Probiotic Potential of Bacillus Subtilis Strain I3: Antagonistic Activity Against Chalkbrood Pathogen and Pesticide Degradation for Enhancing Honeybee Health.

To explore the potential of probiotic candidates beneficial for honeybee health through the modulation of the gut microbiome, bee gut microbes were isolated from bumblebee (Bombus terrestris) and honeybee (Apis mellifera) using diverse media and cultural conditions. A total of 77 bee gut bacteria, classified under the phyla Proteobacteria, Firmicutes, and Actinobacteria, were identified. The antagonistic activity of the isolates against Ascosphaera apis, a fungal pathogen responsible for chalkbrood disease in honeybee larvae, was investigated. The highest growth inhibition percentage against A. apis was demonstrated by Bacillus subtilis strain I3 among the bacterial strains. The presence of antimicrobial peptide genes in the I3 strain was detected using PCR amplification of gene fragments encoding surfactin and fengycin utilizing specific primers. The export of antimicrobial peptides by the I3 strain into growth medium was verified using liquid chromatography coupled with mass spectroscopy. Furthermore, the strain's capabilities for degrading pesticides, used for controlling varroa mites, and its spent growth medium antioxidant activity were substantiated. The survival rate of honeybees infected with (A) apis was investigated after feeding larvae with only medium (fructose + glucose + yeast extract + royal jelly), (B) subtilis I3 strain, A. apis with medium and I3 strain + A. apis with medium. Honeybees receiving the I3 strain + A. apis exhibited a 50% reduction in mortality rate due to I3 strain supplementation under experimental conditions, compared to the control group. In silico molecular docking revealed that fengycin hydrolase from I3 strain effectively interacted with tau-fluvalinate, suggesting its potential in bee health and environmental protection. Further studies are needed to confirm the effects of the I3 strain in different populations of honey bees across several regions to account for genetic and environmental variations.

Assessing potential impact of gut microbiome disruptions on the environmental stress resilience of indoor-reared Bombus terrestris.

Bumblebees are crucial for both natural ecosystems and agriculture, but their decline in distribution and abundance over the past decade is alarming. The global importance of bumblebees in natural ecosystems and agricultural food production cannot be overstated. However, the reported decline over the past decade has led to a surge of interest in understanding and addressing bumblebee population decline. Hence, we aimed to detect disruptions in the gut microbiome of male and worker bumblebees reared indoor and outdoor to assess potential resilience to environmental stress. Using the Illumina MiSeq platform for 16s rRNA amplicon sequencing, we analyzed the gut microbiome of male and worker bees that were raised indoors (designated as the IM and IW group) and those that were raised outdoors (also designated as the OM and OW group). Our results show presence of core bacteria Neisseriaceae, Orbaceae, Lactobacillaceae and Bifidobacteriaceae from indoor reared worker bees. However, a higher abundance of Bifidobacterium and absence of Fructobacillus from indoor reared worker bees was also observed. Indoor-reared male bees had lower diversity and fewer observed OTUs compared to outdoor-reared male bees. Additionally, the relative abundance of Actinobacteriota, Bacteroidota, and Firmicutes was significantly lower in indoor-reared males, while Proteobacteria was significantly increased. Despite this, we did not observe any dysbiosis in the gut microbiota of indoor-reared bumblebees when comparing the role of the gut symbionts among the groups. These results suggest that indoor-reared Bombus terrestris may be resilient to environmental stress when used as outdoor pollinators.

Flavobacterium dauae sp. nov., isolated from rhizosphere soil of a tomato plant.

