Phage SPO1 Protein Gp49 Is a Novel RNA Binding Protein That Is Involved in Host Iron Metabolism.

Bacillus subtilis is a model organism for studying Gram-positive bacteria and serves as a cell factory in the industry for enzyme and chemical production. Additionally, it functions as a probiotic in the gastrointestinal tract, modulating the gut microbiota. Its lytic phage SPO1 is also the most studied phage among the genus Okubovrius, including Bacillus phage SPO1 and Camphawk. One of the notable features of SPO1 is the existence of a "host-takeover module", a cluster of 24 genes which occupies most of the terminal redundancy. Some of the gene products from the module have been characterized, revealing their ability to disrupt host metabolism by inhibiting DNA replication, RNA transcription, cell division, and glycolysis. However, many of the gene products which share limited similarity to known proteins remain under researched. In this study, we highlight the involvement of Gp49, a gene product from the module, in host RNA binding and heme metabolism-no observation has been reported in other phages. Gp49 folds into a structure that does not resemble any protein in the database and has a new putative RNA binding motif. The transcriptome study reveals that Gp49 primarily upregulates host heme synthesis which captures cytosolic iron to facilitate phage development.

Bacillus subtilis KC1 prevents Mycoplasma gallisepticum-induced lung injury by enhancing intestinal Bifidobacterium animalis and regulating indole metabolism in chickens.

It has been reported that dietary administration of Bacillus subtilis KC1 is effective in alleviating lung injury induced by Mycoplasma gallisepticum (MG) infection in chickens. However, the underlying molecular mechanism of B. subtilis KC1 against MG infection is still unclear. The purpose of this study was to determine whether B. subtilis KC1 could alleviate MG infection-induced lung injury in chickens by regulating their gut microbiota. The results of this study indicate that B. subtilis KC1 supplementation has the potential to alleviate MG infection-induced lung injury as reflected by reduced MG colonization, reduced pathologic changes, and decreased production of pro-inflammatory cytokines. In addition, B. subtilis KC1 supplementation was partially effective in alleviating the gut microbiota disorder caused by MG infection. Importantly, B. subtilis KC1 enriched the beneficial Bifidobacterium animalis in gut and thus reversed indole metabolic dysfunction caused by MG infection. B. subtilis KC1 supplementation increased levels of indole, which enhanced aryl hydrocarbon receptor activation, improving barrier function and alleviating lung inflammation caused by MG. Overall, this study indicates that B. subtilis KC1 has a "gut-lung axis" mechanism that can reduce the severity of MG infection by enriching intestinal B. animalis and regulating indole metabolism.

Acute physiological effects following Bacillus subtilis DE111 oral ingestion - a randomised, double blinded, placebo-controlled study.

Previous studies using ileostomy samples from study participants demonstrated that the spore-forming probiotic Bacillus subtilis DE111® can germinate in the small intestine as early as 4 hours after ingestion. Metabolomics, proteomics and sequencing technologies, enabled further analysis of these samples for the presence of hypoglycaemic, hypolipidemic, antioxidant, anti-inflammatory and antihypertensive molecules. In the DE111 treatment group, the polyphenols trigonelline and 2,5-dihydroxybenzoic acid, orotic acid, the non-essential amino acid cystine and the lipokine 12,13-diHome were increased. DE111 also reduced acetylcholine levels in the ileostomy samples, and increased the expression of leucocyte recruiting proteins, antimicrobial peptides and intestinal alkaline phosphatases of the brush border in the small intestine. The combination of B. subtilis DE111 and the diet administered during the study increased the expression of the proteins phosphodiesterase ENPP7, ceramidase ASAH2 and the adipokine Zn-alpha-2-glycoprotein that are involved in fatty acid and lipid metabolism. Acute B. subtilis DE111 ingestion had limited detectable effect on the microbiome, with the main change being its increased presence. These findings support previous data suggesting a beneficial role of DE111 in digestion, metabolism, and immune health that appears to begin within hours of consumption.

Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.

PrkA is an ATP-dependent protease that regulates sporulation in Bacillus subtilis.

In Bacillus subtilis, sporulation is a sequential and highly regulated process. Phosphorylation events by histidine kinases are key points in the phosphorelay that initiates sporulation, but serine/threonine protein kinases also play important auxiliary roles in this regulation. PrkA has been proposed to be a serine protein kinase expressed during the initiation of sporulation and involved in this differentiation process. Additionally, the role of PrkA in sporulation has been previously proposed to be mediated via the transition phase regulator ScoC, which in turn regulates the transcriptional factor σK and its regulon. However, the kinase activity of PrkA has not been clearly demonstrated, and neither its autophosphorylation nor phosphorylated substrates have been unambiguously established in B. subtilis. We demonstrated here that PrkA regulation of ScoC is likely indirect. Following bioinformatic homology searches, we revealed sequence similarities of PrkA with the ATPases associated with diverse cellular activities ATP-dependent Lon protease family. Here, we showed that PrkA is indeed able to hydrolyze α-casein, an exogenous substrate of Lon proteases, in an ATP-dependent manner. We also showed that this ATP-dependent protease activity is essential for PrkA function in sporulation since mutation in the Walker A motif leads to a sporulation defect. Furthermore, we found that PrkA protease activity is tightly regulated by phosphorylation events involving one of the Ser/Thr protein kinases of B. subtilis, PrkC. Taken together, our results clarify the key role of PrkA in the complex process of B. subtilis sporulation.

Bacterial developmental checkpoint that directly monitors cell surface morphogenesis.

Bacillus subtilis spores are encased in two concentric shells: an outer proteinaceous "coat" and an inner peptidoglycan "cortex," separated by a membrane. Cortex assembly depends on coat assembly initiation, but how cells achieve this coordination across the membrane is unclear. Here, we report that the protein SpoVID monitors the polymerization state of the coat basement layer via an extension to a functional intracellular LysM domain that arrests sporulation when coat assembly is initiated improperly. Whereas extracellular LysM domains bind mature peptidoglycan, SpoVID LysM binds to the membrane-bound lipid II peptidoglycan precursor. We propose that improper coat assembly exposes the SpoVID LysM domain, which then sequesters lipid II and prevents cortex assembly. SpoVID defines a widespread group of firmicute proteins with a characteristic N-terminal domain and C-terminal peptidoglycan-binding domains that might combine coat and cortex assembly roles to mediate a developmental checkpoint linking the morphogenesis of two spatially separated supramolecular structures.

Synergistic Protective Effect of Konjac Mannan Oligosaccharides and Bacillus subtilis on Intestinal Epithelial Barrier Dysfunction in Caco-2 Cell Model and Mice Model of Lipopolysaccharide Stimulation.

As the first line of defense against intestinal bacteria and toxins, intestinal epithelial cells are always exposed to bacteria or lipopolysaccharide (LPS), whereas pathogenic bacteria or LPS can cause intestinal epithelial cell damage. Previous studies have shown that konjac mannan oligosaccharides (KMOS) have a positive effect on maintaining intestinal integrity, and Bacillus subtilis (BS) can promote the barrier effect of the intestine. However, it is still unknown whether KMOS and BS have a synergistic protective effect on the intestines. In this study, we used the LPS-induced Caco-2 cell injury model and mouse intestinal injury model to study the synergistic effects of KMOS and BS. Compared with KMOS or BS alone, co-treatment with KMOS and BS significantly enhanced the activity and antioxidant capacity of Caco-2 cell, protected mouse liver and ileum from LPS-induced oxidative damage, and repaired tight junction and mucus barrier damage by up-regulating the expression of Claudin-1, ZO-1 and MUC-2. Our results demonstrate that the combination of KMOS and BS has a synergistic repair effect on inflammatory and oxidative damage of Caco-2 cells and aIIeviates LPS-induced acute intestinal injury in mice.

