Fast and reliable procedure developed to generate soft rot Pectobacteriaceae (Pectobacterium spp. and Dickeya spp.) Tn5 mutants resistant to bacteriophage infection

A simple and fast procedure has been developed to generate soft rot Pectobacteriaceae (SRP: Pectobacterium spp. and Dickeya spp.) Tn5 mutants in genes encoding receptors used by bacteriophages to interact with their hosts, for the follow-up studies. The procedure is inexpensive and does not require any specialized tools and/or dedicated technical support. The neomycin-resistant SRP Tn5 mutants are generated via conjugation with a transposon donor Escherichia coli ST18 strain (requiring 5-aminolevulinic acid (5-ALA) to survive) carrying pFAJ1819-mini-Tn5-neoR. The conjugation is done on solid medium supplemented with 5-ALA. After conjugation bacterial cells are collected, suspended in liquid bacterial medium, added to the suspension containing lytic bacteriophages and incubated for the additional 30 min with shaking (120 rpm). During this stage, the transposon recipients (Pectobacterium spp. and/or Dickeya spp. Tn5 mutants), susceptible to bacteriophage infection are lysed. Likewise, due to the lack of 5-ALA in the growth medium, E. coli ST18 (transposon donor) cells die at this stage. Finally, after incubation, the bacterial mutants with the Tn5 insertions, resistant to phage infection are selected on solid growth medium supplemented with neomycin. The Tn5 insertion sites are sequenced to acquire knowledge about the Tn5-distrupted genes and their putative function in phage-host interactions. The proposed assay allows generation of a number of immediately-available Tn5 mutants expressing phage-resistant phenotypes in a short time (ca. 48 h) that can be later characterized for various other phenotypic features. In this study, as a proof-of-concept, this method has been used to generate Dickeya solani IPO2222 Tn5 mutants resistant to infection caused by the lytic bacteriophage ɸD5.

SENSITIVITY OF PURE CULTURES OF BRADYRHIZOBIUM JAPONICUM TO FUNGICIDES

Objective. Conduct screening of a wide variety of transgenic mutagenesis strains of Bradyrhizobium japonicum nodule bacteria by the sensitivity to Fever, Standak Top, Akanto Plus, Maxym XL, and Benorad fungicides, and obtain Tn5 mutants resistant to different normal rates of the above pesticides. Methods. Microbiological, statistical. Results. Under the conditions of laboratory experiments, Tn5 mutants of B. japonicum, resistant to the production and dual production normal rate of Fever, Standak Top, Akanto Plus, Maxym XL, and Benorad, were selected. It was shown that the active substances of the products with fungicidal activity Fever, Standak Top, Akanto Plus, Maxym XL do not have bactericidal effect on cell viability of the majority of Tn5 mutants obtained as a result of intergeneric conjugation between Escherichia coli S17-1 with different plasmid vectors and strains of B. japonicum 646 and 634b, and only in some cases reduce the intensity of their reproduction. It was established that Tn5 mutants of B. japonicum under study have different sensitivity to the influence of the normal rates of benomil-based Benorad recommended by the manufacturer and twice-increased. Nine Tn5 mutants were evaluated as low-sensitive to the production normal rate of Benorad, 11 were characterized by a higher sensitivity to fungicide, as evidenced by the zones of delayed growth of bacterial lawn around wells with the product over 15 mm. The influence of the double Benorad normal rate on transposon mutants was significantly stronger compared with other fungicides. Three Tn5 mutants, low-sensitive to the influence of the double rate of this product were selected, and delay of the reproduction of cells in these variants of the experiment was 14-15 mm around the wells. Conclusion. The use of bacterial fertilizers for soybeans based on Tn5 mutants of B. japonicum resistant to modern fungicides will help to reduce the consequences of chemical stress on the formation and functioning of symbiotic systems.

The reaction of soybean symbiotic apparatus to losses of water content in leaves and roots, induced by continuous action of drought

