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Background Members of the Bacillus genus produce a large variety of antimicrobial peptides including linear or cyclic lipopeptides and thiopeptides, that often have a broad spectrum of action against Gram-positive and Gram-negative bacteria. We have recently reported that SF214, a marine isolated strain of Bacillus pumilus , produces two different antimicrobials specifically active against either Staphylococcus aureus or Listeria monocytogenes. The anti- Staphylococcus molecule has been previously characterized as a pumilacidin, a nonribosomally synthesized lipopetide composed of a mixture of cyclic heptapeptides linked to fatty acids of variable length. Results Our analysis on the anti- Listeria molecule of B. pumilus SF214 indicated that it is a peptide slightly smaller than 10 kDa, produced during the exponential phase of growth, stable at a wide range of pH conditions and resistant to various chemical treatments. The peptide showed a lytic activity against growing but not resting cells of Listeria monocytogenes and appeared extremely specific being inactive also against L. innocua , a close relative of L. monocytogenes . Conclusions These findings indicate that the B. pumilus peptide is unusual with respect to other antimicrobials both for its time of synthesis and secretion and for its strict specificity against L. monocytogenes . Such specificity, together with its stability, propose this new antimicrobial as a tool for potential biotechnological applications in the fight against the dangerous food-borne pathogen L. monocytogenes.

Bacillus species have demonstrated beneficial effects on intestinal health, production parameters, and immune function in poultry under both standard and disease-challenged conditions. Previously, we found that several novel ingredients (beta-glucan, vegetable protein hydrolysate, and liquid seaweed extract) demonstrated growth stimulation effects on Bifidobacterium lactis and Lactobacillus plantarum , Here, we extended this approach to examine in vitro synbiotic combinations of five novel marine-derived candidate probiotic Bacillus strains to assess their potential for in ovo applications. Beta-glucan enhanced the growth of all candidate Bacillus probiotic strains compared to a glucose control ( p  ≤ 0.05), suggesting a broad-spectrum modulatory role over a 24-h period, with variable magnitudes of response observed between strains. Species specificity was also observed, with lentinus stimulating the Bacillus pumilus but not the Bacillus altitudinis strains. A seaweed extract consistently stimulated the growth of one of the B. altitudinis strains ( p  ≤ 0.05), which, like all of the strains evaluated here, is seaweed-derived. This suggests potential ecological adaptation in substrate utilization. The shared environmental origin may influence substrate specificity and metabolic complementarity between strains and prebiotic candidates. Both B. altitudinis strains also exhibited enhanced growth at almost all time points ( p  ≤ 0.05) when cultured with vegetable protein hydrolysate. Based on these findings, we evaluated the effect of a potential synbiotic formulation comprising one of the B. altitudinis strains and vegetable protein hydrolysate in chickens, in ovo. The components were administered intra-amniotically at embryonic development day 18.5, utilizing a standard vaccination protocol. The hatchability of the chickens was not affected, thereby demonstrating the established dose as safe and applicable for further investigation. Key points • Shared origin of bioactive compounds may enhance probiotic-prebiotic compatibility in vitro • Protein hydrolysate offers a novel alternative to carbohydrate prebiotics • In ovo delivery of Bacillus-based synbiotic formulations offers potential as an early microbiome programming strategy

Biochar is widely used as a soil amendment to increase crop yields. However, the impact of the interaction between the biochar and microbial inoculants (e.g., biofertilizer) on plant nutrient uptake and yield in forage rice is not fully understood. A greenhouse study was conducted to evaluate the synergistic effects of rice-husk biochar and Bacillus pumilus strain TUAT-1 biofertilizer application on growth, yield, and nutrient uptake in two forage rice genotypes; Fukuhibiki and the newly bred line, LTAT-29. Positive effects of biochar and biofertilizer, alone or in a combination, on growth traits, nutrient uptake, and yield components were dependent on the rice genotypes. Biochar and TUAT-1 biofertilizer influenced the overall growth of plants positively and increased straw and above-ground biomass in both genotypes. However, although biochar application significantly increased grain yield in LTAT-29, this was not the case in Fukuhibiki. Biochar and TUAT-1 biofertilizer, either alone or combined, significantly affected plant nutrient uptake but the effect largely depended on rice genotype. Results of this study indicate that biochar amendment and TUAT-1 biofertilizer can enhance forage rice productivity depending on genotypes, and therefore, there is a need to consider plant genetic composition when evaluating the potential for crop response to these soil amendments before application on a commercial scale.