Association between productive parameters and intestinal histomorphological findings in broilers supplemented with probiotics (Saccharomyces cerevisiae, Lactobacillus acidophilus and Bacillus subtilis) / Asociación entre parámetros productivos y hallazgos histomorfológicos en pollos de engorde suplementados con probióticos (Saccharomyces cerevisiae, Lactobacillus acidophilus y Bacillus subtilis)

SUMMARY: This study evaluates the effect of probiotics Saccharomyces cerevisiae, Lactobacillus acidophilus and Bacillus subtilis on production parameters and intestinal histomorphology of broilers of 45 days of age. Eleven 45-day-old Ross 500 broilers were used and classified as control group (CG) (n = 5) or supplemented with probiotics group (n = 8). Histopathological evaluation of duodenum, ileum, and jejunum was performed. The area of the villi height, base and apex were evaluated as well as the size and number of crypts. In addition, mucus production was quantified in different portions of the small intestine. The villi present duodenum of broilers supplemented with probiotics had a greater area (p = 0.0127), a greater basal width (p = 0.0049) and a greater apical width (p = 0.0024), as well as a greater crypt area (p = 0.0189). Significantly higher levels of mucus were noted in the duodenum (p = 0.0480) and jejunum (p = 0.0480) of broilers supplemented with probiotics. We suggest that probiotic supplementation improve the intestinal nutrients absorption.

RESUMEN: Este estudio evalúa el efecto del uso de probióticos como: Saccharomyces cerevisiae, Lactobacillus acidophilus, Bacillus subtilis en parámetros productivos e histomorfologia intestinal de pollos de engorde de 45 días de edad. Fueron usados 11, los cuales fueron clasificados en grupo control (CG) (n = 5) y grupo suplementado con probióticos (PG) (n = 8). Fue realizado análisis histopatológico de secciones de duodeno, íleon y yeyuno. Fue evaluado ancho, altura y área del ápice de la vellosidad, área y número de criptas. Además, fue estimada la producción de moco en los diferentes segmentos del intestino delgado. Fue observada mayor área de la vellosidad en duodeno, PG (p = 0.0127), ancho basal mayor en PG (p = 0.0049) ancho apical mayor en PG (p = 0.0024), así como mayor área de criptas en PG (p = 0.0189). No fueron encontradas diferencias significativas respecto a los segmentos de yeyuno e íleon. PG presentó mayor producción de moco en duodeno (p = 0.0480) y en yeyuno (p = 0.0480). Concluimos que la suplementación con probióticos en pollos de engorde genera cambios en la histomorfologia intestinal, evidenciables en áreas apicales y basales de las vellosidades intestinales. Soporte financiero: Dirección General de Investigaciones - Universidad de los Llanos.

Control of stripe rust of wheat using indigenous endophytic bacteria at seedling and adult plant stage.

