Broad-range antagonistic rhizobacteria Pseudomonas fluorescens and Serratia plymuthica suppress Agrobacterium crown gall tumours on tomato plants.

AIM: To examine the biocontrol activity of broad-range antagonists Serratia plymuthica IC1270, Pseudomonas fluorescens Q8r1-96 and P. fluorescens B-4117 against tumourigenic strains of Agrobacterium tumefaciens and A. vitis. METHODS AND RESULTS: Under greenhouse conditions, the antagonists, applied via root soak prior to injecting Agrobacterium strains into the wounded stems, significantly suppressed tumour development on tomato seedlings. A derivative of P. fluorescens Q8r1-96 tagged with a gfp reporter, as well as P. fluorescens B-4117 and S. plymuthica IC1270 marked with rifampicin resistance, stably persisted in tomato tissues for at least 1 month. Mutants of P. fluorescens Q8r1-96 and S. plymuthica IC1270 deficient in 2,4-diacetylphloroglucinol or pyrrolnitrin production, respectively, also proficiently suppressed the tumour development, indicating that these antibiotics are not responsible for the observed biocontrol effect on crown gall disease. The volatile organic compounds (VOCs) produced by the tested P. fluorescens and S. plymuthica strains inhibited the growth of A. tumefaciens and A. vitis strains in vitro. Solid-phase microextraction-gas chromatography-mass spectrometry analysis revealed dimethyl disulfide (DMDS) as the major headspace volatile produced by S. plymuthica IC1270; it strongly suppressed Agrobacterium growth in vitro and was emitted by tomato plants treated with S. plymuthica IC1270. 1-Undecene was the main volatile emitted by the examined P. fluorescens strains, with other volatiles, including DMDS, being detected in only relatively low quantities. CONCLUSIONS: S. plymuthica IC1270, P. fluorescens B-4117 and P. fluorescens Q8r1-96 can be used as novel biocontrol agents of pathogenic Agrobacterium. VOCs, and specifically DMDS, might be involved in the suppression of oncogenicity in tomato plants. However, the role of specific volatiles in the biocontrol activity remains to be elucidated. SIGNIFICANCE AND IMPACT OF THE STUDY: The advantage of applying these antagonists lies in their multiple activities against a number of plant pathogens, including Agrobacterium.

[Involvement of the global regulators GrrS, RpoS, and SplIR in formation of biofilms in Serratia plymuthica].

Most bacteria exist in the natural environment as biofilms, multicellular communities attached to hard surfaces. Biofilms have a characteristic architecture and are enclosed in the exopolymer matrix. Bacterial cells in biofilms are extremely resistant to antibacterial factors. It was shown in this work that the GrrA/GrrS system of global regulators of gene expression and the sigma S subunit of RNA polymerase (RpoS) play a significant role in positive regulation of biofilm formation in the rhizospheric bacterium Serratia plymuthica IC1270. Inactivation of grrS and rpoS genes resulted in an up to six-to-sevenfold and four-to-fivefold reduction in biofilm formation, respectively. Mutations in the grrS gene decreased the capacity of the bacterium for swarming motility. The splIR Quorum Sensing (QS) system was shown to negatively influence the biofilm formation. Transfer of the recombinant plasmid containing cloned genes splI/splR of S. plymuthica HRO-C48 into S. plymuthica IC1270 cells led to a twofold decrease of their ability to form biofilms. Inactivation of the splI gene coding for the synthase of N-acyl-homoserine lactones in S. plymuthica HRO-C48 resulted in a 2-2.5-fold increase in the level of biofilm formation, whereas the inclusion of plasmid carrying the cloned splI/splR genes into these mutant cells restored the biofilm formation to the normal level. The results obtained demonstrate that the formation of biofilms in S. plymuthica is positively regulated by the GrrA/GrrS and RpoS global regulators and is negatively regulated by the SplIR QS system.

[Structure and expression of gene vfr in Pseudomonas chlororaphis 449].

