Tn5 Mutant Research Articles

RpoS activates formation of Salmonella Typhi biofilms and drives persistence in the gall bladder.

The development of strategies for targeting the asymptomatic carriage of Salmonella Typhi in chronic typhoid patients has suffered owing to our basic lack of understanding of the molecular mechanisms that enable the formation of S. Typhi biofilms. Traditionally, studies have relied on cholesterol-attached biofilms formed by a closely related serovar, Typhimurium, to mimic multicellular Typhi communities formed on human gallstones. In long-term infections, S. Typhi adopts the biofilm lifestyle to persist in vivo and survive in the carrier state, ultimately leading to the spread of infections via the fecal-oral route of transmission. In the present work, we studied S. Typhi biofilms directly, applied targeted as well as genome-wide genetic approaches to uncover unique biofilm components that do not conform to the CsgD-dependent pathway established in S. Typhimurium. We undertook a genome-wide Tn5 mutation screen in H58, a clinically relevant multidrug resistance strain of S. Typhi, in gallstone-mimicking conditions. We generated New Generation Sequencing libraries based on the ClickSeq technology to identify the key regulators, IraP and RpoS, and the matrix components Sth fimbriae, Vi capsule and lipopolysaccharide. We discovered that the starvation sigma factor, RpoS, was required for the transcriptional activation of matrix-encoding genes in vitro, and for S. Typhi colonization in persistent infections in vivo, using a heterologous fish larval model. An rpoS null mutant failed to colonize the gall bladder in chronic zebrafish infections. Overall, our work uncovered a novel RpoS-driven, CsgD-independent paradigm for the formation of cholesterol-attached Typhi biofilms, and emphasized the role(s) of stress signaling pathways for adaptation in chronic infections. Our identification of the biofilm regulators in S. Typhi paves the way for the development of drugs against typhoid carriage, which will ultimately control the increased incidence of gall bladder cancer in typhoid carriers.

Regulatory mechanism of C4-dicarboxylates in cyclo (Phe-Pro) production

Cyclo (Phe-Pro) (cFP), a cyclic dipeptide with notable antifungal, antibacterial, and antiviral properties, shows great promise for biological control of plant diseases. Produced as a byproduct by non-ribosomal peptide synthetases (NRPS), the regulatory mechanism of cFP biosynthesis remains unclear. In a screening test of 997 Tn5 mutants of Burkholderia seminalis strain R456, we identified eight mutants with enhanced antagonistic effects against Fusarium graminearum (Fg). Among these, mutant 88’s culture filtrate contained cFP, confirmed through HPLC and LC-MS, which actively inhibited Fg. The gene disrupted in mutant 88 is part of the Dct transport system (Dct-A, -B, -D), responsible for C4-dicarboxylate transport. Knockout mutants of Dct genes exhibited higher cFP levels than the wild type, whereas complementary strains showed no significant difference. Additionally, the presence of exogenous C4-dicarboxylates reduced cFP production in wild type R456, indicating that these substrates negatively regulate cFP synthesis. Given that cFP synthesis is related to NRPS, we previously identified an NRPS cluster in R456, horizontally transferred from algae. Specifically, knocking out gene 2061 within this NRPS cluster significantly reduced cFP production. A Fur box binding site was predicted upstream of gene 2061, and yeast one-hybrid assays confirmed Fur protein binding, which increased with additional C4-dicarboxylates. Knockout of the Fur gene led to up-regulation of gene 2061 and increased cFP production, suggesting that C4-dicarboxylates suppress cFP synthesis by enhancing Fur-mediated repression of gene 2061.

Transposon sequencing reveals metabolic pathways essential for Mycobacterium tuberculosis infection.