Stripe rust (caused by Puccinia striiformis tritici) is one of the most devastating diseases of wheat. The most effective ways to control stripe rust are the use of resistant cultivars and the timely use of an appropriate dose of fungicide. However, the changing nature of rust pathogen outwits the use of resistant cultivars, and the use of a fungicide is associated with environmental problems. To control the disease without sacrificing the environment, we screened 16 endophytic bacteria, which were isolated from stripe rust-resistant wheat cultivars in our previous study, for their biocontrol potential. A total of 5 bacterial strains Serratia marcescens 3A, Bacillus megaterium 6A, Paneibacillus xylanexedens 7A, Bacillus subtilis 11A, and Staphyloccus agentis 15A showed significant inhibition of Puccinia striiformis f. sp. tritici (Pst) urediniospores germination. Two formulations i.e., fermented liquid with bacterial cell (FLBC) and fermented liquid without bacterial cells (FL) of each bacterial strain, were evaluated against the urediniospores germination. Formulations of five selected endophytic bacteria strains significantly inhibited the uredinioospores germination in the lab experiments. It was further confirmed on seedlings of Pakistani susceptible wheat cultivar Inqilab-91 in the greenhouse, as well as in semi-field conditions. FLBC and FL formulations applied 24 h before Pst inoculation (hbi) displayed a protective mode. The efficacy of FLBC was between 34.45 and 87.77%, while the efficacy of FL was between 39.27 and 85.16% when applied 24 hbi. The inoculated wheat cultivar Inqilab-91 was also tested under semi-field conditions during the 2017-2018 cropping season at the adult plant stage. The strains Bacillus megaterium 6A and Paneibacillus xylanexedens 7A alone significantly reduced the disease severity of stripe rust with the efficacy of 65.16% and 61.11% for the FLBC in protective effect, while 46.07% and 44.47% in curative effect, respectively. Inoculated seedlings of Inqilab-91 showed higher activities of antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL). The treated seedlings also showed higher expressions of pathogenesis-related (PR) protein genes, antifungal protein (PR-1), ß-1,3-endoglucanases (PR-2), endochitinases (PR-4), peroxidase (PR-9), and ribonuclease-like proteins (PR-10). These results indicated that endophytic bacteria have the biocontrol potential, which can be used to manage stripe rust disease. High production antioxidant enzymes, as well as high expression of PR protein genes, might be crucial in triggering the host defense mechanism against Pst.

Whole genome sequence insight of two plant growth-promoting bacteria (B. subtilis BS87 and B. megaterium BM89) isolated and characterized from sugarcane rhizosphere depicting better crop yield potentiality.

Since sugarcane is a ratoon crop, genome analysis of plant growth-promoting bacteria that exist in its soil rhizosphere, can provide opportunity to better understand their characteristics and use of such bacteria in turn, may especially improve perennial crop productivity. In the present study, genome of two bacterial strains, one each of B. megaterium (BM89) and B. subtilis (BS87), isolated and reported earlier (Chandra et al., 2018), were sequenced and characterized. Though both strains have demonstrated plant growth promoting properties and enhanced in-vitro plant growth responses, functional annotation and analysis of genes indicated superiority of BS87 as it possessed more plant growth promotion attributable genes over BM89. Apart from some common genes, trehalose metabolism, glycine betaine production, peroxidases, super oxide dismutase, cold shock proteins and phenazine production associated genes were selectively identified in BS87 genome indicating better plant growth performances and survival potential under harsh environmental conditions. Genes for chitinase, d-cysteine desulfhydrase and γ-aminobutyric acid (GABA), as found in BM89, propose its selective utilization in defense and bio-control measures. Concomitant with better settlings' growth, scanning electron micrographs indicated these isolated and characterized bacteria exhibiting healthy colonization within root of sugarcane crop. Kegg pathways' assignment also revealed added pathways namely carbohydrate and amino acid metabolism attached to B. subtilis strain BS87, a preferable candidate for bio-fertilizer and its utilization to promote growth of both plant and ratoon crops of sugarcane usually experiencing harsh environmental conditions.

Bifunctional mechanisms of autophagy and apoptosis regulations in melanoma from Bacillus subtilis natto fermentation extract.

Melanoma is one of the most dangerous malignant epidermal cancers. Natto freeze-drying extract (NFDE) and natto water extract (NWE) were isolated from natto, soybeans fermented by Bacillus subtilis natto, which were assessed as potential anti-melanoma agents. Cell cytotoxicity assays revealed significant anti-melanoma effects of NFDE and NWE in a dose-dependent manner, and exhibited low influences on normal skin cells, including Hs68, HaCaT and adipose tissue-derived stem cells (ADSCs), respectively. Through a flow cytometer assay and autophagy acridine orange staining, the cellular death phenomenon shifted from autophagy to apoptosis with the increased dosages. Reactive oxygen species (ROS) were enhanced using DCFDA (2,7-dichlorodihydrofluorescein diacetate) staining when melanoma cells were treated with the extract. NFDE and NWE treatments increase the oxidative stress of cancer cells and cause apoptosis by inhibiting AMP-activated protein kinase (AMPK). NFDE and NWE were considered to play a critical role in cell death through ROS adjustment, autophagy regulation and apoptosis promotion.