Gene vfr previously described only in Pseudomonas aeruginosa was cloned, identified, and sequenced in cells of Pseudomonas chlororaphis 449; 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 partially complementary to mutation at crp gene in cells of E. coli AM306 enhancing ten times synthesis of CRP protein-dependent beta-galactosidase. 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.

Phytase activity and its regulation in a rhizospheric strain of Serratia plymuthica.

Serratia plymuthica strain IC1270 isolated from the rhizosphere, possessing antagonistic activity towards a wide range of plant-pathogenic fungi, is able to hydrolyze phytate. Phytase activity was found intracellularly, while no activity was detected in the culture liquid. Optimum activity was found at pH 4-5; it completely disappeared at pH > 7.0 and 2.5. Phytase production was practically absent in the exponential phase and reached a maximum in the late stationary phase. Mutations of genes grrA and grrS, encoding GacA/GacS-like 2-component global regulatory system, or in gene rpoS encoding the sigma factor RpoS subunit of RNA polymerase, led to a deficiency in phytase production. Introduction into mutants of the respective wild-type genes cloned into the wide-range plasmid pJFF224-NX under the control of the bacteriophage T4 gene 32 promoter complemented this deficiency. This is the first report implicating the GacA/GacS global regulators and RpoS factor in phytase production in bacteria.

[Quorum sensing systems of regulation, synthesis of phenazine antibiotics, and antifungal (corrected) activity in rhizospheric bacterium Pseudomonas chlororaphis 449].

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: PhzIR 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.

Activity of Serratia plymuthica IC1270 gene chiA promoter region in Escherichia coli mutants deficient in global regulators of transcription.

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 .

Molecular mechanisms underlying carotenogenesis in the chromoplast: multilevel regulation of carotenoid-associated genes

In nature, carotenoid function and mode of action are highly determined by the neighboring protein and lipid molecules. Therefore an understanding of the proteins' involvement in carotenoid sequestration would be of great help in elucidating carotenoid function in vivo. Based on a study of the expression of chromoplast-specific carotenoid-associated genes from cucumber corolla (CHRC and CHRD), a working model is presented wherein two major regulatory factors control carotenoid sequestration within the chromoplasts: (i) floral tissue-specific transcriptional regulators of chromoplasto- genesis; and (ii) post-transcriptional regulators related to the amount/type of sequestered carotenoids. This model is supported by the major role transcriptional regulation was found to play in the temporal and spatial expression of the CHRC gene, and by the fact that phytohormones such as gibberellic acid (GA3), abscisic acid and ethylene also acted as transcriptional regulators of CHRC expression. The primary response to GA3 was localized within the CHRC promoter to a 290 bp fragment. Furthermore, we demonstrated strong down-regulation of CHRC expression at post-transcriptional and translational/post-translational levels resulting from inhibition of carotenoid biosynthesis, thus revealing a close link between carotenoid biosynthetic and sequestration machineries.

Carotenoid sequestration in plants: the role of carotenoid-associated proteins.

In plants, carotenoid accumulation and sequestration take place within chloroplasts and chromoplasts. In the chloroplast, practically all carotenoids are associated with chlorophyll-binding proteins, whereas chromoplasts have developed a unique mechanism to sequester carotenoids within specific lipoprotein structures. Recent research into the existence of a group of homologous genes that encode carotenoid-associated proteins that aid in the generation of carotenoid-lipoprotein structures in chromoplasts, offers a new framework for elucidating the carotenoid sequestration mechanism.

CHRC, encoding a chromoplast-specific carotenoid-associated protein, is an early gibberellic acid-responsive gene.