New drugs are needed to shorten and simplify treatment of tuberculosis caused by Mycobacterium tuberculosis. Metabolic pathways that M. tuberculosis requires for growth or survival during infection represent potential targets for anti-tubercular drug development. Genes and metabolic pathways essential for M. tuberculosis growth in standard laboratory culture conditions have been defined by genome-wide genetic screens. However, whether M. tuberculosis requires these essential genes during infection has not been comprehensively explored because mutant strains cannot be generated using standard methods. Here we show that M. tuberculosis requires the phenylalanine (Phe) and de novo purine and thiamine biosynthetic pathways for mammalian infection. We used a defined collection of M. tuberculosis transposon (Tn) mutants in essential genes, which we generated using a custom nutrient-rich medium, and transposon sequencing (Tn-seq) to identify multiple central metabolic pathways required for fitness in a mouse infection model. We confirmed by individual retesting and complementation that mutations in pheA (Phe biosynthesis) or purF (purine and thiamine biosynthesis) cause death of M. tuberculosis in the absence of nutrient supplementation in vitro and strong attenuation in infected mice. Our findings show that Tn-seq with defined Tn mutant pools can be used to identify M. tuberculosis genes required during mouse lung infection. Our results also demonstrate that M. tuberculosis requires Phe and purine/thiamine biosynthesis for survival in the host, implicating these metabolic pathways as prime targets for the development of new antibiotics to combat tuberculosis.

Genetic Loci of Plant Pathogenic Dickeya solani IPO 2222 Expressed in Contact with Weed-Host Bittersweet Nightshade (Solanum dulcamara L.) Plants.

Dickeya solani, belonging to the Soft Rot Pectobacteriaceae, are aggressive necrotrophs, exhibiting both a wide geographic distribution and a wide host range that includes many angiosperm orders, both dicot and monocot plants, cultivated under all climatic conditions. Little is known about the infection strategies D. solani employs to infect hosts other than potato (Solanum tuberosum L.). Our earlier study identified D. solani Tn5 mutants induced exclusively by the presence of the weed host S. dulcamara. The current study assessed the identity and virulence contribution of the selected genes mutated by the Tn5 insertions and induced by the presence of S. dulcamara. These genes encode proteins with functions linked to polyketide antibiotics and polysaccharide synthesis, membrane transport, stress response, and sugar and amino acid metabolism. Eight of these genes, encoding UvrY (GacA), tRNA guanosine transglycosylase Tgt, LPS-related WbeA, capsular biosynthesis protein VpsM, DltB alanine export protein, glycosyltransferase, putative transcription regulator YheO/PAS domain-containing protein, and a hypothetical protein, were required for virulence on S. dulcamara plants. The implications of D. solani interaction with a weed host, S. dulcamara, are discussed.

OP27 Oxalyl-CoA decarboxylase is a major and specific virulence factor for Adherent Invasive Escherichia coli

Abstract Background Adherent Invasive E. coli (AIEC) strains are found in one-third of patients with ileal Crohn’s disease (CD). To date, the bacterial genes involved in AIEC survival in the inflammatory environment are still unclear. Here, we systematically identify the genes of AIEC required for gut colonization and survival under the inflammatory conditions present in a mouse model of IBD. Methods We performed a transposon sequencing (TnSeq) approach using the laboratory strain E. coli K12 MG1655 and the AIEC strain LF82. We created two saturated Tn mutant libraries (3x105 mutant per bank) and gavaged them to mice with and without dextran-sodium sulfate (DSS)-induced colitis. We screened for essential genes for each bacterium in both conditions in the Cecum, Colon, Ileum and Jejunum. Results After High-throughput sequencing, we identified oxc gene, encoding for the oxalyl-CoA decarboxylase enzyme, as crucial for the survival of LF82 but not MG1655 in the whole gastrointestinal tract in colitis setting. To confirm our TnSeq results, the effect of oxc isogenic mutant (∆oxc) and complemented strains of LF82 and MG1655 was tested. The inflammation severity was exacerbated in mice colonized with LF82WT or the complemented strain compared to LF82∆oxc. LF82∆oxc strain load decreased after colitis induction while the LF82WT and the complemented strain persisted. In a competition between LF82 WT and ∆oxc, the WT strain was able to outcompete the ∆oxc strain. This altered ability of LF82∆oxc to colonize the gut was associated with decreased bacterial motility and flagella expression. In vitro, we found a decreased resistance of different AIEC strains to oxidative stress and acidic pH compared to a collection of commensal E. coli strains. This difference was abolished in all the ∆oxc AIEC strains we built and tested, demonstrating a crucial role for this gene in the tolerance to oxidative and acidic stress. Finally, lack of oxc also caused a reduction in survival into RAW264.7 macrophages, and an altered pro-inflammatory response of the macrophage, with a significant reduction of TNF-α levels. Conclusion oxc plays a major role in vitro and in vivo for AIEC strains but not for laboratory or commensal strains. Further studies to (i) fully explore this role and (ii) evaluate Oxc as a therapeutic target are currently under investigation.