Molecular and functional identification of a ß-defensin homolog in large yellow croaker (Larimichthys crocea).

ß-defensin (BD) is a cysteine-rich cationic antibacterial peptide that is active against a wide range of bacteria. Here, a ß-defensin homolog (LcBD2) was identified in large yellow croaker (Larimichthys crocea). The open reading frame of LcBD2 contains 195 nucleotides, encoding a protein of 64 amino acids that possesses a typical arrangement of six conserved cysteine residues (C31 , C37 , C41 , C53 , C59 and C60 ). LcBD2 transcripts were constitutively expressed in all examined tissues and significantly increased in head kidney, spleen and gills by Vibrio alginolyticus. The synthetic LcBD2 peptide imparted antimicrobial effects on both Gram-negative bacteria (V. campbellii, V. parahaemolyticus, V. alginolyticus, V. harveyi and Pseudomonas plecoglossicida) and Gram-positive bacteria (Bacillus subtilis). We also observed that after treatment with synthetic LcBD2 peptide, numerous blisters appeared on the membrane of P. plecoglossicida, which in turn may result in cell membrane breakage and bacterial death. Moreover, the synthetic LcBD2 peptide significantly upregulated the expression levels of TNF-α2, IL-1ß and CXCL8_L1 in monocytes/macrophages, while downregulated expression level of IL-10. The LcBD2 peptide also remarkedly enhanced the phagocytosis of monocytes/macrophages. These results indicate that LcBD2 not only protects large yellow croaker against multiple bacterial pathogens but also plays a role in activation of monocytes/macrophages.

Bacillus subtilis Inhibits Viral Hemorrhagic Septicemia Virus Infection in Olive Flounder (Paralichthys olivaceus) Intestinal Epithelial Cells.

Viral hemorrhagic septicemia virus (VHSV) is a highly pathogenic virus that infects a wide range of host fish species causing high economic losses in aquaculture. Epithelial cells in mucosal organs are target sites for VHSV entry into fish. To protect fish against VHSV infection, there is a need to develop antiviral compounds able to prevent establishment of infection at portals of virus entry into fish. Bacillus subtilis is a probiotic with excellent antiviral properties, of which one of its secretions, surfactin, has been shown to inhibit viral infections in mammals. Herein, we demonstrate its ability to prevent VHSV infection in olive flounder (Paralichthys olivaceus) intestinal epithelial cells (IECs) and infection in internal organs. Our findings show inhibition of VHSV infection in IECs by B. subtilis and surfactin. In addition, our findings showed inhibition of VHSV in Epithelioma Papulosum Cyprini (EPC) cells inoculated with intestinal homogenates from the fish pretreated with B. subtilis by oral exposure, while the untreated fish had cytopathic effects (CPE) caused by VHSV infection in the intestines at 48 h after the VHSV challenge. At 96 h post-challenge, samples from the untreated fish had CPE from head kidney and spleen homogenates and no CPE were observed in the intestinal homogenates, while the B. subtilis-pretreated fish had no CPE in all organs. These findings demonstrate that inhibition of VHSV infection at portals of virus entry in the intestines culminated in prevention of infection in internal organs. In summary, our results show that B. subtilis has the potential to prevent VHSV infection in fish and that its use as a probiotic in aquaculture has the potential to serve as an antiviral therapeutic agent against different viral infections.

Bacillus subtilis-based probiotic promotes bone growth by inhibition of inflammation in broilers subjected to cyclic heating episodes.

