Regulation of superoxide dismutase activity in soybean plants by inoculating seeds with rhizobia containing nanoparticles of metal carboxylates under conditions of different water supply

Soybean is one of the most profitable advanced crops in agricultural production in Ukraine and the world as a whole. Therefore, studies of means of regulation and increase in the adaptive capacity of soybeans in symbiosis with nodule bacteria under the action of unfavourable environmental factors are relevant and should be aimed at the use of complex bacterial compositions involving modern nanotechnological approaches. Nanocarboxylates of ferrum, molybdenum and germanium metals were used as components of rhizobia inoculation suspension for soybean seed treatment to study the effectiveness of their complex effect on the regulation of the activity of the key antioxidant enzyme superoxide dismutase in plants under drought. Various symbiotic systems were used, which included soybean plants and inoculation suspensions based on the active, virulent Tn5-mutant Bradyrhizobium japonicum B1-20 by adding nanoparticles of ferrum, germanium and molybdenum carboxylates to the culture medium in a ratio of 1: 1000. Citric acid was the chelator. A model drought lasting 14 days was created during the period of active fixation of atmospheric molecular nitrogen by root nodules of soybeans in the budding and flowering stages, by means of controlled watering of plants to 30% of the total moisture content. In the stage of bean formation, watering of plants was resumed to the optimal level – 60% of the total moisture content. The control was soybean plants, the seeds of which were inoculated with a suspension of rhizobia without the addition of chelated metals. The following research methods were used in the work – microbiological, physiological and biochemical. According to the results, it was found that when nanoparticles of carboxylates of ferrum, molybdenum and germanium were added to the inoculation suspension of rhizobia, there was an increase in superoxide dismutase activity in root nodules and a decrease in soybean leaves under optimal water supply conditions of plants. This indicates the initial changes in the activity of the antioxidant enzyme in these symbiotic systems, induced by the influence of chelated metals in combination with the rhizobia of the active Tn5-mutant B. japonicum B1-20. Prolonged drought induced an increase in the overall level of superoxide dismutase activity in soybean nodules and leaves, compared to plants grown under optimal watering conditions. The symbiotic system formed by soybeans and B. japonicum with molybdenum carboxylate nanoparticles was the most sensitive to long-term drought exposure, compared to two other soybean-rhizobial symbioses using ferrum and germanium nanocarboxylates. This was manifested in the unstable reaction of the enzyme to the action of drought – suppression or intensification of the level of its activity in the root nodules and leaves of soybeans inoculated with rhizobia containing molybdenum carboxylate nanoparticles. In symbiotic systems with the participation of germanium and ferrum nanocarboxylates, slight changes were revealed in superoxide dismutase activity in root nodules and leaves of plants during drought and restoration of enzyme activity to the level of plants with optimal watering after water stress. It is concluded that the addition to the culture medium of rhizobia Tn5-mutant B1-20 of nanocarboxylates of germanium or ferrum is an effective means of regulating the activity of the antioxidant enzyme superoxide dismutase in soybean root nodules and leaves, which can contribute to an increase in the protective properties and adaptation of plants to the action of dehydration.

NirA is an alternative nitrite reductase fromPseudomonas aeruginosawith potential as an anti-virulence target

ABSTRACTThe opportunistic pathogenPseudomonas aeruginosaproduces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, anti-virulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treatP. aeruginosainfections. To identifyP. aeruginosagenes required for virulence in multiple hosts, a random library of Tn5 mutants in PAO1-L was previously screenedin vitrofor those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we have identified a Tn5 mutant with an insertion in PA4130 showing reduced levels in a number of virulence traitsin vitro. Construction of an isogenic mutant in this gene presented similar results as those from the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models ofDrosophila melanogaster,Caenorhabditis elegansas well as decreased toxicity in human cell lines. This mutant also presented an 80% increased survival in murine acute and agar-bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulphite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this protein is a nitrite reductase operating in a siroheme and 4Fe-4S dependant manner. The preference for nitrite and the production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named as NirA.