Decabromodiphenyl ether (BDE-209) is a brominated flame retardant and a priority contaminant. Currently, little information is available about its significance in the environment, specifically about its susceptibility to aerobic biotransformation at low temperature. In this work, five phylogenetically diverse BDE-209-degrading bacterial strains were isolated from river sediments of northern China. These strains were distributed among four different genera— Acinetobacter , Pseudomonas , Bacillus and Staphylococcus . All five isolates were capable of growing on BDE-209, among which two isolates show better growth. By detailed morphological, physiological, and biochemical characteristics and 16S rDNA sequence analysis, the two strains were identified and named as Staphylococcus haemolyticus LY1 and Bacillus pumilus LY2. The two bacteria can grow in mineral salt medium containing BDE-209 substrate across the temperatures ranging from 2.5 to 35 °C, with an optimum temperature of 25 °C which could be considered as psychrotrophs accordingly. The degradation experiment showed that more than 70.6 and 85.5 % of 0.5 mg/L BDE-209 were degraded and the highest mineralization efficiencies of 29.8 and 39.2 % were achieved for 0.5 mg/L BDE-209 by S. haemolyticus LY1 and B. pumilus LY2, respectively. To the best of our knowledge, this is the first demonstration for the biodegradation of BDE-209 by two psychrotrophic bacteria isolated from environment.

Background Bacillus pumilus can secret abundant extracellular enzymes, and may be used as a potential host for the industrial production of enzymes. It is necessary to understand the metabolic processes during cellular growth. Here, an RNA-seq based transcriptome analysis was applied to examine B. pumilus BA06 across various growth stages to reveal metabolic changes under two conditions. Results Based on the gene expression levels, changes to metabolism pathways that were specific to various growth phases were enriched by KEGG analysis. Upon entry into the transition from the exponential growth phase, striking changes were revealed that included down-regulation of the tricarboxylic acid cycle, oxidative phosphorylation, flagellar assembly, and chemotaxis signaling. In contrast, the expression of stress-responding genes was induced when entering the transition phase, suggesting that the cell may suffer from stress during this growth stage. As expected, up-regulation of sporulation-related genes was continuous during the stationary growth phase, which was consistent with the observed sporulation. However, the expression pattern of the various extracellular proteases was different, suggesting that the regulatory mechanism may be distinct for various proteases. In addition, two protein secretion pathways were enriched with genes responsive to the observed protein secretion in B. pumilus . However, the expression of some genes that encode sporulation-related proteins and extracellular proteases was delayed by the addition of gelatin to the minimal medium. Conclusions The transcriptome data depict global alterations in the genome-wide transcriptome across the various growth phases, which will enable an understanding of the physiology and phenotype of B. pumilus through gene expression.

Because of the emergence of antibiotic resistance, we must investigate new antibiotical agents. The present study was designed to find new compounds with antibacterial activity from metabolites of Bacillus pumilus . We found that the concentrated fermentation broth of Bacillus pumilus has antibacterial property. By high-performance liquid chromatography (HPLC), three compounds with antibacterial activity were first isolated from the ethyl acetate layer of fermentation broth of Bacillus pumilus . And then their structures were identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. According to the data, the compound 1, compound 2, and compound 3 were determined to be 3,4-dipentylhexane-2,5-diol, 1,1′-(4,5-dibutylcyclohexane-1,2-diyl)bis(ethan-1-ol), and 1,1′-(4,5-dibutyl-3,6-dimethylcyclohexane-1,2-diyl)bis(ethan-1-one). And all of them exhibited potent inhibitory effects against a panel of pathogenic bacteria including Staphylococcus aureus ATCC6538, Micrococcus luteus CMCC28001, Variant Salmonella gallinarum CVCC79207, Pasteurella multocida CVCC474, Swine Salmonella , Salmonella enterica ATCC13076, Swine Escherichia coli K88 , Chicken Escherichia coli O78. Given its antibacterial activity, 3,4-dipentylhexane-2,5-diol, 1,1′-(4,5-dibutylcyclohexane-1,2-diyl)bis(ethan-1-ol), 1,1′-(4,5-dibutyl-3,6-dimethylcyclohexane-1,2-diyl)bis(ethan-1-one) are assumed to be promising agents for further development as antibacterial agents.

Background Bacillus pumilus is a Gram-positive and endospore-forming bacterium broadly existing in a variety of environmental niches. Because it produces and secrets many industrially useful enzymes, a lot of studies have been done to understand the underlying mechanisms. Among them, scoC was originally identified as a pleiotropic transcription factor negatively regulating protease production and sporulation in B. subtilis . Nevertheless, its role in B. pumilus largely remains unknown. Results In this study we successfully disrupted scoC gene in B. pumilus BA06 and found increased total extracellular protease activity in scoC mutant strain. Surprisingly, we also found that scoC disruption reduced cell motility possibly by affecting flagella formation. To better understand the underlying mechanism, we performed transcriptome analysis with RNA sequencing. The result showed that more than one thousand genes were alternated at transcriptional level across multiple growth phases, and among them the largest number of differentially expressed genes (DEGs) were identified at the transition time point (12 h) between the exponential growth and the stationary growth phases. In accordance with the altered phenotype, many protease genes especially the aprE gene encoding alkaline protease were transcriptionally regulated. In contrast to the finding in B. subtilis , the aprN gene encoding neutral protease was transcriptionally downregulated in B. pumilus , implicating that scoC plays strain-specific roles. Conclusions The pleiotropic transcription factor ScoC plays multiple roles in various cellular processes in B. pumilus, some of which were previously reported in B. subtilis . The supervising finding is the identification of ScoC as a positive regulator for flagella formation and bacterial motility. Our transcriptome data may provide hints to understand the underlying mechanism.

