pseudomonas and Xanthomonas from different geographical zones of Russia and the former USSR were analyzed for the presence of the AHL producers. Screening was conducted by using a test system based on the mutant strain Chromobacterium violaceum, which was unable to synthesize AHL but produced a pigment violacein in the presence of exogenous AHL. The AHL-like compounds proved to be formed by 9.7% of the studied bacteria. Various Pseudomonas species differed in the capacity to synthesize this compounds. In at least a half of the isolated P. aureofaciens and P. aeruginosa, an intense AHL production was observed, whereas the AHL-producers were far less frequent among the P. fluorescens, P. chlororaphis, P. lemonnieri, P. geniculata, and P. putida. None of the 41 Xanthomonas maltophilia strains examined synthesized AHL.
Genetika 05/2002; 38(4):568-70. · 0.44 Impact Factor
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ABSTRACT: Signaling molecules assigned to N-acyl-homoserine-lactones (AHL) serve as autoinducers for the genes controlling the quorum sensing regulatory system. In many gram-negative bacteria, AHL are the key factors responsible for density-dependent regulation of exoenzyme and secondary metabolite production; they also participate in interaction between bacteria and higher organisms. The soil and rhisosphere bacteria Pseudomonasand Xanthomonas from different geographical zones of Russia and the former USSR were analyzed for the presence of the AHL producers. Screening was conducted by using a test system based on the mutant strain Chromobacterium violaceum, which was unable to synthesize AHL but produced a pigment violacein in the presence of exogenous AHL. The AHL-like compounds proved to be formed by 9.7% of the studied bacteria. Various Pseudomonas species differed in the capacity to synthesize these compounds. In at least a half of the isolated P. aureofaciens andP. aeruginosa strains, an intense AHL production was observed, whereas the AHL-producers were far less frequent among the P. fluorescens, P. chlororaphis, P. lemonnieri,P. geniculata,and P. putidastrains. None of the 41 Xanthomonas maltophilia strains examined synthesized AHL.
Russian Journal of Genetics 03/2002; 38(4):467-469. · 0.43 Impact Factor
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ABSTRACT: The level of transcription from the promoter of the microcin C51 operon (Pmcc) depends on the growth phase of Escherichia coli cells: transcription proceeds with low efficiency at the exponential phase of growth and with higher efficiency when growth of cells is delayed during entry into the stationary phase. The functioning of Pmcc was studied in cells grown in different media by a single-copy construct, which contained the cloned promoter region of the microcin C51 operon and the promoterless lac operon. A decrease in the rate of cell growth caused by changes in the sole carbon source in minimal medium correlated with an increase in the level of transcription from the Pmcc promoter at the exponential phase of growth; the expression of Pmcc-lac during cell entry into the stationary phase was higher under unfavorable medium conditions. The use of composite rich media impaired this feature. The addition of l-leucine (100 micrograms/ml) to the medium decreased the expression of Pmcc-lac in wild-type cells carrying the delta lrp mutation. A further increase in leucine concentration and the presence of other amino acids in the medium enhanced transcription that started from Pmcc during cell entry into the stationary growth phase. The capacity of the Pmcc promoter and of the wild-type lacZ gene promoter was virtually the same upon IPTG induction. A mutation in the ompR gene did not markedly influence transcription started from Pmcc.
Genetika 09/2001; 37(8):1055-62. · 0.44 Impact Factor
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ABSTRACT: The level of transcription from the promoter of the microcin C51 operon (P
mcc
) depends on the growth phase of Escherichia colicells: transcription proceeds with low efficiency at the exponential phase of growth and with higher efficiency when growth of cells is delayed during entry into the stationary phase. The functioning of P
mcc
was studied in cells grown in different media by a single-copy construct, which contained the cloned promoter region of the microcin C51 operon and the promoterless lacoperon. A decrease in the rate of cell growth caused by changes in the sole carbon source in minimal medium correlated with an increase in the level of transcription from the P
mcc
promoter at the exponential phase of growth; the expression of P
mcc
–lacduring cell entry into the stationary phase was higher under less favorable medium conditions. The use of composite rich media impaired this feature. The addition of l-leucine (100 g/ml) to the medium decreased the expression of P
mcc
–lacin wild-type cells and those carrying the lrpmutation. A further increase in leucine concentration and the presence of other amino acids in the medium enhanced transcription that started from P
mcc
during cell entry into the stationary growth phase. The capacity of the P
mcc
promoter and of the wild-type lacZgene promoter upon IPTG induction was virtually the same. A mutation in the ompRgene did not markedly influence transcription started from P
mcc
.