A bacterial strain, designated TCH3-2T, was isolated from the rhizosphere of tomato plant grown at Dong-A University Agricultural Experiment Station, Republic of Korea. The strain was Gram-stain-negative, obligate aerobic, orange yellow-coloured, motile by gliding and short rod-shaped. Strain TCH3-2 T only grew on 1/2 tryptic soy agar and Luria-Bertani agar among the media tested, with optimum growth at 28 °C and pH 7. Salt of 1 % NaCl was necessary to support the growth of TCH3-2T. Strain TCH3-2T produced flexirubin-type pigments. The predominant cellular fatty acids were iso-C15 : 0 (55.6 %), iso-C17 : 0 3-OH (17.9 %), summed feature 9 (comprising C16 : 0 10-methyl and/or iso-C17 : 1 ω9c; 10.5 %), iso-C15 : 0 3-OH (4.8 %) and anteiso-C15 : 0 (2.3 %). The major menaquinone was menaquinone-6 and the major polar lipids were phosphatidylethanolamine, five unknown aminolipids and three unknown lipids. Phylogenetic analysis based on 16S rRNA sequences indicated that TCH3-2T was closely related to Flavobacterium ummariense DS-12T (95.16 %), Flavobacterium marinum SW105T (95.14 %) and Flavobacterium viscosus YIM 102796T (94.54 %). The draft genome of TCH3-2T comprised ca. 2.8 Mb with a G+C content of 34.61 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between TCH3-2T and closely related Flavobacterium species showed that it belongs to a distinct species. Furthermore, the results of morphological, physiological and biochemical tests allowed further phenotypic differentiation of TCH3-2T from its closest relatives. Thus, chemotaxonomic characteristics together with phylogenetic affiliation illustrate that TCH3-2T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium dauae sp. nov. (type strain TCH3-2T=KACC 19054T=JCM 34025T) is proposed.

Complete Genome Sequence of a Novel Bacteriophage RpY1 Infecting Ralstonia solanacearum Strains.

Ralstonia solanacearum species complex is deleterious plant pathogenic bacteria causing bacterial wilt in the members of solanaceous crops and the bacterial wilt is difficult to control. Bacteriophages-based biocontrol is an environmentally friendly and promising strategy to control bacterial plant diseases. In this study, we isolated 72 phages from the various crop cultivated soils in Korea using five different strains of R. solanacearum. Among 72 phages, phage RpY1 was selected for further study based on the specificity of the targeted host. This phage was identified as a member of Podoviridae with a head measuring 60-70 nm in length and short tail according to the morphology of transmission electron microscopy images. The genome size of phage RpY1 is 43,284 bp with G + C content of 61.4% and 53 open reading frames (ORFs), including 18 annotated ORFs and 35 hypothetical proteins. This phage genome showed no homology to the genome of known phages except for the DU_RP_II phage infecting R. solanacearum; however, the host range of phage RpY1 is much narrower than that of DU_RP_II.

Dissection of plant microbiota and plant-microbiome interactions.

Plants rooted in soil have intimate associations with a diverse array of soil microorganisms. While the microbial diversity of soil is enormous, the predominant bacterial phyla associated with plants include Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Plants supply nutrient niches for microbes, and microbes support plant functions such as plant growth, development, and stress tolerance. The interdependent interaction between the host plant and its microbes sculpts the plant microbiota. Plant and microbiome interactions are a good model system for understanding the traits in eukaryotic organisms from a holobiont perspective. The holobiont concept of plants, as a consequence of co-evolution of plant host and microbiota, treats plants as a discrete ecological unit assembled with their microbiota. Dissection of plant-microbiome interactions is highly complicated; however, some reductionist approaches are useful, such as the synthetic community method in a gnotobiotic system. Deciphering the interactions between plant and microbiome by this reductionist approach could lead to better elucidation of the functions of microbiota in plants. In addition, analysis of microbial communities' interactions would further enhance our understanding of coordinated plant microbiota functions. Ultimately, better understanding of plantmicrobiome interactions could be translated to improvements in plant productivity.

Contribution of the murI Gene Encoding Glutamate Racemase in the Motility and Virulence of Ralstonia solanacearum.

Bacterial traits for virulence of Ralstonia solanacearum causing lethal wilt in plants were extensively studied but are not yet fully understood. Other than the known virulence factors of Ralstonia solanacearum, this study aimed to identify the novel gene(s) contributing to bacterial virulence of R. solanacearum. Among the transposon-inserted mutants that were previously generated, we selected mutant SL341F12 strain produced exopolysaccharide equivalent to wild type strain but showed reduced virulence compared to wild type. In this mutant, a transposon was found to disrupt the murI gene encoding glutamate racemase which converts L-glutamate to D-glutamate. SL341F12 lost its motility, and its virulence in the tomato plant was markedly diminished compared to that of the wild type. The altered phenotypes of SL341F12 were restored by introducing a full-length murI gene. The expression of genes required for flagella assembly was significantly reduced in SL341F12 compared to that of the wild type or complemented strain, indicating that the loss of bacterial motility in the mutant was due to reduced flagella assembly. A dramatic reduction of the mutant population compared to its wild type was apparent in planta (i.e., root) than its wild type but not in soil and rhizosphere. This may contribute to the impaired virulence in the mutant strain. Accordingly, we concluded that murI in R. solanacearum may be involved in controlling flagella assembly and consequently, the mutation affects bacterial motility and virulence.