CHRC, a corolla-specific carotenoid-associated protein, is a major component of carotenoid-lipoprotein complexes in Cucumis sativus chromoplasts. Using an in vitro flower bud culture system that mimics in vivo flower development, CHRC mRNA levels in corollas were shown to be specifically up-regulated by gibberellic acid. The response to gibberellic acid was very rapid (within 20 min) and insensitive to protein synthesis inhibition by cycloheximide. Abscisic acid, known to antagonize gibberellin in many developmental systems, strongly down-regulated CHRC mRNA levels. The gibberellin synthesis inhibitor paclobutrazol exhibited a similar negative effect on CHRC expression. Inclusion of exogenous gibberellic acid into the in vitro bud culture system with the paclobutrazol not only prevented the CHRC mRNA down-regulation, it up-regulated transcript accumulation to the level of gibberellic acid-treated corollas. CHRC mRNA accumulation in response to gibberellic acid displayed a dose-dependent increase up to 10(-4) M gibberellic acid. The up-regulation could be detected with as little as 10(-7) M gibberellic acid. Based on these data, we suggest that CHRC is the first structural gene identified to date whose expression is regulated by gibberellic acid in a primary fashion. The critical role of the rapid response of CHRC to gibberellic acid in aiding carotenoid sequestration while preserving chromoplast structural organization is discussed.

Isolation and regulation of accumulation of a minor chromoplast-specific protein from cucumber corollas.

The differentiation of chloroplasts to chromoplasts in cucumber (Cucumis sativus L.) corollas parallels flower development. Chromoplast biogenesis involves chlorophyll degradation, carotenoid accumulation, and the appearance of a new set of proteins. To study factors involved in chromoplast biogenesis in floral tissues, a minor (in abundance) protein of about 14 kD, CHRD (chromoplast protein D), was isolated from cucumber corolla chromoplasts. Immunological characterization revealed that the protein is chromoplast-specific and that its steady-state level in corollas increases in parallel to flower development. The protein was not detected in cucumber leaves or fruits. Immunological analysis of corollas and fruits from variety of other plants also did not reveal cross-reactivity with the CHRD protein antisera. Using an in vitro bud culture system, we analyzed the effect of phytohormones on CHRD expression. Gibberellic acid rapidly enhanced, whereas paclobutrazol down-regulated, the steady-state level of CHRD. Ethylene also down-regulated the protein's steady-state level. It is suggested that hormonal control of chromoplastogenesis is tightly regulated at the tissue/organ level and that mainly developmental signals control carotenoid accumulation in nonphotosynthetic tissues.

Molecular cloning of a carotenoid-associated protein from Cucumis sativus corollas: homologous genes involved in carotenoid sequestration in chromoplasts.

Chromoplasts are carotenoid-accumulating plastids found in the corollas and fruits of many higher plants. In most cases, the pigment in these plastids is accumulated with the aid of carotenoid-associated proteins located within unique structures. This paper reports the isolation and characterization of the cDNA (CHRC) from Cucumis sativus corollas which encodes the chromoplast-specific carotenoid-associated protein CHRC. The transit peptide cleavage site was determined and, using a chloroplast uptake system, it is shown that CHRC can be post-translationally targeted to these plastids where it is peripherally associated with thylakoids. Analysis of CHRC transcript level in Cucumis sativus revealed its temporal and tissue-specific regulation: the transcript was detected only in corollas, where its level increased in parallel to flower development, peaking just before anthesis. CHRC shares significant homology (59%) with the gene coding for fibrillin-a protein in Capsicum annuum red fruits whose function is essentially identical to that of CHRC. A CHRC fragment including the potential active site of the protein was used as a probe in Northern blot analyses of floral and fruit tissues from various plants containing chromoplasts of different types: CHRC homologs of similar sizes were revealed in all cases. The existence of a group of homologous genes coding for chromoplast-specific proteins which aid in the sequestration of carotenoids within specific structures is proposed.

RecP, a new minor pathway of general recombination in Escherichia coli encoded by plasmid R1drd-19.

