In-Cheol Yeo

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.

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.

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.

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.

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.

Isolation of Carotenoid-producing yeast, Rhodosporidium babjevae JI-1, and evaluation of cell extract toxicity against rat hepatic cells

Carotenoid-producing yeast Rhodosporidium babjevae JI-1 was isolated from citrus fruit peel. The red pigment produced by R. babjevae JI-1 was confirmed as β-carotene. Viability of rat hepatocytes treated with R. babjevae JI-1 cell extract at 20–1,000 μg/mL was about 85.1±15.1–100±14.5%.

Identification of Microorganisms, Cladosporium sp. and Sterigmatomyces sp., Proliferated on the Surface of Traditional Soy Sauce, and the Effect of NaCl Concentration on Their Enzymatic Activity

Two strains, traditionally referred to as rock flower (Bawhi-kot) and buckwheat flower (Memil-kot or Chile- Kot), were isolated from stored traditional soy sauce and were identified by using the 18S ITS1/4 region sequences. The rock flower strain showed 99% of similarity with Cladosporium sp. and buckwheat flower strain was 99% identical with yeast Sterigmatomyces halophilus. Both strains were tentatively named Cladosporium sp. NK1 and Sterigmatomyces halophilus NK2, respectively. The optimal growth pHs and temperatures of both strains in a YPD broth medium were in the range of pH 5.0 to 7.0 and 22 to 27 o C. Both strains were able to grow in more than 20% of NaCl. In the enzyme activity assay, high protease activity of Cladosporium sp. NK1 and S. halophilus NK2 were obtained in YPD containing 10% of NaCl. High amylase activities of both stains were in 15% and 5% of NaCl, respectively. Lipase activity was, however, not detected in both strains.

Heterologous Expression of Polygalacturonase Genes Isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris

AbstactThe 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, Km, were confirmed to be 0.94 and 0.84 mM, respectively. In the presence of Mn2+, the activity of PG1 and PG2 were increased to 160.8 and 146.4% of normal levels, respectively. In contrast, Cu2+ and Fe3+ acted as strong inhibitors to the PGs.