Design of a CRISPR-Cas system to increase resistance of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> to bacteriophage SPP1

Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (cas) genes form an adaptive prokaryotic immune system which provides acquired resistance against viruses and plasmids. Bacillus subtilis presently is the best-characterized laboratory model for Gram-positive bacteria and also widely used for industrial production of enzymes, vitamins and antibiotics. In this study, we show that type II-A CRISPR-Cas system from Streptococcus thermophilus can be transferred into B. subtilis and provides heterologous protection against phage infection. We engineered a heterologous host by cloning S. thermophilus Cas9 and a spacer targeting bacteriophage SPP1 into the chromosome of B. subtilis, which does not harbor its own CRISPR-Cas systems. We found that the heterologous CRISPR-Cas system is functionally active in B. subtilis and provides resistance against bacteriophage SPP1 infection. The high efficiency of the acquired immunity against phage could be useful in generation of biotechnologically important B. subtilis strains with engineered chromosomes.     

Impact of Chemical,Organic and Bio-Fertilizers Application on Bell Pepper, <Emphasis Type="Italic">Capsicum annuum</Emphasis> L. and Biological Parameters of <Emphasis Type="Italic">Myzus persicae</Emphasis> (Sulzer) (Hem.: Aphididae)

Myzus persicae (Sulzer) is a polyphagous aphid that causes chlorosis, necrosis, stunting, and reduce growth rate of the host plants. In this research, the effects of Zinc sulfate and vermicompost (30%), Bacillus subtilis, Pseudomonas fluorescens, Glomus intraradices, G. intraradices × B. subtilis, and G. intraradices × P. fluorescens compared to control was investigated on the growth characters of Capsicum annuum L. and biological parameters of M. persicae. Different fertilizers caused a significant effect on growth characters of C. annuum and biological parameters of M. persicae. The highest plant growth was observed on Zinc sulfate and B. subtilis treated plants, and the lowest was on control. Increase in the amount of specific leaf area (SLA) (0.502 mm² mg^?1) was significantly higher in the B. subtilis than other fertilizer treatments. The longest (10.3 days) and the shortest (5.3 days) developmental times of M. persicae nymphs were observed on 30% vermicompost and Zinc sulfate treatments, respectively. The lowest adult longevity periods of M. persicae (11.2 and 11.3 days) were observed on G. intraradices × B. subtilis and 30% vermicompost treatments, respectively, and the longest ones (16.4 days) on Zinc sulfate. The highest rate of nymphal mortality and the lowest amount of nymphal growth index (NGI) were recorded on 30% vermicompost. The nymphs reared on Zinc sulfate treatment had the lowest rate of nymphal mortality and the highest amount of NGI. Thus, amending the soil with 30% vermicompost had a significantly negative effect on the biological parameters of M. persicae that can be used as an ecological control tactic for this pest.     

Defense Responses and Changes in Symbiotic Gut Microflora in the Colorado Potato Beetle <Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis> under the Effect of Endophytic Bacteria from the Genus Bacillus

Phytophagous insects and host plants have a complex of microsymbionts and make up a united co-evolving system with them. Microsymbiotic complexes are actively involved in stress responses of macrosymbionts. We established that a treatment of potato plants with endophytic bacterial strains Bacillus thuringiensis var. thuringiensis-5689, B. th. var. kurstaki-5351, and Bacillus subtilis 26D decreased the survival rate of the plant feeder, Colorado potato beetle Leptinotarsa decemlineata Say. The B. th. strains suppressed phenoloxidase and acetylcholinesterase activities in the beetle hemolymph. An antagonistic relationship was found between endophytic bacteria B. subtilis 26D and beetle symbiotic bacteria from the genera Acinetobacter and Enterobacter, with the former being able to suppress the growth of endophytic colonies. The recombinant B. subtilis strain 26D Cry, containing the B. th. var. kurstaki δ-endotoxin cry1Ia gene, combined the ability of the original B. subtilis 26D strain to suppress the development of beetle symbionts and immune responses with a production of the Cry toxin, thus leading to a high mortality of the phytophage.     

A novel targeted/untargeted GC-Orbitrap metabolomics methodology applied to <Emphasis Type="Italic">Candida albicans</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilms

Introduction

Combined infections from Candida albicans and Staphylococcus aureus are a leading cause of death in the developed world. Evidence suggests that Candida enhances the virulence of Staphylococcus—hyphae penetrate through tissue barriers, while S. aureus tightly associates with the hyphae to obtain entry to the host organism. Indeed, in a biofilm state, C. albicans enhances the antimicrobial resistance characteristics of S. aureus. The association of these microorganisms is also associated with significantly increased morbidity and mortality. Due to this tight association we hypothesised that metabolic effects were also in evidence.

