RNA-Seq Analysis of Antibiotic-Producing Bacillus subtilis SC-8 Reveals a Role for Small Peptides in Controlling PapR Signaling.

Bacillus subtilis SC-8 (BSSC8) shows a narrow antimicrobial activity against the Bacillus cereus group. Previously, B. cereus-derived PapR as a signal peptide to stimulate PlcR, which plays a significant role in regulating the transcription of virulence factors, was assumed to stimulate antibiotic production in BSSC8. To better understand the functional role of PapR in the antibiotic production of BSSC8 and the interspecies interaction, the global transcriptomic profiling of BSSC8 was investigated using RNA-Seq in this study. Small peptides derived from B. cereus wild type (WTBC) and a papR-deleted mutant strain (MTBC) were individually supplied to BSSC8 cultures, and changes in global transcription levels were compared by RNA-Seq. In the presence of WTBC small peptides, more genes (80.9%) were significantly upregulated than in cells exposed to MTBC small peptides. Specifically, 48.8 and 83.4% of genes involved in glycolysis and the TCA cycle, respectively, showed changes in transcription levels in response to small peptides from both strains. Of the genes showing the alterations, 35.0% (glycolysis) and 60.0% (TCA cycle) of transcripts were significantly regulated only in response to WTBC-derived small peptides. Furthermore, the expression of biosynthetic genes encoding several known antibiotics in BSSC8 was further decreased in response to WTBC small peptides.

Hypoglycemic Effect of Opuntia ficus-indica var. saboten Is Due to Enhanced Peripheral Glucose Uptake through Activation of AMPK/p38 MAPK Pathway.

Opuntia ficus-indica var. saboten (OFS) has been used in traditional medicine for centuries to treat several illnesses, including diabetes. However, detailed mechanisms underlying hypoglycemic effects remain unclear. In this study, the mechanism underlying the hypoglycemic activity of OFS was evaluated using in vitro and in vivo systems. OFS treatment inhibited α-glucosidase activity and intestinal glucose absorption assessed by Na⁺-dependent glucose uptake using brush border membrane vesicles. AMP-activated protein kinase (AMPK) is widely recognized as an important regulator of glucose transport in skeletal muscle, and p38 mitogen-activated protein kinase (MAPK) has been proposed to be a component of AMPK-mediated signaling. In the present study, OFS dose-dependently increased glucose uptake in L6 muscle cells. The AMPK and p38 MAPK phosphorylations were stimulated by OFS, and inhibitors of AMPK (compound C) and p38 MAPK (SB203580) abolished the effects of OFS. Furthermore, OFS increased glucose transporter 4 (GLUT4) translocation to the plasma membrane. OFS administration (1 g/kg and 2 g/kg body weight) in db/db mice dose-dependently ameliorated hyperglycemia, hyperinsulinemia, and glucose tolerance. Insulin resistance assessed by homeostasis model assessment of insulin resistance and quantitative insulin sensitivity check index were also dose-dependently improved with OFS treatment. OFS administration improved pancreatic function through increased ß-cell mass in db/db mice. These findings suggest that OFS acts by inhibiting glucose absorption from the intestine and enhancing glucose uptake from insulin-sensitive muscle cells through the AMPK/p38 MAPK signaling pathway.

Antimicrobial peptides of the genus Bacillus: a new era for antibiotics.

The rapid onset of resistance reduces the efficacy of most conventional antimicrobial drugs and is a general cause of concern for human well-being. Thus, there is great demand for a continuous supply of novel antibiotics to combat this problem. Bacteria-derived antimicrobial peptides (AMPs) have long been used as food preservatives; moreover, prior to the development of conventional antibiotics, these AMPs served as an efficient source of antibiotics. Recently, peptides produced by members of the genus Bacillus were shown to have a broad spectrum of antimicrobial activity against pathogenic microbes. Bacillus-derived AMPs can be synthesized both ribosomally and nonribosomally and can be classified according to peptide biosynthesis, structure, and molecular weight. The precise mechanism of action of these AMPs is not yet clear; however, one proposed mechanism is that these AMPs kill bacteria by forming channels in and (or) disrupting the bacterial cell wall. Bacillus-derived AMPs have potential in the pharmaceutical industry, as well as the food and agricultural sectors. Here, we focus on Bacillus-derived AMPs as a novel alternative approach to antibacterial drug development. We also provide an overview of the biosynthesis, mechanisms of action, applications, and effectiveness of different AMPs produced by members of the Bacillus genus, including several recently identified novel AMPs.