The Transcription Regulator GntR/HutC Regulates Biofilm Formation, Motility and Stress Tolerance in Lysobacter capsici X2-3.

Lysobacter capsici X2-3, a plant growth-promoting rhizobacteria (PGPR), was isolated from wheat rhizosphere and has inhibitory effects against a wide range of pathogens. One important characteristic of L. capsici is its ability to produce diverse antibiotics and lytic enzymes. The GntR family of transcription factors is a common transcription factor superfamily in bacteria that has fundamental roles in bacterial metabolism regulation. However, the GntR family transcription factor in Lysobacter has not been identified. In this study, to obtain an understanding of the GntR/HutC gene function in L. capsici X2-3, a random Tn5-insertion mutant library of X2-3 was constructed to select genes showing pleiotropic effects on phenotype. We identified a Tn5 mutant with an insertion in LC4356 that showed reduced biofilm levels, and sequence analysis indicated that the inserted gene encodes a GntR/HutC family transcription regulator. Furthermore, the LC4356 mutant showed reduced extracellular polysaccharide (EPS) production, diminished twitching motility and decreased survival under UV radiation and high-temperature. The RT‒qPCR results indicated that the pentose phosphate pathway-related genes G6PDH, 6PGL and PGDH were upregulated in the LC4356 mutant. Thus, since L. capsici is an efficient biocontrol agent for crop protection, our findings provide fundamental insights into GntR/HutC and will be worthwhile to improve PGPR biocontrol efficacy.

Genetic Manipulation of Corynebacterium mastitidis to Better Understand the Ocular Microbiome.

Corynebacterium spp. are Gram-positive bacteria commonly associated with the ocular surface. Corynebacterium mastitidis was isolated from mouse eyes and was demonstrated to induce a beneficial immune response that can protect the eye from pathogenic infection. Because eye-relevant Corynebacterium spp. are not well described, we generated a C. mast transposon (Tn) mutant library to gain a better understanding of the nature of eye-colonizing bacteria. Tn mutagenesis was performed with a custom Tn5-based transposon that incorporated a promoterless gene for the fluorescent protein mCherry. We screened our library using flow cytometry and enzymatic assays to identify useful mutants that demonstrate the utility of our approach. Fluorescence-activated cell sorting (FACS) of mCherry+ bacteria allowed us to identify a highly fluorescent mutant that was detectable on the murine ocular surface using microscopy. We also identified a functional knockout that was unable to hydrolyze urea, UreaseKO. Although uric acid is an antimicrobial factor produced in tears, UreaseKO bacterium maintained an ability to colonize the eye, suggesting that urea hydrolysis is not required for colonization. In vitro and in vivo, both mutants maintained the potential to stimulate protective immunity as compared to wild-type C. mast. In sum, we describe a method to genetically modify an eye-colonizing microbe, C. mast. Furthermore, the procedures outlined here will allow for the continued development of genetic tools for modifying ocular Corynebacterium spp., which will lead to a more complete understanding of the interactions between the microbiome and host immunity at the ocular surface.

Genome-wide transposon mutagenesis analysis of Burkholderia pseudomallei reveals essential genes for in vitro and in vivo survival.