Russian Journal of Genetics 07/2001; 37(8):876-883. · 0.43 Impact Factor
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ABSTRACT: Pseudomonas aureofaciens B-4117 and P. fluorescens CR330D inhibited the growth of a wide range of plant pathogens, including Agrobacterium tumefaciens when tested on agar media. In a series of nursery-based trials with natural pathogen inoculum, application of either B-4117 or CR330D significantly reduced the incidence and severity of crown gall on grapevine and raspberry caused by A. tumefaciens. The extent of disease control depended upon the variety tested. Both bacteria reduced disease during seedling root production and grafting. Disease incidence on root cuttings of three grapevine varieties was reduced by 56-80% and the disease severity index (DSI) was decreased by 75-86%. Depending on the scion variety, the number of healthy rooted grafts increased 2 - 3.5 fold, while the DSI was reduced 1.5 - 3 fold. The results suggest that there is potential in using these antagonists to diminish the influence of latent rootstock infection on graft sensitivity to crown gall. Pre-treatment of rooted raspberry seedlings with P. aureofaciens B-4117 prevented the development of crown galls caused by A. tumefaciens strain K24, or by a mixture of A. tumefaciens pathogenic strains previously isolated from raspberry. Both Pseudomonas spp. persisted on the root surfaces of inoculated vine cuttings and in non-sterile soil. The advantages of using the antagonistic bacteria as biocontrol agents of crown gall are discussed.
Biocontrol Science and Technology 01/1998; 8:45-57. · 0.92 Impact Factor
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ABSTRACT: Cloning of plasmid genes for the synthesis of two peptide broad-spectrum antibiotics-microcins B2 and B27- and for host cell immunity to their action was performed. Recombinant plasmids containing these genes were designated pBE108 and pVB27, respectively. Deletional derivatives of plasmid pBE108 and mutant plasmids were obtained via transposon Tn5 insertions, which did not determine production of microcin B2 and immunity to it. Phenotypic study and physical mapping of these plasmids demonstrated that a 4.2-kb DNA fragment is responsible for B2 microcin production; immunity is provided by a 1.4-kb DNA fragment. A 5-kb DNA fragment is necessary for microcin B27 synthesis and expression of immunity to its action. Homology between these fragments and with plasmid DNA for the synthesis of microcin B17 and immunity to it was found. Homology between plasmid genes determining synthesis of type B and C microcins and host cell immunity to them was not observed. The production of B27 microcin is controlled by the product of the ompR gene; B2 microcin synthesis does not depend on this product. The mutations recA and lexA increase the susceptibility of Escherichia coli cells to the action of microcins B2 and B27 but not microcin C51.
Genetika 07/1994; 30(6):731-9. · 0.44 Impact Factor
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ABSTRACT: A screening of 11956 enterobacteria isolates resulted in selection of seven active microcin-producing strains. The microcins were shown to be peptides or their derivatives with a rather broad spectrum of activity, mainly against Gram-negative bacteria. According to cross-immunity criteria, the microcins studied belonged to two of the previously suggested types, B (five strains) and C (two strains). Those of type B could be further classified into two subtypes on the account of differences in the spectrum of antibacterial activity. In five cases out of seven the microcin-producing ability has been attributed to plasmids that the strains harboured. The effect of microcins on sensitive cells was shown to depend on ompR and ompF gene products.
FEMS Microbiology Letters 09/1993; 111(2-3):269-74. · 2.04 Impact Factor
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ABSTRACT: As a result of screening among 11956 enterobacteria strains isolated from feces of normal children, grown-ups and lambs, seven active microcin-producing strains were obtained. The microcins were shown to be peptides or their derivatives with a low molecular weight (less than 10,000) and a broad spectrum of activity, mainly against gram-negative bacteria. According to cross immunity criteria the microcins studied belonged to two different types. Those of type I could be further classified into two subtypes on the account of difference in the spectrum of antibacterial activity. In 5 cases out of 7 the microcin-producing ability and immunity to microcins have been attributed to plasmids that the strains harboured. The effect of microcins on sensitive cells depended on ompR and ompF gene products.
Genetika 06/1993; 29(5):768-76. · 0.44 Impact Factor
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ABSTRACT: A new microcin produced by an Citrobacter R51 strain has been detected. This antibiotic has been shown to inhibit the growth of a number of Gram-negative as well as Gram-positive strains of bacteria on the minimal medium plates. The properties of partially purified microcin were characterized. Constitutive synthesis of microcin is determined by a conjugative plasmid. The genes of microcin synthesis and immunity were cloned on a plasmid and plasmid vehicles. A physical map of the 12 kb fragment coding for the production of microcin R51 and immunity to this antibiotic is presented.
Molekuliarnaia genetika, mikrobiologiia i virusologiia 06/1990;