Objectives

To explore the interaction, we used a novel GC-Orbitrap-based mass spectrometer, the Q Exactive GC, which combines the high peak capacity and chromatographic resolution of gas chromatography with the sub-ppm mass accuracy of an Orbitrap system. This allows the capability to leverage the widely available electron ionisation libraries for untargeted applications, along with expanding accurate mass libraries and targeted matches based around authentic standards.

Methods

Optimised C. albicans and S. aureus mono- and co-cultured biofilms were analysed using the new instrument in addition to the fresh and spent bacterial growth media.

Results

The targeted analysis experiment was based around 36 sugars and sugar phosphates, 22 amino acids and five organic acids. Untargeted analysis resulted in the detection of 465 features from fresh and spent medium and 405 from biofilm samples. Three significantly changing compounds that matched to high scoring library fragment patterns were chosen for validation.

Conclusion

Evaluation of the results demonstrates that the Q Exactive GC is suitable for metabolomics analysis using a targeted/untargeted methodology. Many of the results were as expected: e.g. rapid consumption of glucose and fructose from the medium regardless of the cell type. Modulation of sugar-phosphate levels also suggest that the pentose phosphate pathway could be enhanced in the cells from co-cultured biofilms. Untargeted metabolomics results suggested significant production of cell-wall biosynthesis components and the consumption of non-proteinaceous amino-acids.

     

<Emphasis Type="Italic">Bacillus subtilis</Emphasis> and Vermicompost Suppress Damping-off Disease in Psyllium through Nitric Oxide-Dependent Signaling System

Fusarium oxysporum is one of pathogens causing the damping-off disease of Plantago psyllium in Iran. A greenhouse experiment was conducted to assess the effect of Bacillus subtilis and vermicompost singly and in combination on control of Fusarium–induced damping-off in psyllium. The results showed that vermicompost or B. subtilis, significantly increased the growth of psyllium seedlings and both were effective biocontrol agents against F. oxysporum. Among treatments at least damping-off incidence was recorded in combination of 50% vermicompost and B. subtilis. Results for the first time exhibited that vermicompost as well as B. subtilis induced systemic resistance through nitric oxide (NO) signaling and their combined application further than their individual treatments induced development of plant defense related enzymes including β-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and the activities of antioxidant enzymes (ascorbate peroxidase, catalase, superoxide dismutase and peroxidase) and also more effectively reduced lipid peroxidation in psyllium leaves. These findings suggested potential of B. subtilis in promoting plant growth as well as inducing systemic resistance in the host plants, was enhanced by vermicompost application.     

Polyphasic taxonomic analysis of <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain C6—the antagonist of phytopathogenic microorganisms

Polyphasic taxonomic analysis was carried out for Bacillus sp. strain C6, as the antagonist of phytopathogenic bacteria and micromycetes. The combination of cultural, morphological, physiological, and biochemical properties of the strain has enabled researchers to refer it to the Bacillus subtilis group. It has been shown that the fatty acids of the strain’s cell walls were predominantly represented by branched iso- and anteiso-C15:0 and C17:0 fatty acids (over 85%), which was typical for the Bacillus amyloliquefaciens species. The molecular genetic analysis carried out on the nucleotide sequence of the 16S rRNA gene, and the profiling of polymorphic nucleotides have enabled researchers to refer the strain in question to Bacillus amyloliquefaciens subsp. plantarum.     

A metabolomics and proteomics study of the <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> in the grass carp fermentation

Background

Lactobacillus plantarum, a versatile lactic acid-fermenting bacterium, isolated from the traditional pickles in Ningbo of China, was chosen for grass carp fermentation, which could also improve the flavor of grass carp. We here explored the central metabolic pathways of L. plantarum by using metabolomic approach, and further proved the potential for metabolomics combined with proteomics approaches for the basic research on the changes of metabolites and the corresponding fermentation mechanism of L. plantarum fermentation.

Results

This study provides a cellular material footprinting of more than 77 metabolites and 27 proteins in L. plantarum during the grass carp fermentation. Compared to control group, cells displayed higher levels of proteins associated with glycolysis and nucleotide synthesis, whereas increased levels of serine, ornithine, aspartic acid, 2-piperidinecarboxylic acid, and fumarate, along with decreased levels of alanine, glycine, threonine, tryptophan, and lysine.

Conclusions

Our results may provide a deeper understanding of L. plantarum fermentation mechanism based on metabolomics and proteomic analysis and facilitate future investigations into the characterization of L. plantarum during the grass carp fermentation.