EFFECT OF FUNGICIDES ON THE EFFICIENCY OF SOYBEAN INOCULATION WITH PESTICIDE-RESISTANT NODULE BACTERIA

Objective. Evaluate the efficiency of soybean inoculation with pesticide-resistant nodule bacteria Bradyrhizobium japonicum under the influence of pre-sowing seed dressing with Maxym XL,Standak Top and Fever and preventive spraying of plants with the fungicide Akanto Plus. Methods. Physiological, microbiological, gas chromatographic, vegetation experiment, statistical.Results. The number and weight of nodules formed on soybean roots during the growing seasonunder the action of the prothioconazole-based fungicide Fever were reported to be lower comparedwith the control and other variants of experiment. Pesticides Maxym XL and Standak Top had a lesspronounced toxic effect on the formation of legume-rhizobial symbiosis with the participation offungicide-resistant B. japonicum B78 Tn5 mutant compared to Fever. A decrease in the level of N2assimilation by 10.9–41.1 % in the three-leaf phase was found with the combined use of seed pesticides and nodule bacteria included in the study, relative to the variant with inoculation only. However, during the growing season, the negative effect of fungicidal preparations on the functioning oflegume-rhizobial symbiosis decreased. Conclusion. Under the conditions of the model vegetationexperiment with the concomitant use of Standak Top and Fever and bacterization, a decrease in individual plant productivity by 7.0 and 14.1 %, respectively, has been reported. The complex actionof the pesticide Maxym XL and inoculation with pesticide-resistant B. japonicum B78 transposonmutant, contributed to the maximum increase in yield by 27.2 %. The obtained data confirm the expediency of selection of fungicides and nitrogen-fixing microorganisms for concomitant use to ensure high economic and protective effects.

A DeoR-Type Transcription Regulator Is Required for Sugar-Induced Expression of Type III Secretion-Encoding Genes in Pseudomonas syringae pv. tomato DC3000

The type III secretion system (T3SS) of plant-pathogenic Pseudomonas syringae is essential for virulence. Genes encoding the T3SS are not constitutively expressed and must be induced upon infection. Plant-derived metabolites, including sugars such as fructose and sucrose, are inducers of T3SS-encoding genes, yet the molecular mechanisms underlying perception of these host signals by P. syringae are unknown. Here, we report that sugar-induced expression of type III secretion A (setA), predicted to encode a DeoR-type transcription factor, is required for maximal sugar-induced expression of T3SS-associated genes in P. syringae DC3000. From a Tn5 transposon mutagenesis screen, we identified two independent mutants with insertions in setA. When both setA::Tn5 mutants were cultured in minimal medium containing fructose, genes encoding the T3SS master regulator HrpL and effector AvrRpm1 were expressed at lower levels relative to that of a wild-type strain. Decreased hrpL and avrRpm1 expression also occurred in a setA::Tn5 mutant in response to glucose, sucrose, galactose, and mannitol, demonstrating that setA is genetically required for T3SS induction by many different sugars. Expression of upstream regulators hrpR/S and rpoN was not altered in setA::Tn5, indicating that SetA positively regulates hrpL expression independently of increased transcription of these genes. In addition to decreased response to defined sugar signals, a setA::Tn5 mutant had decreased T3SS deployment during infection and was compromised in its ability to grow in planta and cause disease. These data suggest that SetA is necessary for P. syringae to effectively respond to T3SS-inducing sugar signals encountered during infection.

Capsular Polysaccharide Is a Receptor of a Clostridium perfringens Bacteriophage CPS1

Clostridium perfringens is a Gram-positive, anaerobic, and spore forming bacterium that is widely distributed in the environment and one of the most common causes of foodborne illnesses. Bacteriophages are regarded as one of the most promising alternatives to antibiotics in controlling antibiotic-resistant pathogenic bacteria. Here we isolated a virulent C. perfringens phage, CPS1, and analysis of its whole genome and morphology revealed a small genome (19 kbps) and a short noncontractile tail, suggesting that CPS1 can be classified as a member of Picovirinae, a subfamily of Podoviridae. To determine the host receptor of CPS1, the EZ-Tn5 random transposon mutant library of C. perfringens ATCC 13124 was constructed and screened for resistance to CPS1 infection. Analysis of the CPS1-resistant mutants revealed that the CPF_0486 was disrupted by Tn5. The CPF_0486 was annotated as galE, a gene encoding UDP-glucose 4-epimerase (GalE). However, biochemical analyses demonstrated that the encoded protein possessed dual activities of GalE and UDP-N-acetylglucosamine 4-epimerase (Gne). We found that the CPF_0486::Tn5 mutant produced a reduced amount of capsular polysaccharides (CPS) compared with the wild type. We also discovered that glucosamine and galactosamine could competitively inhibit host adsorption of CPS1. These results suggest that CPS acts as a receptor for this phage.