Burkholderia pseudomallei, a soil-dwelling microbe that infects humans and animals is the cause of the fatal disease melioidosis. The molecular mechanisms that underlie B. pseudomallei's versatility to survive within a broad range of environments are still not well defined. We used the genome-wide screening tool TraDIS (Transposon Directed Insertion-site Sequencing) to identify B. pseudomallei essential genes. Transposon-flanking regions were sequenced and gene essentiality was assessed based on the frequency of transposon insertions within each gene. Transposon mutants were grown in LB and M9 minimal medium to determine conditionally essential genes required for growth under laboratory conditions. The Caenorhabditis elegans infection model was used to assess genes associated with in vivo B. pseudomallei survival. Transposon mutants were fed to the worms, recovered from worm intestines, and sequenced. Two selected mutants were constructed and evaluated for the bacteria's ability to survive and proliferate in the nematode intestinal lumen. Approximately 500,000 transposon-insertion mutants of B. pseudomallei strain R15 were generated. A total of 848,811 unique transposon insertion sites were identified in the B. pseudomallei R15 genome and 492 genes carrying low insertion frequencies were predicted to be essential. A total of 96 genes specifically required to support growth under nutrient-depleted conditions were identified. Genes most likely to be involved in B. pseudomallei survival and adaptation in the C. elegans intestinal lumen, were identified. When compared to wild type B. pseudomallei, a Tn5 mutant of bpsl2988 exhibited reduced survival in the worm intestine, was attenuated in C. elegans killing and showed decreased colonization in the organs of infected mice. The B. pseudomallei conditional essential proteins should provide further insights into the bacteria's niche adaptation, pathogenesis, and virulence.

Transposon mutagenesis of Rickettsia felis sca1 confers a distinct phenotype during flea infection.

Since its recognition in 1994 as the causative agent of human flea-borne spotted fever, Rickettsia felis, has been detected worldwide in over 40 different arthropod species. The cat flea, Ctenocephalides felis, is a well-described biological vector of R. felis. Unique to insect-borne rickettsiae, R. felis can employ multiple routes of infection including inoculation via salivary secretions and potentially infectious flea feces into the skin of vertebrate hosts. Yet, little is known of the molecular interactions governing flea infection and subsequent transmission of R. felis. While the obligate intracellular nature of rickettsiae has hampered the function of large-scale mutagenesis strategies, studies have shown the efficiency of mariner-based transposon systems in Rickettsiales. Thus, this study aimed to assess R. felis genetic mutants in a flea transmission model to elucidate genes involved in vector infection. A Himar1 transposase was used to generate R. felis transformants, in which subsequent genome sequencing revealed a transposon insertion near the 3' end of sca1. Alterations in sca1 expression resulted in unique infection phenotypes. While the R. felis sca1::tn mutant portrayed enhanced growth kinetics compared to R. felis wild-type during in vitro culture, rickettsial loads were significantly reduced during flea infection. As a consequence of decreased rickettsial loads within infected donor fleas, R. felis sca1::tn exhibited limited transmission potential. Thus, the use of a biologically relevant model provides evidence of a defective phenotype associated with R. felis sca1::tn during flea infection.

Elucidation of Essential Genes and Mutant Fitness during Adaptation toward Nitrogen Fixation Conditions in the Endophyte Azoarcus olearius BH72 Revealed by Tn-Seq.

Azoarcus olearius BH72 is a diazotrophic model endophyte that contributes fixed nitrogen to its host plant, Kallar grass, and expresses nitrogenase genes endophytically. Despite extensive studies on biological nitrogen fixation (BNF) of diazotrophic endophytes, little is known about global genetic players involved in survival under respective physiological conditions. Here, we report a global genomic screen for putatively essential genes of A. olearius employing Tn5 transposon mutagenesis with a modified transposon combined with high-throughput sequencing (Tn-Seq). A large Tn5 master library of ~6 × 105 insertion mutants of strain BH72 was obtained. Next-generation sequencing identified 183,437 unique insertion sites into the 4,376,040-bp genome, displaying one insertion every 24 bp on average. Applying stringent criteria, we describe 616 genes as putatively essential for growth on rich medium. COG (Clusters of Orthologous Groups) assignment of the 564 identified protein-coding genes revealed enrichment of genes related to core cellular functions and cell viability. To mimic gradual adaptations toward BNF conditions, the Tn5 mutant library was grown aerobically in synthetic medium or microaerobically on either combined or atmospheric nitrogen. Enrichment and depletion analysis of Tn5 mutants not only demonstrated the role of BNF- and metabolism-related proteins but also revealed that, strikingly, many genes relevant for plant-microbe interactions decrease bacterial competitiveness in pure culture, such type IV pilus- and bacterial envelope-associated genes. IMPORTANCE A constantly growing world population and the daunting challenge of climate change demand new strategies in agricultural crop production. Intensive usage of chemical fertilizers, overloading the world's fields with organic input, threaten terrestrial and marine ecosystems as well as human health. Long overlooked, the beneficial interaction of endophytic bacteria and grasses has attracted ever-growing interest in research in the last decade. Capable of biological nitrogen fixation, diazotrophic endophytes not only provide a valuable source of combined nitrogen but also are known for diverse plant growth-promoting effects, thereby contributing to plant productivity. Elucidation of an essential gene set for a prominent model endophyte such as A. olearius BH72 provides us with powerful insights into its basic lifestyle. Knowledge about genes detrimental or advantageous under defined physiological conditions may point out a way of manipulating key steps in the bacterium's lifestyle and plant interaction toward a more sustainable agriculture.

