April 21, 2020  |  

Plantibacter flavus, Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens Endophytes Provide Host-Specific Growth Promotion of Arabidopsis thaliana, Basil, Lettuce, and Bok Choy Plants.

A collection of bacterial endophytes isolated from stem tissues of plants growing in soils highly contaminated with petroleum hydrocarbons were screened for plant growth-promoting capabilities. Twenty-seven endophytic isolates significantly improved the growth of Arabidopsis thaliana plants in comparison to that of uninoculated control plants. The five most beneficial isolates, one strain each of Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens and two strains of Plantibacter flavus were further examined for growth promotion in Arabidopsis, lettuce, basil, and bok choy plants. Host-specific plant growth promotion was observed when plants were inoculated with the five bacterial strains. P. flavus strain M251 increased the total biomass and total root length of Arabidopsis plants by 4.7 and 5.8 times, respectively, over that of control plants and improved lettuce and basil root growth, while P. flavus strain M259 promoted Arabidopsis shoot and root growth, lettuce and basil root growth, and bok choy shoot growth. A genome comparison between P. flavus strains M251 and M259 showed that both genomes contain up to 70 actinobacterial putative plant-associated genes and genes involved in known plant-beneficial pathways, such as those for auxin and cytokinin biosynthesis and 1-aminocyclopropane-1-carboxylate deaminase production. This study provides evidence of direct plant growth promotion by Plantibacter flavusIMPORTANCE The discovery of new plant growth-promoting bacteria is necessary for the continued development of biofertilizers, which are environmentally friendly and cost-efficient alternatives to conventional chemical fertilizers. Biofertilizer effects on plant growth can be inconsistent due to the complexity of plant-microbe interactions, as the same bacteria can be beneficial to the growth of some plant species and neutral or detrimental to others. We examined a set of bacterial endophytes isolated from plants growing in a unique petroleum-contaminated environment to discover plant growth-promoting bacteria. We show that strains of Plantibacter flavus exhibit strain-specific plant growth-promoting effects on four different plant species.Copyright © 2019 American Society for Microbiology.

April 21, 2020  |  

Information about variations in multiple copies of bacterial 16S rRNA genes may aid in species identification.

Variable region analysis of 16S rRNA gene sequences is the most common tool in bacterial taxonomic studies. Although used for distinguishing bacterial species, its use remains limited due to the presence of variable copy numbers with sequence variation in the genomes. In this study, 16S rRNA gene sequences, obtained from completely assembled whole genome and Sanger electrophoresis sequencing of cloned PCR products from Serratia fonticola GS2, were compared. Sanger sequencing produced a combination of sequences from multiple copies of 16S rRNA genes. To determine whether the variant copies of 16S rRNA genes affected Sanger sequencing, two ratios (5:5 and 8:2) with different concentrations of cloned 16S rRNA genes were used; it was observed that the greater the number of copies with similar sequences the higher its chance of amplification. Effect of multiple copies for taxonomic classification of 16S rRNA gene sequences was investigated using the strain GS2 as a model. 16S rRNA copies with the maximum variation had 99.42% minimum pairwise similarity and this did not have an effect on species identification. Thus, PCR products from genomes containing variable 16S rRNA gene copies can provide sufficient information for species identification except from species which have high similarity of sequences in their 16S rRNA gene copies like the case of Bacillus thuringiensis and Bacillus cereus. In silico analysis of 1,616 bacterial genomes from long-read sequencing was also done. The average minimum pairwise similarity for each phylum was reported with their average genome size and average “unique copies” of 16S rRNA genes and we found that the phyla Proteobacteria and Firmicutes showed the highest amount of variation in their copies of their 16S rRNA genes. Overall, our results shed light on how the variations in the multiple copies of the 16S rRNA genes of bacteria can aid in appropriate species identification.

April 21, 2020  |  

Genomic and Functional Characterization of the Endophytic Bacillus subtilis 7PJ-16 Strain, a Potential Biocontrol Agent of Mulberry Fruit Sclerotiniose.