Heat stress as an environmental stressor causes abnormal bone remodeling and microarchitectural deterioration. The objective of this study was to investigate the effects of a Bacillus subtilis-based probiotic on bone mass of broilers subjected to cycling high ambient temperature. One hundred and twenty 1-day-old Ross 708 male broiler chicks were randomly assigned to 2 dietary treatments (12 pens per treatment): control diet and the control diet plus 250-ppm probiotic consisting of 3 strains of Bacillus subtilis. Room temperature was gradually decreased from 35°C on day 1 by 0.5°C/d until day 15, when ambient temperature was increased from 28°C to 32°C for 10 h (07:00 h-17:00 h) daily until day 44. Samples of blood, leg bones (tibia and femur), and brains (raphe nuclei and hypothalamus) were collected at day 43, while latency to lie test was conducted at day 44. Compared with controls, probiotic supplementation increased bone mineral content, weight, size, weight to length index, and reduced robusticity index in the tibia and femur (P < 0.05) of broilers subjected to heat stress. Serum concentrations of c-terminal telopeptide of type I collagen (CTX) were reduced (P = 0.02) by the probiotic supplementation, while ionized calcium, phosphate, and osteocalcin were not affected (P > 0.05). Moreover, tumor necrosis factor-α (TNF-α) in probiotic fed broilers was decreased (P = 0.003) without changes of plasma interleukin (IL)-6, IL-10, interferon-γ, and corticosterone concentrations. There were no treatment effects on the concentrations of peripheral serotonin and central serotonin and catecholamines (norepinephrine, epinephrine, and dopamine) as well as their metabolites. These results may indicate that dietary supplementation of Bacillus subtilis-based probiotic increases bone growth in broilers under a cyclic heating episode probably via inhibition of bone resorption, resulting from downregulation of the circulating TNF-α and CTX. Dietary probiotic supplementation may be a management strategy for increasing skeletal health of broilers under hot weather.

Bacillus subtilis Modulates Its Usage of Biofilm-Bound Iron in Response to Environmental Iron Availability.

Iron (Fe) is one of the most important micronutrients for most life forms on earth. While abundant in soil, Fe bioavailability in oxic soil is very low. Under environmental conditions, bacteria need to acquire sufficient Fe to sustain growth while limiting the energy cost of siderophore synthesis. Biofilm formation might mitigate this Fe stress, since it was shown to accumulate Fe in certain Gram-negative bacteria and that this Fe could be mobilized for uptake. However, it is still unclear if, and to what extent, the amount of Fe accumulated in the biofilm can sustain growth and if the mobilization of this local Fe pool is modulated by the availability of environmental Fe (i.e., Fe outside the biofilm matrix). Here, we use a nondomesticated strain of the ubiquitous biofilm-forming soil bacterium Bacillus subtilis and stable Fe isotopes to precisely evaluate the origin of Fe during growth in the presence of tannic acid and hydroxides, used as proxies for different environmental conditions. We report that this B. subtilis strain can accumulate a large quantity of Fe in the biofilm, largely exceeding Fe associated with cells. We also report that only a fraction of biofilm-bound Fe is available for uptake in the absence of other sources of Fe in the vicinity of the biofilm. We observed that the availability of environmental Fe modulates the usage of this pool of biofilm-bound Fe. Finally, our data suggest that consumption of biofilm-bound Fe relates to the efficacy of B. subtilis to transport Fe from the environment to the biofilm, possibly through siderophores.IMPORTANCE Recent pieces of evidence suggest that Fe bound to the biofilm could assume at least two important functions, a local source of Fe for uptake and a support to extracellular metabolism, such as extracellular electron transfer. Our results show that B. subtilis can use biofilm-bound Fe for uptake only if it does not compromise Fe homeostasis of the biofilm, i.e., maintains a minimum Fe concentration in the biofilm for extracellular purposes. We propose a theoretical framework based on our results and recent literature to explain how B. subtilis manages biofilm-bound Fe and Fe uptake in response to environmental Fe availability. These results provide important insights into the management of biofilm-bound and environmental Fe by B. subtilis in response to Fe stress.