Identification of essential genes in Mycobacterium avium subsp. paratuberculosis genome for persistence in dairy calves.

To cause disease Mycobacterium avium subsp. paratuberculosis needs to enter mammalian cells, arrest phagosomal maturation and manipulate the host immune system. The genetic basis of the bacterial capacity to achieve these outcomes remains largely unknown. Identifying these genes would allow us to gain a deeper understanding of MAP's pathogenesis and potentially develop a live attenuated Johne's disease vaccine by knocking out these genes. MAP genes demonstrated to be essential for colonization in the natural host, ruminants, are unknown. Genome-wide transposon mutagenesis and high-throughput sequencing were combined to evaluate the essentiality of each coding region in the bacterial genome to survive in dairy calves. A saturated library of 3,852 MAP Tn mutants, with insertions in 56% of TA sites, interrupting 88% of genes, was created using a MycoMarT7 phagemid containing a mariner transposon. Six calves were inoculated with a high dose of a library of MAP mutants, 1011 CFUs, (input) at 2 weeks of age. Following 2 months of incubation, MAP cells were isolated from the ileum, jejunum, and their associated lymph nodes of calves, resulting in approximately 100,000 colonies grown on solid media across 6 animals (output). Targeted next-generation sequencing was used to identify the disrupted genes in all the mutants in the input pool and the output pool recovered from the tissues to identify in vivo essential genes. Statistical analysis for the determination of essential genes was performed by a Hidden Markov Model (HMM), categorizing genes into essential genes that are devoid of insertions and growth-defect genes whose disruption impairs the growth of the organism. Sequence analysis identified 430 in vivo essential and 260 in vivo growth-defect genes. Gene ontology enrichment analysis of the in vivo essential and growth-defect genes with the highest reduction in the tissues revealed a high representation of genes involved in metabolism and respiration, cell wall and cell processing, virulence, and information pathway processes. This study has systematically identified essential genes for the growth and persistence of MAP in the natural host body.

Phage Resistance Reduced the Pathogenicity of Xanthomonas oryzae pv. oryzae on Rice.

Plants grow together with microbes that have both negative and positive impacts on the host, while prokaryotes are in turn also hosts for viruses, co-evolving together in a complex interrelationship. Most research focuses on the interaction of either bacterial pathogens interacting with the plant host, or the impact on viruses on their pathogenic bacterial hosts. Few studies have investigated the co-evolution of bacterial pathogens with their host plants as well as with their bacterial viruses. In this work, we aimed to identify the genes that were associated with both phage sensitivity and host pathogenicity of the bacterium Xanthomonas oryzae pv. oryzae (Xoo), which is the most important bacterial rice pathogen. Using the Tn5 transposon mutation technology, we created a library of Xoo strain C2 comprising 4524 mutants, which were subsequently tested for phage infectability. The phage infection tests showed that less than 1% of the mutants (n = 36) were resistant to phage infection, which was attributed to the Tn5 insertion in 19 genes. Interestingly, three out of 19 genes that conveyed resistance to the phage resulted in reduced pathogenicity to rice seedlings compared to the wild type. We identified three genes involved in both phage infection and bacterial virulence, which were studied by knockout mutants and complementation experiments. All of the three knockout mutants were resistant to infection by phage X2, while the complemented strains restored the susceptibility to the bacterial virus. Surprisingly, the genes are also essential for pathogenicity, which we confirmed by single knockout mutants corresponding to the Tn5 mutants. All three genes are involved in lipopolysaccharide synthesis, thus changing the cell envelope surface molecule composition. Our work shows a possible balance in terms of the connection between bacterial virulence and phage resistance, supporting the deployment of phages for the biocontrol of plant pathogens.