The reaction of the soybean symbiotic apparatus inoculated with Bradyrhizobium japonicum strains and Tn5 mutants, which were different in efficiency, was studied for the loss of water content in leaves and roots induced by prolonged drought, as well as the seed productivity of the formed symbiotic systems. To conduct researches were used microbiological, physiological, biochemical methods, gas chromatography and spectrophotometry. The objects of the study were selected symbiotic systems formed with the participation of soybean plants and strains B. japonicum 646 (active, virulent) and 604k (inactive, highly virulent), as well as Tn5-mutants – B1-20 (active, virulent) and 107 (low-active, virulent) obtained by the method of transposon mutagenesis in the department of symbiotic nitrogen fixation at the Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine. Before sowing, sterilized with 70 % ethanol and washed under running water for 1 h, the seeds were inoculated with suspensions of nodules bacteria (the titre of the suspension was 108 cells in 1 ml). The combined model drought was created during 12 days by stopping watering of plants up to 40 % of full moisture content starting from the stage of two true leaves and gradual transfer of watering to 30 % of full moisture content in the stage of three true leaves and budding - the beginning of flowering. After the stopping of drought, the moisture content of the substrate was adjusted to 60 % of full moisture content (watering recovery) into the stage of mass flowering. Control plants were inoculated by rhizobium culture, as well as plants without inoculation, which grew for optimal watering. It was investigated that in symbiotic systems formed with the participation of soybean and the active strain B. japonicum (646) and Tn5-mutant (B1-20) there was no significant reduction in the water content of plants under drought conditions and the effective work of the symbiotic apparatus was recorded, which contributed to the preservation of seed productivity. Ineffective symbiotic systems observed significant losses in water content and inhibition of the process of nodulation (strain 604k) and nitrogen fixation (Tn5-mutants 107), which was accompanied by significant losses of soybean crop yields. As a result of the research, it was concluded that in soil-climatic conditions with insufficient rainfall and frequent droughts, effective symbiotic systems should be used, which will promote the optimal functioning of the symbiotic apparatus and preserve the seed productivity of soybeans by adaptive regulation of water balance and fixation of molecular nitrogen of the atmosphere. The study of the functioning of leguminous plants in symbiosis with strains of nodule bacteria is important for finding effective symbiotic systems that are able to realize their adaptive potential for the effects of stress factors, in particular drought. Effective symbiotic relationships are the main source of nitrogen fixation in terrestrial ecosystems, which will reduce the need to enrich the soil with chemical compounds and provide additional economic and environmental advantage.

Nonspecific Symbiosis Between Sophora flavescens and Different Rhizobia

We explored the genetic basis of the promiscuous symbiosis of Sophora flavescens with diverse rhizobia. To determine the impact of Nod factors (NFs) on the symbiosis of S. flavescens, nodulation-related gene mutants of representative rhizobial strains were generated. Strains with mutations in common nodulation genes (nodC, nodM, and nodE) failed to nodulate S. flavescens, indicating that the promiscuous nodulation of this plant is strictly dependent on the basic NF structure. Mutations of the NF decoration genes nodH, nodS, nodZ, and noeI did not affect the nodulation of S. flavescens, but these mutations affected the nitrogen-fixation efficiency of nodules. Wild-type Bradyrhizobium diazoefficiens USDA110 cannot nodulate S. flavescens, but we obtained 14 Tn5 mutants of B. diazoefficiens that nodulated S. flavescens. This suggested that the mutations had disrupted a negative regulator that prevents nodulation of S. flavescens, leading to nonspecific nodulation. For Ensifer fredii CCBAU 45436 mutants, the minimal NF structure was sufficient for nodulation of soybean and S. flavescens. In summary, the mechanism of promiscuous symbiosis of S. flavescens with rhizobia might be related to its nonspecific recognition of NF structures, and the host specificity of rhizobia may also be controlled by currently unknown nodulation-related genes.

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.

Identification of Loci of Pseudomonas syringae pv. actinidiae Involved in Lipolytic Activity and Their Role in Colonization of Kiwifruit Leaves

Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae, an emerging pathogen of kiwifruit plants, has recently brought about major economic losses worldwide. Genetic studies on virulence functions of P. syringae pv. actinidiae have not yet been reported and there is little experimental data regarding bacterial genes involved in pathogenesis. In this study, we performed a genetic screen in order to identify transposon mutants altered in the lipolytic activity because it is known that mechanisms of regulation, production, and secretion of enzymes often play crucial roles in virulence of plant pathogens. We aimed to identify the set of secretion and global regulatory loci that control lipolytic activity and also play important roles in in planta fitness. Our screen for altered lipolytic activity phenotype identified a total of 58 Tn5 transposon mutants. Mapping all these Tn5 mutants revealed that the transposons were inserted in genes that play roles in cell division, chemotaxis, metabolism, movement, recombination, regulation, signal transduction, and transport as well as a few unknown functions. Several of these identified P. syringae pv. actinidiae Tn5 mutants, notably the functions affected in phosphomannomutase AlgC, lipid A biosynthesis acyltransferase, glutamate–cysteine ligase, and the type IV pilus protein PilI, were also found affected in in planta survival and/or growth in kiwifruit plants. The results of the genetic screen and identification of novel loci involved in in planta fitness of P. syringae pv. actinidiae are presented and discussed.