Plasmid R1drd-19 markedly improves the recombination deficiency of recB and recBrecC mutants of Escherichia coli K12 as measured by Hfr crosses and increases their resistance to uv inactivation. The effect correlates with the production of an ATP-dependent ds DNA exonuclease in recB/R1drd-19 cells. This paper further investigates the suppressive effect of plasmid R1drd-19 on the recB mutation of E. coli. The gene(s) responsible for the effect was localized to the 13.1-kb EcoRI-C fragment of the resistance transfer factor (RTF) portion of R1drd-19. The plasmid-encoded activity does not merely replace the RecBCD enzyme failure but differs in several significant ways. It promotes a hyper-recombinogenic phenotype, as judged by the phenomenon of super oligomerization of the tester pACYC184 plasmid in recB/R1drd-19 cells and two inter- and intramolecular plasmid recombination test systems. It is probably not inhibited by lambda Gam protein and does not restrict plating of T4gp2 mutant. No significant homology between the E. coli chromosomal fragment carrying recBrecCrecD genes and the EcoRI-C fragment of R1drd-19 was observed. It is suggested that the plasmid-encoded recombination activity is involved in a new minor recombination pathway (designated RecP, for Plasmid). RecP resembles in some traits the RecBCD-independent pathways RecE and RecF but differs in activity and perhaps substrate specificity from the main RecBCD pathway.

[A comparative study of the functions of recBCD-nuclease from Escherichia coli and "recombinase", encoded by plasmid R1drd-19]. / Sravnitel'noe issledovanie funktsii recBCD-nukleazy Escherichia coli i "recombinazy", kodiruemoi plazmidoi R1drd-19.

The RecBCD nuclease of Escherichia coli and "recombinase" determined by R1drd-19 plasmid (the latter is able to replace at least partially the indicated cellular enzyme) were shown to differ from each other in some essential features. The product encoded by the plasmid as distinct from RecBCD nuclease practically is not sensitive to inhibition by GamS protein of the lambda phage. Earlier, it was found that the presence of R1drd-19 plasmid in the recBC cells restores the level of the total ATP-dependent exonuclease activity because of appearance in such cells of a new exonuclease activity also ATP-dependent. The exonuclease activity determined by R1drd-19 plasmid was found to differ from the corresponding activity of the RecBCD enzyme. The plasmid enzyme was able to prevent reproduction of T4g2- mutant on recBC cells. The ability of the plasmid "recombinase" to some stimulation of intrachromosomal recombination in recA mutant witness to incomplete RecA-dependence of its function. No significant homology was registered between Escherichia coli DNA fragment containing the recB, recC, recD genes and the EcoRI-C-fragment of R1drd-19 carrying the sequences responsible for recombination and repair functions of the plasmid.

[Plasmid R1drd-19-mediated superoligomerization of the plasmid pACYC184in the recB mutant of Escherichia coli K12]. / Obuslovlennaia plazmidoi R1drd-19 sverkholigomerizatsiia plazmidy pACYC184 v mutante recB Escherichia coli K12.

It was found that monomers of the pACYC184 plasmid undergo superoligomerization in a recB mutant of Escherichia coli K12 which is deficient in ATP-dependent RecBC nuclease and carries the drug resistance plasmid R1drd-19. The observed effect is specifically related to the ability of R1drd-19 to determine an ATP-dependent exonucleolytic activity which is functionally similar but not identical to the RecBC nuclease. The oligomerization of pACYC184 is accompanied by the formation of high-order circular structures, and this leads to elimination of the plasmid from cells growing under non-selective conditions.

[Identification of the plasmid R1drd-19 region complementing the mutant phenotype recB- in Escherichia coli K12 strain]. / Identifikatsiia raiona plazmidy R1drd-19, komplementiruiushchego mutantnyi fenotip recB- shtamma Escherichia coli K12.

Plasmid R1-19 and its copy number mutants markedly increase the recombinational efficiency of a recB- strain of E. coli K12 and its resistance to the lethal action of UV and mitomycin C. These effects are associated with the appearance of a new ATP-dependent exonuclease activity in recB- cells known to be deficient in the ATP-dependent exonuclease V. Using hybrid plasmids carrying different EcoRI fragments of R1-19 (in the pSF124 vector), the gene(s) responsible for effect of R1-19 in recB-cells were localized in the EcoRI-C fragment (8.5 MD) belonging to the RTF portion of R1-19. Expression of the gene(s) in hybrid plasmids depends on the orientation of EcoRI-C fragment in the vector. The copy number of the EcoRI-C fragment was not strictly correlated with the degree of expression of the effects in the recB- mutant.