The Loss of focA Gene Increases the Ability of Salmonella Enteritidis to Exit from Macrophages and Boosts Early Extraintestinal Spread for Systemic Infection in a Mouse Model.

Salmonella Enteritidis (SE) can spread from the intestines to cause systemic infection, mainly involving macrophages. Intramacrophage Salmonella exits and reinfects neighboring cells, leading to severe disease. Salmonella genes involved in exiting from macrophages are not well understood or fully identified. A focA::Tn5 mutant was identified by an in vitro assay, with increased ability to exit from macrophages. A defined SEΔfocA mutant and its complemented derivative strain, SEΔfocA::focA, were constructed to confirm this phenotype. Although the lethal ability of focA mutants was similar to that of the parental SE in mice, it was isolated earlier from the liver and spleen than the parental SE. focA mutants induced higher levels of proinflammatory IL-12 and TNF-α compared with the parental SE and SEΔfocA::focA. focA mutants showed higher cytotoxicity and lower formate concentrations than SE and SEΔfocA::focA, whereas there was no change in pyroptosis, apoptosis and flagella formation ability. These current data suggest that the focA gene plays an important role in regulating intramacrophage Salmonella exiting and extraintestinal spread in mice, although the specific mechanism requires further in-depth studies.

Resistance of Dickeya solani strain IPO 2222 to lytic bacteriophage ΦD5 results in fitness tradeoffs for the bacterium during infection

Resistance to bacteriophage infections protects bacteria in phage-replete environments, enabling them to survive and multiply in the presence of their viral predators. However, such resistance may confer costs for strains, reducing their ecological fitness as expressed as competitiveness for resources or virulence or both. There is limited knowledge about such costs paid by phage-resistant plant pathogenic bacteria in their natural habitats. This study analyzed the costs of phage resistance paid by the phytopathogenic pectinolytic bacterium Dickeya solani both in vitro and in potato (Solanum tuberosum L.) plants. Thirteen Tn5 mutants of D. solani IPO 2222 were identified that exhibited resistance to infection by lytic bacteriophage vB_Dsol_D5 (ΦD5). The genes disrupted in these mutants encoded proteins involved in the synthesis of bacterial envelope components (viz. LPS, EPS and capsule). Although phage resistance did not affect most of the phenotypes of ΦD5-resistant D. solani such as growth rate, production of effectors, swimming and swarming motility, use of various carbon and nitrogen sources and biofilm formation evaluated in vitro, all phage resistant mutants were significantly compromised in their ability to survive on leaf surfaces as well as to grow within and cause disease symptoms in potato plants.

Screening for Virulence-Related Genes via a Transposon Mutant Library of Streptococcus suis Serotype 2 Using a Galleria mellonella Larvae Infection Model.

Streptococcus suis (S. suis) is a zoonotic bacterial pathogen causing lethal infections in pigs and humans. Identification of virulence-related genes (VRGs) is of great importance in understanding the pathobiology of a bacterial pathogen. To identify novel VRGs, a transposon (Tn) mutant library of S. suis strain SC19 was constructed in this study. The insertion sites of approximately 1700 mutants were identified by Tn-seq, which involved 417 different genes. A total of 32 attenuated strains were identified from the library by using a Galleria mellonella larvae infection model, and 30 novel VRGs were discovered, including transcription regulators, transporters, hypothetical proteins, etc. An isogenic deletion mutant of hxtR gene (ΔhxtR) and its complementary strain (CΔhxtR) were constructed, and their virulence was compared with the wild-type strain in G. mellonella larvae and mice, which showed that disruption of hxtR significantly attenuated the virulence. Moreover, the ΔhxtR strain displayed a reduced survival ability in whole blood, increased sensitivity to phagocytosis, increased chain length, and growth defect. Taken together, this study performed a high throughput screening for VRGs of S. suis using a G. mellonella larvae model and further characterized a novel critical virulence factor.