     

<Emphasis Type="Italic">Flavobacterium ureilyticum</Emphasis> sp. nov., a novel urea hydrolysing bacterium isolated from stream bank soil

A novel bacterium designated S-42^T was isolated from stream bank soil. Cells were found to be aerobic, Gram staining-negative, oxidase-positive, catalase-negative, non-motile, non-spore-forming, rod-shaped, and yellow-pigmented. The strain can grow at 15–35 °C, pH 6.0–10.0, and at 0.5% (w/v) NaCl concentration. Urea was hydrolysed. Flexirubin-type pigments were absent. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain S-42^T formed a lineage within the family Flavobacteriaceae of the phylum Bacteroidetes that is distinct from various species of the genus Flavobacterium, including Flavobacterium maotaiense T9^T (97.6% sequence similarity), Flavobacterium hibernum ATCC 51468^T (97.4%), and Flavobacterium granuli Kw05^T (97.1%). The 16S rRNA gene sequences identity between strain S-42^T and other members of the genus Flavobacterium were < 97.0%. Strain S-42^T contains MK-6 as sole respiratory quinone. The major polar lipids were identified as phosphatidylethanolamine and an unidentified aminolipid. The major cellular fatty acids were identified as iso-C_15:0, summed feature 3 (C_16:1ω7c and/or C_{16: 1}ω6c), C_16:0, anteiso-C_15:0, iso-C_17:0 3-OH, iso-C_15:0 3-OH, and iso-C_15:1 G. The DNA G?+?C content of the strain was 35.8 mol%. The polyphasic characterization indicated that strain S-42^T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium ureilyticum sp. nov. is proposed. The type strain is S-42^T (=?KEMB 9005-537^T?=?KACC 19115^T?=?NBRC 112683^T).     

Integrated control of tobacco black shank by combined use of riboflavin and <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain Tpb55

We investigated the effect of riboflavin on the biocontrol activity of Bacillus subtilis Tpb55 against Phytophthora nicotianae (Pn), which causes tobacco black shank. Riboflavin (0.2 mg ml^?1) significantly improved the biocontrol activity of Tpb55 (2.0 × 10⁸ cfu ml^?1). Riboflavin (0.02–0.5 mg ml^?1) alone could not significantly inhibit Pn growth. However, it enhanced the B. subtilis population, both in vitro and in tobacco roots and significantly increased the activity of defense enzymes, peroxidase, catalase, superoxide dismutase, and β-1,3-glucanase, in the roots of B. subtilis-treated tobacco seedlings. Our results indicate that riboflavin can stimulate the growth of B. subtilis Tpb55 and induce resistance to Pn in tobacco plants. These findings should boost the prospects for practical application of B. subtilis Tpb55 as a biocontrol agent against black shank of tobacco.     

Enhanced expression of ginsenoside biosynthetic genes and in vitro ginsenoside production in elicited <Emphasis Type="Italic">Panax sikkimensis</Emphasis> (Ban) cell suspensions

Dual metabolite, i.e., ginsenoside and anthocyanin, co-accumulating cell suspensions of Panax sikkimensis were subjected to elicitation with culture filtrates of Serratia marcescens (SD 21), Bacillus subtilis (FL11), Trichoderma atroviridae (TA), and T. harzianum (TH) at 1.25% and 2.5% v/v for 1- and 3-week duration. The fungal-derived elicitors (TA and TH) did not significantly affect biomass accumulation; however, bacterial elicitors (SD 21 and FL11), especially SD 21, led to comparable loss in biomass growth. In terms of ginsenoside content, differential responses were observed. A maximum of 3.2-fold increase (222.2 mg/L) in total ginsenoside content was observed with the use of 2.5% v/v TH culture filtrate for 1 week. Similar ginsenoside accumulation was observed with the use of 1-week treatment with 2.5% v/v SD 21 culture filtrate (189.3 mg/L) with a 10-fold increase in intracellular Rg2 biosynthesis (31 mg/L). Real-time PCR analysis of key ginsenoside biosynthesis genes, i.e., FPS, SQS, DDS, PPDS, and PPTS, revealed prominent upregulation of particularly PPTS expression (20–23-fold), accounting for the observed enhancement in protopanaxatriol ginsenosides. However, none of the elicitors led to successful enhancement in in vitro anthocyanin accumulation as compared to control values.     