Bacillus sp. 7PJ-16, an endophytic bacterium isolated from a healthy mulberry stem and previously identified as Bacillus tequilensis 7PJ-16, exhibits strong antifungal activity and has the capacity to promote plant growth. This strain was studied for its effectiveness as a biocontrol agent to reduce mulberry fruit sclerotiniose in the field and as a growth-promoting agent for mulberry in the greenhouse. In field studies, the cell suspension and supernatant of strain 7PJ-16 exhibited biocontrol efficacy and the lowest disease incidence was reduced down to only 0.80%. In greenhouse experiments, the cell suspension (1.0?×?106 and 1.0?×?105 CFU/mL) and the cell-free supernatant (100-fold and 1000-fold dilution) stimulated mulberry seed germination and promoted mulberry seedling growth. In addition, to accurately identify the 7PJ-16 strain and further explore the mechanisms of its antifungal and growth-promoting properties, the complete genome of this strain was sequenced and annotated. The 7PJ-16 genome is comprised of two circular plasmids and a 4,209,045-bp circular chromosome, containing 4492 protein-coding genes and 116 RNA genes. This strain was ultimately designed as Bacillus subtilis based on core genome sequence analyses using a phylogenomic approach. In this genome, we identified a series of gene clusters that function in the synthesis of non-ribosomal peptides (surfactin, fengycin, bacillibactin, and bacilysin) as well as the ribosome-dependent synthesis of tasA and bacteriocins (subtilin, subtilosin A), which are responsible for the biosynthesis of numerous antimicrobial metabolites. Additionally, several genes with function that promote plant growth, such as indole-3-acetic acid biosynthesis, the production of volatile substances, and siderophores synthesis, were also identified. The information described in this study has established a good foundation for understanding the beneficial interactions between endophytes and host plants, and facilitates the further application of B. subtilis 7PJ-16 as an agricultural biofertilizer and biocontrol agent.

April 21, 2020  |  

Characterization of a Novel Insecticidal Protein Cry9Cb1 from Bacillus thuringiensis.

In recent decades, there have been increasing reports of insect resistance in Bacillus thuringiensis (Bt) crops. Alternative use of Cry toxins, with high insecticidal activity and different mechanisms of action, may be an important strategy to manage this resistance. Cry9 protein, with high toxicity to the lepidopteran pests and no cross-resistance with commercial Cry1 proteins, is a valuable relevant resource. A novel insecticidal protein, MP1489, subsequently named as Cry9Cb1, with 88% amino acid sequence identity with Cry9Ca1, was identified from Bt strain SP663; it exhibited high insecticidal activity against Plutella xylostella, Ostrinia furnacalis, and Chilo suppressalis and no cross-resistance with Cry1Fa in Ostrinia furnacalis. Its minimal active fragments against Plutella xylostella and Ostrinia furnacalis were identified to be 72T-657V and 68D-655A, respectively; food-safety assessment showed no sequence homology with any known allergen and rapid degradation and inactivation by both heat and the gastrointestinal environment. Therefore, Cry9Cb1 is proposed to have a brilliant prospect as an insecticidal protein in agriculture.

April 21, 2020  |  

A First Study of the Virulence Potential of a Bacillus subtilis Isolate From Deep-Sea Hydrothermal Vent.

Bacillus subtilis is the best studied Gram-positive bacterium, primarily as a model of cell differentiation and industrial exploitation. To date, little is known about the virulence of B. subtilis. In this study, we examined the virulence potential of a B. subtilis strain (G7) isolated from the Iheya North hydrothermal field of Okinawa Trough. G7 is aerobic, motile, endospore-forming, and requires NaCl for growth. The genome of G7 is composed of one circular chromosome of 4,216,133 base pairs with an average GC content of 43.72%. G7 contains 4,416 coding genes, 27.5% of which could not be annotated, and the remaining 72.5% were annotated with known or predicted functions in 25 different COG categories. Ten sets of 23S, 5S, and 16S ribosomal RNA operons, 86 tRNA and 14 sRNA genes, 50 tandem repeats, 41 mini-satellites, one microsatellite, and 42 transposons were identified in G7. Comparing to the genome of the B. subtilis wild type strain NCIB 3610T, G7 genome contains many genomic translocations, inversions, and insertions, and twice the amount of genomic Islands (GIs), with 42.5% of GI genes encoding hypothetical proteins. G7 possesses abundant putative virulence genes associated with adhesion, invasion, dissemination, anti-phagocytosis, and intracellular survival. Experimental studies showed that G7 was able to cause mortality in fish and mice following intramuscular/intraperitoneal injection, resist the killing effect of serum complement, and replicate in mouse macrophages and fish peripheral blood leukocytes. Taken together, our study indicates that G7 is a B. subtilis isolate with unique genetic features and can be lethal to vertebrate animals once being introduced into the animals by artificial means. These results provide the first insight into the potential harmfulness of deep-sea B. subtilis.

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