Inability to Catabolize Rhamnose by Sinorhizobium meliloti Rm1021 Affects Competition for Nodule Occupancy.

Rhizobium leguminosarum strains unable to grow on rhamnose as a sole carbon source are less competitive for nodule occupancy. To determine if the ability to use rhamnose as a sole carbon source affects competition for nodule occupancy in Sinorhizobium meliloti, Tn5 mutants unable to use rhamnose as a sole carbon source were isolated. S. meliloti mutations affecting rhamnose utilization were found in two operons syntenous to those of R. leguminosarum. Although the S. meliloti Tn5 mutants were complemented using an R. leguminosarum cosmid that contains the entire wild-type rhamnose catabolic locus, complementation did not occur if the cosmids carried Tn5 insertions within the locus. Through a series of heterologous complementation experiments, enzyme assays, gene fusion, and transport experiments, we show that the S. meliloti regulator, RhaR, is dominant to its R. leguminosarum counterpart. In addition, the data support the hypothesis that the R. leguminosarum kinase is capable of directly phosphorylating rhamnose and rhamnulose, whereas the S. meliloti kinase does not possess rhamnose kinase activity. In nodule competition assays, S. meliloti mutants incapable of rhamnose transport were shown to be less competitive than the wild-type and had a decreased ability to bind plant roots in the presence of rhamnose. The data suggests that rhamnose catabolism is a general determinant in competition for nodule occupancy that spans across rhizobial species.

High-throughput identification of genes influencing the competitive ability to obtain nutrients and performance of biocontrol in Pseudomonas putida JBC17

Elucidating underlying mechanisms of biocontrol agents (BCAs) could aid in selecting potent BCAs and increasing their biocontrol efficacy. Nutrient competition is an important biocontrol mechanism; however, essential nutrient sources, and contributing genes for nutrient competition still remain to be explored. Pseudomonas putida JBC17 (JBC17WT) suppressed green mold in satsuma mandarins by inhibiting conidial germination of Penicillium digitatum via nutrient competition. To analyze genes essential for biocontrol performance of JBC17WT, we generated a transposon (Tn)-mediated mutant library and selected mutants with the ability to suppress conidial germination. Several mutants in the genes of flagella-formation, including fliR, fliH, and flgG, increased biocontrol performance and enhanced inhibition of conidial germination. They lost swimming motility, exhibited increased growth and rapid carbon and nitrogen utilization than the wild type under nutrient-poor conditions. The nutrient competition assay using polytetrafluoroethylene cylinders revealed that conidial germination was inhibited by nutrient absorption under nutrient-poor conditions. In addition, genes, including amidohydrolase (ytcJ), tonB-dependent receptor (cirA), argininosuccinate synthase (argG), D-3-phosphoglycerate dehydrogenase (serA), and chaperone protein (dnaJ), were involved in the inhibition of conidial germination. The results of this study indicate that rapid and continuous absorption of nutrients by JBC17WT restrict nutrient availability for conidial germination on nutrient-limited fruit surfaces, thereby decreasing the chances of fungal spores infecting fruits. The high-throughput analysis of Tn mutants of this study highlighted the importance of nutrient competition and the genes that influence biocontrol ability, which contributes to the development of biocontrol applications.

The influence of seed-dressings on the formation and functioning of symbiotic soybean systems with fungicide-resistant rhizobia