L-Asparaginase activity analysis, ansZ gene identification and anticancer activity of a new Bacillus subtilis isolated from sponges of the Red Sea.

This study describes the isolation of various marine bacteriafrom sponges collected from the Red Sea (Saudi Arabia) andL-asparaginase (anti-cancer enzyme) production from bacterialisolates. The 16S rDNA based phylogenetic analysis revealed thatthe isolate WSA3 was a Bacillus subtilis. Its partial-length genesequence was submitted to GenBank under the accession numberMK072695. The new B. subtilis strain harbored the exact size(1128 bp) of the new L-asparaginase (ansZ) gene as confirmedby PCR and in gel visualization, which was submitted to the NCBIdatabase (accession number MN566442). The molecular weightof partially purified L-asparaginase was determined as 45 kDa bySDS-PAGE. In addition, the enzyme L-asparaginase did not showglutaminase activity which is very important from a medical pointof view. Moreover, 100 µg/mL of the partially purified B. subtilis Lasparaginaseshowed promising anti-cancer activities when testedagainst three cancer cell lines (HCT-116, MCF-7, and HepG2).

Channels modestly impact compartment-specific ATP levels during Bacillus subtilis sporulation and a rise in the mother cell ATP level is not necessary for Pro-σK cleavage.

Starvation of Bacillus subtilis initiates endosporulation involving formation of mother cell (MC) and forespore (FS) compartments. During engulfment, the MC membrane migrates around the FS and protein channels connect the two compartments. The channels are necessary for postengulfment FS gene expression, which relieves inhibition of SpoIVFB, an intramembrane protease that cleaves Pro-σK , releasing σK into the MC. SpoIVFB has an ATP-binding domain exposed to the MC cytoplasm, but the role of ATP in regulating Pro-σK cleavage has been unclear, as has the impact of the channels on MC and FS ATP levels. Using luciferase produced separately in each compartment to measure relative ATP concentrations during sporulation, we found that the MC ATP concentration rises about twofold coincident with increasing cleavage of Pro-σK , and the FS ATP concentration does not decline. Mutants lacking a channel protein or defective in channel protein turnover exhibited modest and varied effects on ATP levels, which suggested that low ATP concentration does not explain the lack of postengulfment FS gene expression in channel mutants. Furthermore, a rise in the MC ATP level was not necessary for Pro-σK cleavage by SpoIVFB, based on analysis of mutants that bypass the need for relief of SpoIVFB inhibition.

A protein phosphorylation module patterns the Bacillus subtilis spore outer coat.

Assembly of the Bacillus subtilis spore coat involves over 80 proteins which self-organize into a basal layer, a lamellar inner coat, a striated electrodense outer coat and a more external crust. CotB is an abundant component of the outer coat. The C-terminal moiety of CotB, SKRB , formed by serine-rich repeats, is polyphosphorylated by the Ser/Thr kinase CotH. We show that another coat protein, CotG, with a central serine-repeat region, SKRG , interacts with the C-terminal moiety of CotB and promotes its phosphorylation by CotH in vivo and in a heterologous system. CotG itself is phosphorylated by CotH but phosphorylation is enhanced in the absence of CotB. Spores of a strain producing an inactive form of CotH, like those formed by a cotG deletion mutant, lack the pattern of electrondense outer coat striations, but retain the crust. In contrast, deletion of the SKRB region, has no major impact on outer coat structure. Thus, phosphorylation of CotG by CotH is a key factor establishing the structure of the outer coat. The presence of the cotB/cotH/cotG cluster in several species closely related to B. subtilis hints at the importance of this protein phosphorylation module in the morphogenesis of the spore surface layers.

Effects of an F18 enterotoxigenic Escherichia coli challenge on growth performance, immunological status, and gastrointestinal structure of weaned pigs and the potential protective effect of direct-fed microbial blends.