[Participation of plasmid F in the replication of the chromosome of an Hfr strain of Escherichia coli K-12]. / Uchastie plazmidy F v replikatsii khromosomy Hfr shtamma Escherichia coli K-12.

In the region of plasmid F DNA with coordinates 52,2-55,8 kb, the chr ("chromosome replication") locus has been revealed. A failure in the functioning of this locus in the integrated plasmid, which leads to a temperature-sensitive disturbance in chromosome replication of the Hfr strain and to the changes in its sensitivity to some membranotropic agents. Integration of an F segment containing the chr+ allele into the chromosome of an F-like derivative of such Hfr strain (retaining a mutant part of the F DNA), results in formation of temperature-resistant clones. In these clones, chromosomal replication is controlled by the plasmid replicon at the elevated temperature. It has been concluded that the F plasmid can control chromosome replication of the dna+ HfrC strain of Escherichia coli K-12 and that the product of the chr gene is a membrane protein involved in chromosomal replication.

[Effect of plasmid R6K on expression of the temperature-sensitive mutation in gene dnaE of Escherichia coli K-12]. / Vliianie plazmidy R6K na proiavlenie temperaturchuvstvitel'noi mutatsii v gene dnaE u Escherichia coli K-12.

The multicopy, conjugative R6K plasmid is responsible for the increase in the number of temperature-resistant (Tr) clones formed by Escherichia coli K-12 E486 strain, carrying a ts mutation dnaE486 in the gene coding for DNA polymerase III. The effect observed is not due to the mutator action of R6K or a mutation in the plasmid and requires the intactness of the host recA function. Tr derivatives MG488 and MG492, isolated under non-permissive condition from the strain E486(R6K), still possess the ts allele dnaE486 in the chromosome. Both Tr derivatives are more resistant to UV-irradiation and the characterized with a different level of spontaneous and UV-induced mutagenesis as compared to the initial strain E486(R6K). The data obtained suggest that the plasmid R6K is involved in the metabolism of chromosomal DNA of the host.

Plasmid control of recombination of E. coli K12.

The recombination proficiency of three recipient strains of Escherichia coli K12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec- derivatives. The same plasmid was also found to protect different rec- derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.

F plasmid genes involved in the production of recombination-stimulating factor, control of sensitivity to some injurious agents, and chromosome replication in Escherichia coli K-12 HfrC.

Three related F'arg+ plasmids isolated by Guyer and Clark were used to analyze some properties of strain MG751, a recipient derivative of an HfrC mutant (MG7) carrying a previously described pleiotropic mutation in the integrated F plasmid. Strains MG7 and MG751 both failed to produce recombination-stimulating factor, were sensitive to monofunctional alkylating agents and UV irradiation, and were temperature sensitive for growth and DNA synthesis. It was shown that these phenotypes are controlled by F plasmids genes (designated rsf, prt, and rep) that can be separated by deletion mutations occurring on the F plasmid.

[Identification of 3 genes in the F-plasmid of Escherichia coli K-12]. / Identifikatsiia trekh genov v F-plazmide Escherichia coli K-12.

The F'argG plasmid and its two transfer-deficient (tra-) analogues have been used to analyse the pleiotropic effect of a mutation in the integrated F-factor of HfrC strain. This mutation has been shown to disturb the functioning of at least three plasmid genes constituting, probably, a single regulon: the rsf gene determining the production of recombination-stimulating factor via conjugation (RSF); the prt gene responsible for the protective effect of the plasmid against N-methyl-N'-nitro-N-nitrosoguanidine, methyl methanesulphonate and UV-irradiation; the rep gene the product of which can be involved in the control of Hfr-chromosome replication. Possible location and order of the genes in the F-plasmid are discussed.