. The application of bacterial fertilizers, prepared on the basis of active, competitive strains of nitrogen-fixing microorganisms and fungicidal dressings for pre-sowing seed treatment in soybean cultivation technologies is characterized by high biological and economic efficiency. Therefore, it is important to find such combination of chemicals and microbial preparations, for the complex use of which will be maintained their main purpose. The aim of the work was to investigate the formation and functioning of symbiotic soybean systems with fungicide-resistant nodule bacteria Bradyrhizobium japonicum PC09 and B144 under the influence of Standak Top and Fever seed dressings. Differences in the effect of chemical plant protection products on the formation of symbiotic systems of soybean variety Almaz and nitrogen-fixing microorganisms have been established. A decrease by 9.2‒24.1 % in the number of nodules formed on the roots during the soybean growing season with the combined use of Fever with rhizobia bacterization of PC09 strain was revealed. At inoculation the seed with Tn5- mutant B144, use of the same preparation, the decrease of this indicator compared to control plants was 6.5‒32.8 %. An increase in the weight of root nodules by 10.3‒36 % was found under the effect of Standak Top and bacterization by both strains of rhizobia.There was a decrease in the intensity of N2 assimilation compared to control plants by 24.2 and 42.3 % in the stage of three true leaves with the combined use of Fever with inoculation and a gradual decrease of the toxic effect of pesticides on the functioning of symbiotic systems in the budding-flowering stage. At shared treatment of seeds with rhizobia strains B144 and PC09 and Standak Top revealed an increase in nitrogen fixation intensity by 6.2‒25 % in the stage of three true leaves, 12.5‒58 % in the stage of budding-beginning of flowering and by 28.1‒42.3 % in the stage of bean formation. Maintaining high level of nitrogenase activity of legume-rhizobial symbiosis, formed with the participation of pesticide-resistant nodule bacteria with using of seed dressings, will provide plants with available forms of nitrogen, protect sown seeds from soil and seed infections and create conditions for grain production.

Identification of a Cupin Protein Gene Responsible for Pathogenicity, Phage Susceptibility and LPS Synthesis of Acidovorax citrulli.

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King’s B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

Influence of metal nanocarboxylates and different water supply conditions on efficiency of soybean-rhizobial symbiotic systems

Insufficient water supply is one of the main factors that significantly reduce the activity of nitrogen fixation by legume-rhizobial symbiotic systems. That is why comprehensive research on aspects of their resistance to water stress and the search for scientifically substantiated ways to improve the existing ones and develop modern, competitive technologies of growing legumes in arid conditions are becoming especially relevant. The aim of the work was to investigate the processes of formation and functioning of soybean-rhizobial symbiotic systems developed under conditions of different water supply and influence of nanocarboxylates of cobalt, ferum, germanium, chromium, сuprum and molybdenum. The nanoparticles of specified metal nanocarboxylates were used as components of the inoculation suspension of rhizobia of Tn5 mutant B1-20 for soybean seed treatment. A model drought lasting 14 days was created by controlled irrigation. Microbiological and physiological research methods were used in the study. We determined that insufficient water supply caused a significant decrease in the nodulation potential of rhizobia and the intensity of molecular nitrogen fixation by symbiotic systems formed with the participation of soybean plants and nodule bacteria without adding these metal nanocarboxylates to the inoculation suspension. Application of most of the metal nanocarboxylates used as components of the inoculation suspension mitigated the negative impact of stress on the investigated parameters. The study revealed the stimulating effect of cobaltnanocarboxylate on the activity of molecular nitrogen fixation, which was more pronounced in the conditions of insufficient water supply. Symbiotic soybean systems formed with the participation of nodule bacteria containing germaniumcarboxylate nanoparticles were proved to be the least sensitive to the negative impact of insufficient water supply. This was indicated by high rates of nodulation and nitrogen-fixing activity compared with other studied symbiotic systems. We confirmed that the addition of chromium nanocarboxylate to the inoculation suspension of rhizobia provided the highest rates of nodulation and nitrogen-fixing activity of soybean root nodules under optimal growing conditions and, at the same time, had no noticeable positive effect under water stress. We determined that сuprum and molybdenum nanocarboxylates, as components of the inoculation suspension, regardless of the water supply level, had a less notable positive effect on the processes of nodule formation and nitrogen fixation, and in some cases even led to a decrease in the investigated values for control plants. Thus, the study demonstrated that the use of germanium, cobalt and ferum nanocarboxylates as components of the bacterial suspension helped to increase the adaptation of the formed legume-rhizobial symbiotic systems to water stress, as evidenced by the maximum indexes of nodulation and molecular nitrogen fixation in the context of insufficient water supply and recovery of their level to optimal after the stress influence had ended. Based on the results, it was concluded that inoculation of seeds by the complex bacterial preparations made on the basis of Bradyrhizobium japonicum B1-20 with a content of germanium, cobalt and ferum nanocarboxylates in the concentration of 1:1000 can become one of the important means in soybean growing technologies of increasing the nitrogen-fixing potential and resistance of plants to insufficient water supply.