Alteration of Bacterial Wilt Resistance in Tomato Plant by Microbiota Transplant.

Plant-associated microbiota plays an important role in plant disease resistance. Bacterial wilt resistance of tomato is a function of the quantitative trait of tomato plants; however, the mechanism underlying quantitative resistance is unexplored. In this study, we hypothesized that rhizosphere microbiota affects the resistance of tomato plants against soil-borne bacterial wilt caused by Ralstonia solanacearum. This hypothesis was tested using a tomato cultivar grown in a defined soil with various microbiota transplants. The bacterial wilt-resistant Hawaii 7996 tomato cultivar exhibited marked suppression and induction of disease severity after treatment with upland soil-derived and forest soil-derived microbiotas, respectively, whereas the transplants did not affect the disease severity in the susceptible tomato cultivar Moneymaker. The differential resistance of Hawaii 7996 to bacterial wilt was abolished by diluted or heat-killed microbiota transplantation. Microbial community analysis revealed the transplant-specific distinct community structure in the tomato rhizosphere and the significant enrichment of specific microbial operational taxonomic units (OTUs) in the rhizosphere of the upland soil microbiota-treated Hawaii 7996. These results suggest that the specific transplanted microbiota alters the bacterial wilt resistance in the resistant cultivar potentially through a priority effect.

A triclosan-resistance protein from the soil metagenome is a novel enoyl-acyl carrier protein reductase: Structure-guided functional analysis.

The synthetic biocide triclosan targets enoyl-acyl carrier protein reductase(s) (ENR) in bacterial type II fatty acid biosynthesis. Screening and sequence analyses of the triclosan resistome from the soil metagenome identified a variety of triclosan-resistance ENRs. Interestingly, the mode of triclosan resistance by one hypothetical protein was elusive, mainly due to a lack of sequence similarity with other proteins that mediate triclosan resistance. Here, we carried out a structure-based function prediction of the hypothetical protein, herein referred to as FabMG, and in vivo and in vitro functional analyses. The crystal structure of FabMG showed limited structural homology with FabG and FabI, which are also involved in type II fatty acid synthesis. In vivo complementation and in vitro activity assays indicated that FabMG is functionally a FabI-type ENR that employs NADH as a coenzyme. Variations in the sequence and structure of FabMG are likely responsible for inefficient binding of triclosan, resulting in triclosan resistance. These data unravel a previously uncharacterized FabMG, which is prevalent in various microbes in triclosan-contaminated environments and provide mechanistic insight into triclosan resistance.

Biochemical and Structural Insights Concerning Triclosan Resistance in a Novel YX7K Type Enoyl-Acyl Carrier Protein Reductase from Soil Metagenome.

Enoyl-acyl carrier protein reductase (ENR) catalyzes the last reduction step in the bacterial type II fatty acid biosynthesis cycle. ENRs include FabI, FabL, FabL2, FabK, and FabV. Previously, we reported a unique triclosan (TCL) resistant ENR homolog that was predominant in obligate intracellular pathogenic bacteria and Apicomplexa. Herein, we report the biochemical and structural basis of TCL resistance in this novel ENR. The purified protein revealed NADH-dependent ENR activity and shared similarity to prototypic FabI. Thus, this metagenome-derived ENR was designated FabI2. Unlike other prototypic bacterial ENRs with the YX6K type catalytic domain, FabI2 possessed a unique YX7K type catalytic domain. Computational modeling followed by site-directed mutagenesis revealed that mild resistance (20 µg/ml of minimum inhibitory concentration) of FabI2 to TCL was confined to the relatively less bulky side chain of A128. Substitution of A128 in FabI2 with bulky valine (V128) elevated TCL resistance. Phylogenetic analysis further suggested that the novel FabI2 and prototypical FabI evolved from a common short-chain dehydrogenase reductase family. To our best knowledge, FabI2 is the only known ENR shared by intracellular pathogenic prokaryotes, intracellular pathogenic lower eukaryotes, and a few higher eukaryotes. This suggests that the ENRs of prokaryotes and eukaryotes diverged from a common ancestral ENR of FabI2.