The objective of this experiment was to investigate the impact of an F18 enterotoxigenic Escherichia coli (ETEC) challenge on growth performance, aspects of intestinal function, and selected immune responses of piglets, as well as to evaluate potential protective effects of direct-fed microbial (DFM) blends. Seventy-two weaned piglets (6.4 ± 0.2 kg body weight [BW]; ~21 d of age) were assigned to one of four treatments: 1) NC: Nonchallenged (n = 10), 2) positive challenged control (PC): F18 ETEC-challenged (n = 10), 3) PC + DFM1 (n = 8; three strains of Bacillus amyloliquefaciens; 7.5 × 105 colony-forming units [cfu]/g), or 4) PC + DFM2 (n=8; 2 strains of B. amyloliquefaciens and one strain of Bacillus subtilis; 1.5 × 105 cfu/g). Feed intake and BW were recorded on day 0, 7, and 17. Pigs were sham-infected either with 6 mL phosphate-buffered saline or inoculated with 6 mL F18 ETEC (~1.9 × 109 cfu/mL) on day 7 (0 d postinoculation [dpi]). All ETEC-challenged pigs were confirmed to be genetically susceptible to F18. Pigs had ad libitum access to feed and water throughout the 17-d trial. Fecal scores were visually ranked and rectal temperatures were recorded daily. To evaluate ETEC shedding, fecal swabs were collected on dpi 0, 1, 2, 3, 5, 7, and 10. Blood samples were collected on dpi 0, 1, 2, 4, 7, and 10. Ileal tissues were collected at necropsy on dpi 10. All challenged treatments had lower final BW, decreased average daily gain (ADG), and average daily feed intake (ADFI) during the 10-d postchallenge period (P < 0.01). The DFM2 treatment increased E. coli shedding on dpi 2 and decreased iton dpi 7 (P < 0.05) compared with the PC. Rectal temperature decreased across all challenged treatments (P < 0.01). Ileal mRNA abundance of occludin (OCLN) and zonula occludens-1 (ZO-1) decreased in PC and DFM1 compared with NC (P < 0.05). Pigs fed DFM2 had intermediate ileal mRNA abundance of OCLN and increased ZO-1 mRNA compared with pigs in PC (P < 0.05). Interleukin 8 (IL-8) increased in the plasma of PC and DFM2 on dpi 2 compared with NC (P < 0.05). Mucosal IL-8 increased in PC compared with NC (P < 0.05). All challenged treatments tended to have elevated tumor necrosis factor-α (TNF-α) mRNA abundance compared with NC (P < 0.10). Challenged pigs had reduced secretory immunoglobulin A and villus height compared with NC pigs (P < 0.05). The impact of an ETEC challenge on intestinal function and the immune system has been revealed, information critical to developing improved treatment regimes.

Effect of Bacillus subtilis on corrosion behavior of 10MnNiCrCu steel in marine environment.

Bacillus widely exists in wet natural environment such as soil, water and air, and is often studied as one of representative microorganisms for microbiologically influenced corrosion(MIC) research. In this paper, the growth curve of Bacillus subtilis isolated from marine environment was determined by turbidimetry and its effect on corrosion behavior of 10MnNiCrCu steel was studied by open circuit potential, AC impedance, polarization curve and scanning electron microscopy(SEM). The results showed that with the change of the growth curve of Bacillus subtilis(BS), the open circuit potential(Eocp) shifted positively and then negatively, and the charge transfer resistance shown by AC impedance was much lower than that of the sterile system, increasing first and then decreasing. The polarization curves showed that the corrosion current density in BS medium was obviously higher than that in sterile system. The corrosion morphology observation showed that although a biofilm by BS developed on the steel surface, the localized corrosion of 10MnNiCrCu steel was aggravated due to the acidness of the metabolite itself and the biofilm with access for electrolyte ions.