RNA-seq analysis of antibiotic-producing Bacillus subtilis SC-8 in response to signal peptide PapR of Bacillus cereus.

Bacillus subtilis SC-8 produces an antibiotic that has narrow antagonistic activity against bacteria in the Bacillus cereus group. In B. cereus group bacteria, peptide-activating PlcR (PapR) plays a significant role in regulating the transcription of virulence factors. When B. subtilis SC-8 and B. cereus are co-cultured, PapR is assumed to stimulate antibiotic production by B. subtilis SC-8. To better understand the effect of PapR on this interspecies interaction, the global transcriptome profile of B. subtilis SC-8 was analyzed in the presence of PapR. Significant changes were detected in 12.8 % of the total transcripts. Genes related to amino acid transport and metabolism (16.5 %) and transcription (15 %) were mainly upregulated, whereas genes involved in carbohydrate transport and metabolism (12.7 %) were markedly downregulated. The expression of genes related to transcription, including several transcriptional regulators and proteins involved in tRNA biosynthesis, was increased. The expression levels of genes associated with several transport systems, such as antibiotic, cobalt, and iron complex transporters, was also significantly altered. Among the downregulated genes were transcripts associated with spore formation, the subtilosin A gene cluster, and nitrogen metabolism.

Heterologous expression of polygalacturonase genes isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris.

ABSTACT: The objective of this work was to isolate the polygalacturonase genes of Galactomyces citri-aurantii IJ-1 harvested from rotten citrus peels and to heterologously express these genes in Pichia pastoris. Two polygalacturonase (PG) genes from G. citri-aurantii IJ-1 were obtained and tentatively named PG1 and PG2. The genes were cloned into pPICZαC, and expressed in Pichia pastoris strain GS115 with a native signal peptide or the α-factor secretion signal peptide of Saccharomyces cerevisiae. All of the recombinant proteins were successfully secreted into the culture media and confirmed as a single band with a molecular weight of 35 to 38 kDa by SDS-PAGE. The specific enzyme activities of recombinant PG1 and PG2 purified by His-tag affinity resin were 4,749 and 6,719 U/mg, respectively, with an optimal pH and temperature of pH 4.0 and 50°C. The Michaelis-Menten kinetic constants for PG1 and PG2, K (m), were confirmed to be 0.94 and 0.84 mM, respectively. In the presence of Mn(2+), the activity of PG1 and PG2 were increased to 160.8 and 146.4% of normal levels, respectively. In contrast, Cu(2+) and Fe(3+) acted as strong inhibitors to the PGs.

Interspecies interaction of signal peptide PapR secreted by Bacillus cereus and its effect on production of antimicrobial peptide.

This study was carried out to investigate the interspecies interaction of PapR peptide secreted by Bacillus cereus on production of BSAP-254, an antimicrobial peptide produced by Bacillus subtilis SC-8 isolated from the Korean fermented soybean paste and exhibited narrow antagonistic activity against the B. cereus group, but not against other foodborne pathogens. PapR is a signal peptide that activates PlcR, which is a pleiotropic regulator controlling the expression of various virulence factors in B. cereus. When B. subtilis SC-8 was co-cultured with B. cereus, it completely inhibited the growth of B. cereus within 12 h, and the rate of BSAP-254 production was increased 34.2% at 12 h. Furthermore, 5 µM of synthetic PapR peptide added to the culture of B. subtilis SC-8 increased the rate of BSAP-254 production up to 59.7%. The growth of B. subtilis SC-8, however, was not significantly different with or without the addition of PapR. When B. cereus papR mutant was co-cultured with B. subtilis SC-8, the growth of the mutant was not inhibited and the rate of BSAP-254 production was decreased by 45%.

Characterization of Lactic Acid Bacteria Isolated from Sauce-type Kimchi.