Culturing Simpler and Bacterial Wilt Suppressive Microbial Communities from Tomato Rhizosphere.

Plant phenotype is affected by a community of associated microorganisms which requires dissection of the functional fraction. In this study, we aimed to culture the functionally active fraction of an upland soil microbiome, which can suppress tomato bacterial wilt. The microbiome fraction (MF) from the rhizosphere of Hawaii 7996 treated with an upland soil or forest soil MF was successively cultured in a designed modified M9 (MM9) medium partially mimicking the nutrient composition of tomato root exudates. Bacterial cells were harvested to amplify V3 and V4 regions of 16S rRNA gene for QIIME based sequence analysis and were also treated to Hawaii 7996 prior to Ralstonia solanacearum inoculation. The disease progress indicated that the upland MM9 1st transfer suppressed the bacterial wilt. Community analysis revealed that species richness was declined by successive cultivation of the MF. The upland MM9 1st transfer harbored population of phylum Proteobacteria (98.12%), Bacteriodetes (0.69%), Firmicutes (0.51%), Actinobacteria (0.08%), unidentified (0.54%), Cyanobacteria (0.01%), FBP (0.001%), OD1 (0.001%), Acidobacteria (0.005%). The family Enterobacteriaceae of Proteobacteria was the dominant member (86.76%) of the total population of which genus Enterobacter composed 86.76% making it a potential candidate to suppress bacterial wilt. The results suggest that this mixed culture approach is feasible to harvest microorganisms which may function as biocontrol agents.

Unique gene Pmhyp controlling melanization of pycnidia in Phoma medicaginis.

Phoma medicaginis (syn. Ascochyta medicaginicola Qchen & L. Cai) causes spring black stem and leaf spot of alfalfa and the model legume Medicago truncatula. Phoma medicaginis produces uninucleate conidia in melanized pycnidia and is genetically tractable through Agrobacterium tumefaciens-mediated transformation (ATMT), which can result in insertional mutants. One T-DNA-tagged mutant, P1A17 produced conidia in non-melanized (hyaline) pycnidia. Pycnidial melanization recovered if the mutant was supplemented with melanin precursors or allowed to age. DNA sequences flanking the insertion did not predict any disrupted open reading frames (ORF) unless a Coccidioides prediction algorithm was used. Pmhyp gene was expressed in the wild type, but not the mutant, and has not been annotated in any genomes, to date. Expression of two conserved genes flanking the T-DNA disrupted Pmhyp was unchanged from the wild type. Knockout of Pmhyp strain displayed same cultural phenotype (non-melanized pycnidia). Complementation of Pmhyp strains with wild type PmHYP partially recovered pycnidial melanization. Both knockout and complementation transformants were confirmed using RT-PCR and southern blot analysis. Taken together, PmHYP appears to be a novel regulator of pycnidium specific melanization.

Rhizosphere microbiome structure alters to enable wilt resistance in tomato.

Tomato variety Hawaii 7996 is resistant to the soil-borne pathogen Ralstonia solanacearum, whereas the Moneymaker variety is susceptible to the pathogen. To evaluate whether plant-associated microorganisms have a role in disease resistance, we analyzed the rhizosphere microbiomes of both varieties in a mesocosm experiment. Microbiome structures differed between the two cultivars. Transplantation of rhizosphere microbiota from resistant plants suppressed disease symptoms in susceptible plants. Comparative analyses of rhizosphere metagenomes from resistant and susceptible plants enabled the identification and assembly of a flavobacterial genome that was far more abundant in the resistant plant rhizosphere microbiome than in that of the susceptible plant. We cultivated this flavobacterium, named TRM1, and found that it could suppress R. solanacearum-disease development in a susceptible plant in pot experiments. Our findings reveal a role for native microbiota in protecting plants from microbial pathogens, and our approach charts a path toward the development of probiotics to ameliorate plant diseases.