The NtrY/NtrX System of Sinorhizobium meliloti GR4 Regulates Motility, EPS I Production, and Nitrogen Metabolism but Is Dispensable for Symbiotic Nitrogen Fixation

Sinorhizobium meliloti can translocate over surfaces. However, little is known about the regulatory mechanisms that control this trait and its relevance for establishing symbiosis with alfalfa plants. To gain insights into this field, we isolated Tn5 mutants of S. meliloti GR4 with impaired surface motility. In mutant strain GRS577, the transposon interrupted the ntrY gene encoding the sensor kinase of the NtrY/NtrX two-component regulatory system. GRS577 is impaired in flagella synthesis and overproduces succinoglycan, which is responsible for increased biofilm formation. The mutant also shows altered cell morphology and higher susceptibility to salt stress. GRS577 induces nitrogen-fixing nodules in alfalfa but exhibits decreased competitive nodulation. Complementation experiments indicate that both ntrY and ntrX account for all the phenotypes displayed by the ntrY::Tn5 mutant. Ectopic overexpression of VisNR, the motility master regulator, was sufficient to rescue motility and competitive nodulation of the transposant. A transcriptome profiling of GRS577 confirmed differential expression of exo and flagellar genes, and led to the demonstration that NtrY/NtrX allows for optimal expression of denitrification and nifA genes under microoxic conditions in response to nitrogen compounds. This study extends our knowledge of the complex role played by NtrY/NtrX in S. meliloti.

QUORUM QUENCHING POTENTIALS OF PROBIOTIC ENTEROCOCCUS DURANS LAB38 AGAINST METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS

Objective: he focus of this study was to explore the nuance strategy to combat the virulence factors of the pathogens by probiotic Enterococcus durans LAB38Methods: Probiotic attributes was determined by bile salt tolerance (0.5%) and Artemia gnotobiotic assay. Quorum sensing (QS) inhibitory activity of the supernatant and ethyl acetate (EA) extract of LAB38 was evaluated by using the indicator strains, includes Chromobacterium violaceum CV026 (mini-Tn5 mutant of ATCC 31532), methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA). Reporter strains, Vibrio harveyi BB170 (luxN mutant), BB886 (luxP mutant) and Escherichia coli pSB401 (pACYC184- derived) were used for bioluminescence-based target specificity analysis. GC-MS analysis of EA extract was performed using standard protocol. Results: LAB38 has shown bile salt tolerance and positive probiotic effect towards Artemia salina. In addition, 100 µg/ml EA extract has significantly reduced the violacein production (37 ± 1.4%) in CV026, biofilm formation in MRSA (94 ± 0.9 %) and PA (22 ± 0.08%). Further, 200 µg/ml of EA extract has shown inhibition against both autoinducer-1 and autoinducer-2 mediated QS system. Bioluminescence inhibition is directly proportional to the time of exposure. GC-MS result revealed that bromine, sulphur containing molecule and azulene derivative were found in the EA extract.Conclusion This is the first report on probiotic Enterococcus durans for quorum quenching activity. Hence, the bacterium could be used for future therapeutics application. Keywords: Autoinducer, Biofilm, Methicillin resistant Staphylococcus aureus, Quorum quenching, Probiotics, CV026.

Type IV Pili Are Required for Virulence, Twitching Motility, and Biofilm Formation of Acidovorax avenae subsp. citrulli

Acidovorax avenae subsp. citrulli is the causal agent of bacterial fruit blotch (BFB), a threatening disease of watermelon, melon, and other cucurbits. Despite the economic importance of BFB, relatively little is known about basic aspects of the pathogen's biology and the molecular basis of its interaction with host plants. To identify A. avenae subsp. citrulli genes associated with pathogenicity, we generated a transposon (Tn5) mutant library on the background of strain M6, a group I strain of A. avenae subsp. citrulli, and screened it for reduced virulence by seed-transmission assays with melon. Here, we report the identification of a Tn5 mutant with reduced virulence that is impaired in pilM, which encodes a protein involved in assembly of type IV pili (TFP). Further characterization of this mutant revealed that A. avenae subsp. citrulli requires TFP for twitching motility and wild-type levels of biofilm formation. Significant reductions in virulence and biofilm formation as well as abolishment of twitching were also observed in insertional mutants affected in other TFP genes. We also provide the first evidence that group I strains of A. avenae subsp. citrulli can colonize and move through host xylem vessels.

INVESTIGATION OF SYMBIOTIC PROPERTIES OF TN5-MUTANTS SOYBEAN NODULE BACTERIA

Effectiveness forming and functioning of nitrogen-fixing systems created by soybean (Glycine max (L.) Merr.) and Tn5-mutants of Bradyrhizobium japonicum 646 with different symbiotic properties were studied in green house experiments. Inoculation of seeds by Tn5- mutants 107 and 113 with high virulence, but low activities, lead to decrease of the biometric properties and enhanced yield of plant seeds in comparison with control strains wild 646 and production 634б. Tn5- mutant 118-8 had the low nodulation and nitrogen fixing activities. Inoculation of seeds by high activity mutants 21-2 and 17-2 got reliable increase grain yield of plants in comparison with control. Consequently, this Tn5-mutants may to be used as perspective initial material for the next selection productive nodulate bacteria strains.