Antibiotic activity of bacterial endobionts of basidiomycete fruit bodies

Bacterial strains (93 isolates) capable of growth on full-strength nutrient media were isolated from 86 fungal fruit bodies collected in the Moscow region. Antimicrobial activity of the endobiont isolates against 12 bacterial and fungal test strains (including drug-resistant ones) was studied in submerged cultures. Most of the strains (84.9%) were found to produce antibiotic compounds with different antimicrobial properties, including antifungal activity in 18.3% of the strains. Morphological characteristics and analysis of the 16S rRNA gene sequences were used to determine the taxonomic position of 16 bacterial strains of the following 10 species: Bacillus subtilis, Ewingella americana, Pseudomonas sp., Stenotrophomonas maltophilia, as well as Achromobacter spanius, B. licheniformis, Hafnia paralvei, Micrococcus terreus, Nocardia coeliaca, and St. rhizophila, which have not been previously known to be endobionts of basidiomycete fruit bodies. Antimicrobial activity of A. spanius, E. americana, H. paralvei, M. terreus, N. coeliaca, and St. rhizophila has not been reported previously. Complex mechanisms of symbiotic relations between fungi and bacteria, including those associated with antibiotic formation, probably developed in the course of co-evolution.     

A novel series of C<Subscript>18</Subscript>–C<Subscript>22</Subscript><Emphasis Type="Italic">trans</Emphasis> ω3 PUFA from Northern and Southern Hemisphere strains of the marine haptophyte <Emphasis Type="Italic">Imantonia rotunda</Emphasis>

A series of novel C₁₈–C₂₂ trans ω3 polyunsaturated fatty acids (PUFA) with a single trans double bond in the ω3 position was found in Northern and Southern Hemisphere strains of the marine haptophyte Imantonia rotunda. The novel ω3 PUFA were identified as 18:3(9c,12c,15t) (0.2–1.8 % of total fatty acids), 18:4(6c,9c,12c,15t) (1.9–4.1 %), 18:5 (3c,6c,9c,12c,15t) (0.7–8.8 %), 20:5(5c,8c,11c,14c,17t) (1.2–4.1 %) and 22:6(4c,7c,10c,13c,16c,19t) (0.3–4.3 %), and were accompanied by larger proportions of the all cis isomers: 18:3ω3(9,12,15) (2.7–3.5 %), 18:4ω3(6,9,12,15) (9.3–14.3 %), 18:5ω3(3,6,9,12,15) (7.8–18.5 %), 20:5ω3(5,8,11,14,17) (3.2–3.9 %), 22:5ω3(7,10,13,16,19) (0.1–0.3 %) and 22:6ω3(4,7,10,13,16,19) (2.3–5.2 %). GC analysis of FAME using a non-polar column did not reveal the trans isomers as they coeluted with the all cis PUFA. However, GC using a polar column resolved the trans PUFA from the all cis PUFA, with the trans isomers eluting before the all cis isomers. GC-MS of FAME fractionated by argentation solid-phase chromatography confirmed the molecular ions of all components. FAME were derivatized to form 4,4-dimethyloxazoline (DMOX) derivatives, and GC-MS revealed the same double bond positions for each trans and cis FAME. The results suggest that the ω3 trans double bond originated from the Δ15/ω3 desaturation of 18:2(9c,12c), suggesting that this desaturase has dual cis/trans activity in these species. These results indicate that 18:3(9c,12c,15?t) was the precursor trans isomer produced for the trans series and further desaturation by the common Δ6 desaturase to produce the trans tetraene and successive elongations and desaturations led to the subsequent series of trans ω3 PUFA isomers. To our knowledge, this is the first report of these trans ω3 isomers occurring in strains of I. rotunda. These trans ω3 PUFA may be used as biomarkers in marine food webs for this species and with their unique structure may be biologically active.     

<Emphasis Type="Italic">Spirosoma metallicus</Emphasis> sp. nov., isolated from an automobile air conditioning system

A Gram-stain-negative and orangish yellow-pigmented bacterial strain, designated PR1014K^T, was isolated from an automobile evaporator core collected in Korea. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain PR1014K^T was related with the members of the genus Spirosoma (94.7–90.2%) and closely related with Spirosoma lacussanchae CPCC 100624^T (94.7%), Spirosoma knui 15J8-12^T (94.3%), and Spirosoma soli MIMBbqt12^T (93.3%). The strain grew at 15–40°C (optimum, 25°C), pH 6.5–7.0 (optimum, 6.5) and 0–1% (w/v) NaCl (optimum, 0%). The predominant fatty acids were summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c), C_16:0, iso-C_15:0, C_16:1 ω5c, and iso-C_17:0 3-OH. The major menaquinone was MK-7. The polar lipid profile of the strain indicated that the presence of one phosphatidylethanolamine, one unidentified aminolipid, two unidentified aminophospholipids, and three unidentified lipids. The DNA G+C content of the strain was 47.4 mol%. On the basis of the phenotypic, genotypic and chemotaxonomic characteristics, strain PR1014K^T represents a novel species in the genus Spirosoma, for which the name Spirosoma metallicus sp. nov. (=KACC 17940^T =NBRC 110792^T) is proposed.