Background Culture media containing complex compounds like yeast extract or peptone show numerous disadvantages. The chemical composition of the complex compounds is prone to significant variations from batch to batch and quality control is difficult. Therefore, the use of chemically defined media receives more and more attention in commercial fermentations. This concept results in better reproducibility, it simplifies downstream processing of secreted products and enable rapid scale-up. Culturing bacteria with unknown auxotrophies in chemically defined media is challenging and often not possible without an extensive trial-and-error approach. In this study, a respiration activity monitoring system for shake flasks and its recent version for microtiter plates were used to clarify unknown auxotrophic deficiencies in the model organism Bacillus pumilus DSM 18097. Results Bacillus pumilus DSM 18097 was unable to grow in a mineral medium without the addition of complex compounds. Therefore, a rich chemically defined minimal medium was tested containing basically all vitamins, amino acids and nucleobases, which are essential ingredients of complex components. The strain was successfully cultivated in this medium. By monitoring of the respiration activity, nutrients were supplemented to and omitted from the rich chemically defined medium in a rational way, thus enabling a systematic and fast determination of the auxotrophic deficiencies. Experiments have shown that the investigated strain requires amino acids, especially cysteine or histidine and the vitamin biotin for growth. Conclusions The introduced method allows an efficient and rapid identification of unknown auxotrophic deficiencies and can be used to develop a simple chemically defined tailor-made medium. B. pumilus DSM 18097 was chosen as a model organism to demonstrate the method. However, the method is generally suitable for a wide range of microorganisms. By combining a systematic combinatorial approach based on monitoring the respiration activity with cultivation in microtiter plates, high throughput experiments with high information content can be conducted. This approach facilitates media development, strain characterization and cultivation of fastidious microorganisms in chemically defined minimal media while simultaneously reducing the experimental effort.

In the present work, the local isolate Bacillus pumilus 15.1 has been morphologically and biochemically characterized in order to gain a better understanding of this novel entomopathogenic strain active against Ceratitis capitata. This strain could represent an interesting biothechnological tool for the control of this pest. Here, we report on its nutrient preferences, extracellular enzyme production, motility mechanism, biofilm production, antibiotic suceptibility, natural resistance to chemical and physical insults, and morphology of the vegetative cells and spores. The pathogen was found to be β-hemolytic and susceptible to penicillin, ampicillin, chloramphenicol, gentamicin, kanamycin, rifampicin, tetracycline, and streptomycin. We also report a series of biocide, thermal, and UV treatments that reduce the viability of B. pumilus 15.1 by several orders of magnitude. Heat and chemical treatments kill at least 99.9 % of vegetative cells, but spores were much more resistant. Bleach was the only chemical that was able to completely eliminate B. pumilus 15.1 spores. Compared to the B. subtilis 168 spores, B. pumilus 15.1 spores were between 2.67 and 350 times more resistant to UV radiation while the vegetative cells of B. pumilus 15.1 were almost up to 3 orders of magnitude more resistant than the model strain. We performed electron microscopy for morphological characterization, and we observed geometric structures resembling the parasporal crystal inclusions synthesized by Bacillus thuringiensis. Some of the results obtained here such as the parasporal inclusion bodies produced by B. pumilus 15.1 could potentially represent virulence factors of this novel and potentially interesting strain.

Gladiolus (Gladiolus grandiflorus) is a highly-valued bulbous flower crop that is commercially grown worldwide for its versatile and showy cut flowers. Among the heavy metals, cadmium (Cd) stress has adverse effects on the growth and quality of gladiolus flowers. In this study, plant growth-promoting rhizobacteria (PGPR) strains including Bacillus velezensis JM11 and Bacillus pumilus CPCF54 were evaluated for their potential to mitigate Cd stress in gladiolus. These strains exhibited the ability to produce siderophores, indole-3-acetic acid, and solubilize phosphates. B. velezensis JM11 and B. pumilus CPCF54 application improved the vegetative and ornamental traits of gladiolus under Cd stress condition. Furthermore, the application of JM11 and CPCF54 also improved stomatal conductance, photosynthesis rate, and transpiration rate compared to non-treated Cd-stressed gladiolus plants. The reduction in oxidative stress was noticed as hydrogen peroxide, malondialdehyde, membrane stability index, and electrolyte leakage were decreased in gladiolus plants treated with JM11 and CPCF54 under Cd stress. Moreover, the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were enhanced in response to the application of Bacillus strains compared to control and Cd-treated plants. The expression of CAT1, MnSODM, and POD genes was upregulated in plants treated with JM11 and CPCF54 under Cd stress. In conclusion, JM11 and CPCF54 had the potential to improve plant growth and Cd stress tolerance in gladiolus plants.