Distribution of triclosan-resistant genes in major pathogenic microorganisms revealed by metagenome and genome-wide analysis.

The substantial use of triclosan (TCS) has been aimed to kill pathogenic bacteria, but TCS resistance seems to be prevalent in microbial species and limited knowledge exists about TCS resistance determinants in a majority of pathogenic bacteria. We aimed to evaluate the distribution of TCS resistance determinants in major pathogenic bacteria (N = 231) and to assess the enrichment of potentially pathogenic genera in TCS contaminated environments. A TCS-resistant gene (TRG) database was constructed and experimentally validated to predict TCS resistance in major pathogenic bacteria. Genome-wide in silico analysis was performed to define the distribution of TCS-resistant determinants in major pathogens. Microbiome analysis of TCS contaminated soil samples was also performed to investigate the abundance of TCS-resistant pathogens. We experimentally confirmed that TCS resistance could be accurately predicted using genome-wide in silico analysis against TRG database. Predicted TCS resistant phenotypes were observed in all of the tested bacterial strains (N = 17), and heterologous expression of selected TCS resistant genes from those strains conferred expected levels of TCS resistance in an alternative host Escherichia coli. Moreover, genome-wide analysis revealed that potential TCS resistance determinants were abundant among the majority of human-associated pathogens (79%) and soil-borne plant pathogenic bacteria (98%). These included a variety of enoyl-acyl carrier protein reductase (ENRs) homologues, AcrB efflux pumps, and ENR substitutions. FabI ENR, which is the only known effective target for TCS, was either co-localized with other TCS resistance determinants or had TCS resistance-associated substitutions. Furthermore, microbiome analysis revealed that pathogenic genera with intrinsic TCS-resistant determinants exist in TCS contaminated environments. We conclude that TCS may not be as effective against the majority of bacterial pathogens as previously presumed. Further, the excessive use of this biocide in natural environments may selectively enrich for not only TCS-resistant bacterial pathogens, but possibly for additional resistance to multiple antibiotics.

Soil metagenome-derived 3-hydroxypalmitic acid methyl ester hydrolases suppress extracellular polysaccharide production in Ralstonia solanacearum.

Autoinducers are indispensable for bacterial cell-cell communication. However, due to the reliance on culture-based techniques, few autoinducer-hydrolyzing enzymes are known. In this study, we characterized soil metagenome-derived unique enzymes capable of hydrolyzing 3-hydroxypalmitic acid methyl ester (3-OH PAME), an autoinducer of the plant pathogenic bacterium Ralstonia solanacearum. Among 146 candidate lipolytic clones from a soil metagenome library, 4 unique enzymes capable of hydrolyzing the autoinducer 3-OH PAME, termed ELP86, ELP96, ELP104, and EstDL33, were selected and characterized. Phylogenetic analysis revealed that metagenomic enzymes were novel esterase/lipase candidates as they clustered as novel subfamilies of family I, V, X, and family XI. The purified enzymes displayed various levels of hydrolytic activities towards 3-OH PAME with optimum activity at 40-50 °C and pH 7-10. Interestingly, ELP104 also displayed N-(3-oxohexanoyl)-L-homoserine lactone hydrolysis activity. Heterologous expression of the gene encoding 3-OH PAME hydrolase in R. solanacearum significantly decreased exopolysaccharide production without affecting bacterial growth. mRNA transcription analysis revealed that genes regulated by quorum-sensing, such as phcA and xpsR, were significantly down-regulated in the stationary growth phase of R. solanacearum. Therefore, metagenomic enzymes are capable of quorum-quenching by hydrolyzing the autoinducer 3-OH PAME, which could be used as a biocontrol strategy against bacterial wilt.

A noncanonical poly(A) RNA polymerase gene affects morphology in Phoma medicaginis.