This study was carried out to investigate the isolation and characterization of lactic acid bacteria (LAB) from naturally fermented sauce-type kimchi. Sauce-type kimchi was prepared with fresh, chopped ingredients (Korean cabbage, radish, garlic, ginger, green onion, and red pepper). The two isolated bacteria from sauce-type kimchi were identified as Pediococcus pentosaceus and Lactobacillus brevis by 16S rDNA sequencing and tentatively named Pediococcus sp. IJ-K1 and Lactobacillus sp. IJ-K2, respectively. Pediococcus sp. IJ-K1 was isolated from the early and middle fermentation stages of sauce-type kimchi whereas Lactobacillus sp. IJ-K2 was isolated from the late fermentation stage. The resistance of Pediococcus sp. IJ-K1 and Lactobacillus sp. IJ-K2 to artificial gastric and bile acids led to bacterial survival rates that were 100% and 84.21%, respectively.

Genome sequencing of Bacillus subtilis SC-8, antagonistic to the Bacillus cereus group, isolated from traditional Korean fermented-soybean food.

Bacillus subtilis SC-8 is a Gram-positive bacterium displaying narrow antagonistic activity for the Bacillus cereus group. B. subtilis SC-8 was isolated from Korean traditional fermented-soybean food. Here we report the draft genome sequence of B. subtilis SC-8, including biosynthetic genes for antibiotics that may have beneficial effects for control of food-borne pathogens.

Narrow antagonistic activity of antimicrobial peptide from Bacillus subtilis SCK-2 against Bacillus cereus.

Bacillus subtilis SCK-2, producing an antimicrobial peptide of this study, was isolated from Kyeopjang, the Korean traditional fermented-soybean paste. This strain showed a narrow antagonistic activity as it inhibited Bacillus cereus causing food poisoning in human. The antimicrobial peptide, tentatively named AMP IC-1, was purified, characterized, and compared to BSAP-254, another peptide which was previously recovered from traditionally fermented-soybean paste. AMP IC-1 was found to be more thermally stable than BSAP-254, retained inhibitory activity similar to that of BSAP-254 over wide range of pH values, and was also destroyed by proteolytic enzymes. Two compounds were detected by anti-BSAP-254 polyclonal antibody and showed to contain peptide moieties and aliphatic hydrocarbons by Fourier transform infrared analysis. AMP IC-1 had an identical R(f) value (0.69) on TLC plate and a molecular weight similar to that of BSAP-254 (AMP IC-1, m/z 3401; BSAP-254, m/z 3400 to 3473). AMP IC-1 was found to contain about 33 residues and 13 types of amino acids: Cys, Asp or Asn, Glu or Gln, Ser, Gly, Arg, Thr, Ala, Pro, Val, Ile, Leu, and Lys. Compared to BSAP-254, the molar ratios of Asp or Asn, Ser, Val, and Leu were different and only AMP IC-1 contained Arg, but not Trp. Both compounds showed non-hemolytic activity. A partial synergistic effect against B. cereus was observed in response to treatment when AMP IC-1 and BSAP-254 were administered in combination. Therefore, AMP IC-1 is a possible candidate as an antimicrobial agent to prevent food-borne infectious disease in human caused by B. cereus.

Growth inhibition and induction of stress protein, GroEL, of Bacillus cereus exposed to antibacterial peptide isolated from Bacillus subtilis SC-8.

This study was conducted to investigate the antibacterial effect of BSAP-254 on Bacillus cereus with the induced stress proteins. The BSAP-254 is an antimicrobial peptide isolated from soybean-fermenting bacteria, Bacillus subtilis SC-8. It had a narrow spectrum of activity against B. cereus group. The growth inhibitory effect of BSAP-254 (50 µg/mL) reduced the population of B. cereus from >10(8) to 10(4) colony-forming units per milliliter within 30 min. In B. cereus exposed to BSAP-254, 14 intracellular proteins were differentially expressed as determined by 2-DE coupled with MS. Of the differentially expressed proteins identified, the stress protein GroEL, which is heat shock protein, was induced in B. cereus exposed to antibacterial peptide.

Marinitalea sucinacia gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from tidal flat sediment.

An amber-pigmented, Gram-negative, rod-shaped and aerobic bacterial strain devoid of flagella, designated strain JC2131(T) , was isolated from tidal flat sediment of Dongmak in Ganghwa island, South Korea. Identification was carried out on the basis of polyphasic taxonomy. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate belonged to the family Flavobacteriaceae and showed the highest sequence similarity of 94.5% with Lutibacter litoralis KCCM 42118(T). The predominant cellular fatty acids were iso-C(15:0) (25.9%), iso-C(15:0) 3-OH (20.0%) and iso-C(13:0) (12.7%). Flexirubin-type pigments were absent. The major isoprenoid quinone was MK-6. The DNA G+C content was 43.7 mol%. Several phenotypic and chemotaxonomic properties including growth at pH 6, sea salts requirement, aesculin hydrolysis, carbon utilization, DNA G+C content and fatty acid profiles also differentiated the strain from the related members of the family. Therefore, results from the polyphasic taxonomy study suggested that strain JC2131(T) represents a novel genus and species in the family Flavobacteriaceae for which the name Marinitalea sucinacia gen. nov., sp. nov. is proposed (type strain JC2131(T)=KCTC 12705(T)=JCM 14003(T)).

Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus.

In this study, an effective substance was isolated from Bacillus subtilis SC-8, which was obtained from traditionally fermented soybean paste, cheonggukjang. The substance was purified by HPLC, and its properties were analyzed. It had an adequate antagonistic effect on Bacilluscereus, and its spectrum of activity was narrow. When tested on several gram-negative and gram-positive foodborne pathogenic bacteria such as Salmonella enterica, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes, no antagonistic effect was observed. Applying the derivative from B. subtilis SC-8 within the same genus did not inhibit the growth of major soybean-fermenting bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens. The range of pH stability of the purified antagonistic substance was wide (from 4.0 to >10.0), and the substance was thermally stable up to 60 degrees C. In the various enzyme treatments, the antagonistic activity of the purified substance was reduced with proteinase K, protease, and lipase; its activity was partially destroyed with esterase. Spores of B. cereus did not grow at all in the presence of 5mug/mL of the purified antagonistic substance. The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254 because it absorbed to UV radiation at 254nm.

Characterization of Lactobacillus spp. isolated from the feces of breast-feeding piglets.

Lactobacillus spp., referred to as IJ-1 and IJ-2, were isolated from the feces of breast-feeding piglets and analyzed for probiotic properties. According to the analyses of 16S rDNA sequence, Lactobacillus sp. IJ-1 showed greater than 99% homology with Lactobacillus reuteri DSM 20016(T), and Lactobacillus sp. IJ-2 had greater than 99% homology with the L. gasseri ATCC 33323(T) and L. johnsonii ATCC 33200(T). The pH changes in the culture media of Lactobacillus sp. IJ-1 and Lactobacillus sp. IJ-2 were from 6.5 to 4.2 and 4.6, respectively. Their respective resistance against artificial gastric acid and artificial bile acid led to survival rates of nearly 186+/-44% and 13+/-5%. Neither strain produced the carcinogenic enzyme beta-glucuronidase. Both strains inhibited the growth of pathogenic microorganisms, such as Listeria monocytogenes ATCC 19111, Salmonella enterica KCTC 12401, Salmonella enteritidis ATCC 13076, Staphylococcus aureus KCTC 3881, and Bacillus cereus 3711, within 24 h of growth.

Actibacter sediminis gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from tidal flat sediment.

A yellow-pigmented, Gram-negative, aerobic bacterial strain comprising rod-shaped cells devoid of flagellar and gliding motility, designated strain JC2129(T), was isolated from tidal flat sediment of Dongmak on Ganghwa Island, South Korea. Results from a 16S rRNA gene sequence analysis indicated that the isolate belonged to the family Flavobacteriaceae; the highest level of nucleotide sequence similarity (91.9%) occurred with Polaribacter dokdonensis DSW-5(T). The predominant cellular fatty acids were iso-C(15:0) (19.8%), iso-C(15:1) G (14.0%), iso-C(17:0) 3-OH (13.7%) and iso-C(13:0) (6.4%). Flexirubin-type pigments were absent. The major isoprenoid quinone was MK-6. The DNA G+C content was 43-45 mol%. Data from a polyphasic taxonomy study suggested that the isolate represents a novel genus and species in the family Flavobacteriaceae, for which the name Actibacter sediminis gen. nov., sp. nov. is proposed. The type strain of Actibacter sediminis is JC2129(T) (=KCTC 12704(T) =JCM 14002(T)).