Phoma medicaginis (syn. Ascochyta medicaginicola Qchen & L. Cai) causes spring black stem and leaf spot, an important disease of alfalfa and annual medics. P. medicaginis forms uninucleate conidia in melanized pycnidia and is genetically tractable using Agrobacterium mediated transformation (ATMT), resulting in random integration of T-DNA that occasionally generates pycnidial mutants. The T-DNA tagged mutant, P265 displayed smaller pycnidia and more aerial hyphae than the wild type. A single T-DNA disrupted a putative noncanonical poly(A) RNA polymerase gene, Pmncpap1, which in yeast interacts with ribonucleotide reductase (RNR). As in yeast mutants, P265 showed sensitivity to hydroxyurea (HU), a RNR inhibitor. To characterize the role of Pmncpap1, targeted ΔPmncpap1 mutants were created using a hygromycin selectable marker flanked by 1 Kbp regions of Pmncpap1. ΔPmncpap1 mutants possessed similar morphological features to those of P265. The plasmid for rescue of PmncPAP1, pCAM-Nat1 (nourseothricin selection) was constructed and used to introduce full-length PmncPAP1 into mutants. Rescued P265 showed partial recovery of wild type and the original T-DNA was lost due to homologous integration. To our knowledge, this is the first ncPAP to be examined in a filamentous fungus.

Comparative Genome Analysis of Rathayibacter tritici NCPPB 1953 with Rathayibacter toxicus Strains Can Facilitate Studies on Mechanisms of Nematode Association and Host Infection.

Rathayibacter tritici, which is a Gram positive, plant pathogenic, non-motile, and rod-shaped bacterium, causes spike blight in wheat and barley. For successful pathogenesis, R. tritici is associated with Anguina tritici, a nematode, which produces seed galls (ear cockles) in certain plant varieties and facilitates spread of infection. Despite significant efforts, little research is available on the mechanism of disease or bacteria-nematode association of this bacterium due to lack of genomic information. Here, we report the first complete genome sequence of R. tritici NCPPB 1953 with diverse features of this strain. The whole genome consists of one circular chromosome of 3,354,681 bp with a GC content of 69.48%. A total of 2,979 genes were predicted, comprising 2,866 protein coding genes and 49 RNA genes. The comparative genomic analyses between R. tritici NCPPB 1953 and R. toxicus strains identified 1,052 specific genes in R. tritici NCPPB 1953. Using the BlastKOALA database, we revealed that the flexible genome of R. tritici NCPPB 1953 is highly enriched in 'Environmental Information Processing' system and metabolic processes for diverse substrates. Furthermore, many specific genes of R. tritici NCPPB 1953 are distributed in substrate-binding proteins for extracellular signals including saccharides, lipids, phosphates, amino acids and metallic cations. These data provides clues on rapid and stable colonization of R. tritici for disease mechanism and nematode association.

Identification of a Gene Involved in the Negative Regulation of Pyomelanin Production in Ralstonia solanacearum.

Ralstonia solanacearum causes bacterial wilt in a wide variety of host plant species and produces a melanin-like blackish-brown pigment in stationary phase when grown in minimal medium supplemented with tyrosine. To study melanin production regulation in R. solanacearum, five mutants exhibiting overproduction of melanin-like pigments were selected from a transposon (Tn) insertion mutant library of R. solanacearum SL341. Most of the mutants, except one (SL341T), were not complemented by the original gene or overproduced melanins. SL341T showed Tn insertion in a gene containing a conserved domain of eukaryotic transcription factor. The gene was annotated as a hypothetical protein, given its weak similarity to any known proteins. Upon complementation with its original gene, the mutant strains reverted to their wild-type phenotype. SL341T produced 3-folds more melanin at 72 h post-incubation compared with wild-type SL341 when grown in minimal medium supplemented with tyrosine. The chemical analysis of SL341T cultural filtrate revealed the accumulation of a higher amount of homogentisate, a major precursor of pyomelanin, and a lower amount of dihydroxyphenylalanine, an intermediate of eumelanin, compared with SL341. The expression study showed a relatively higher expression of hppD (encoding hydroxyphenylpyruvate dioxygenase) and lower expression of hmgA (encoding homogentisate dioxygenase) and nagL (encoding maleylacetoacetate isomerase) in SL341T than in SL341. SL341 showed a significantly higher expression of tyrosinase gene compared with SL341T at 48 h post-incubation. These results indicated that R. solanacearum produced both pyomelanin and eumelanin, and the novel hypothetical protein is involved in the negative regulation of melanin production.