Aims: To characterize the plantaricin IIA-1A5 crude extract that biosynthesized by Lactobacillus plantarum IIA-1A5 using corn steep liquor (CSL) based medium. Methodology and results: Lactobacillus plantarum IIA-1A5 was grown in several media containing different components including corn steep liquor (CSL), molasses and MRS (de Man Rogosa Sharpe) as control medium for 24 h at 37 °C. Antibacterial activities of the cell-free supernatant were expressed as diameter of inhibition zones observed by paper disc method. The results showed that CSL medium produced cell-free supernatant of L. plantarum IIA-1A5 with significantly higher antibacterial activity againts Staphylococcus aureus ATCC 25923 (9.81 mm), Lactobacillus monocytogenes ATCC 7644 (9.61 mm), Bacillus cereus (8.97 mm) and Escherichia coli ATCC 25922 (9.23 mm) were not significantly different compared to control MRS broth media (9.59 mm). CSL medium added only with 3% yeast extract and Tween 80 produced supernatant which showed similar antibacterial activity either to 10% molasses or control medium (Medium K and B). The CSL medium was considered more efficient and low cost, therefore this medium was selected for production and characterization of plantaricin IIA-1A5 crude extract. Further characterization performed by SDS PAGE analysis showed that crude plantaricin had molecular weight of approximately 9.9 kDa, higher than that produced in control medium (8.0 kDa). However, both plantaricins were categorized under the same class for small bacteriocin (class II). This study also revealed the plantaricin IIA-1A5 produced in CSL medium was stable to heat and pH and not significantly different compared to control MRS broth media. The antibacterial activity of plantaricin IIA-1A5 crude extract against S. aureus ATCC 25923 (10.09 mm) was not significantly different with 1000 ppm sodium benzoate (9.70 mm) and 300 ppm sodium nitrite (9.82 mm). Conclusion, significance and impact of study The CSL medium produced cell-free supernatant of L. plantarum IIA 1A5 had significant antibacterial activity characterization againts S. aureus ATCC 25923, L. monocytogenes ATCC 7644, B. cereus and E. coli ATCC 25922. Comparison of the inhibition activity of plantaricin IIA-1A5 crude extract against pathogen with synthetic preservatives indicated that plantaricin IIA-1A5 crude extract have the potency to replace synthetic preservatives. CSL based medium is potential to be used for low-cost plantaricin IIA-1A5 production.
Anti-Bacterial Agents--metabolism ; Bacteriocins--metabolism ; Lactobacillus plantarum ; Microbial Viability--drug effects ; Staphylococcus aureus

The objective of this study is to propose a more accurate and faster MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay (MCA) for quantitative measurement of polypeptide bacteriocins in solutions with nisin as an example. After an initial incubation of nisin and indicator bacterium Micrococcus luteus NCIB 8166 in tubes, MTT was added for another incubation period. After that, nisin was quantified by estimating the number of viable bacteria based on measuring the amount of purple formazan produced by cleavage of yellow tetrazolium salt MTT. Then MCA was compared to a standard agar diffusion assay (ADA). The results suggested a high correlation coefficient (r(2)=0.975+/-0.004) between optical density (OD) and the inhibitory effect of nisin on a bacterial strain Micrococcus luteus NCIB 8166 at a range of 0.125-32 IU/ml. The MCA described in this study was very quick. Quantification of nisin took only 7-8 h and the detection limit was at the level of 0.125 IU/ml when compared to 12 IU/ml and 24-28 h for ADA. The MCA provides an accurate and rapid method for quantification of nisin in solutions and is expected to be used for quantification of other antimicrobial substances.
Bacteriocins ; analysis ; metabolism ; Colorimetry ; methods ; Immunodiffusion ; Micrococcus luteus ; metabolism ; Nisin ; Regression Analysis ; Tetrazolium Salts ; analysis ; Thiazoles ; analysis ; Time Factors

Broad-spectrum of conventional antibiotics is one of the key factors that cause antibiotics-resistance of many bacteria. Bacteriocins are regarded as the next generation of antibiotics on account of their narrow-spectrum bactericidal activities. Many attentions have been paid to colicins because they are believed to be safe in regard to human body. In this paper, the genes encoding colicin S4 and its immunity protein were cloned into pQE30 to produce colicin S4 expression vector pQE30-Col S4. Colicin S4 was highly expressed as soluble form in gE colig M15 containing pQE30-Col S4. The yields ranged from 30 mg/L to 50 mg/L. The recombinant colicin S4 with an additional 6 His-tag at its N-terminus was found being similar to the natural colicin S4 in antibacterial activity. It only showed bactericidal activity against E. coli strains, thus makig it attractive to develop this protein as a novel antibiotic with narrow spectrum.
Anti-Bacterial Agents ; biosynthesis ; Bacteriocins ; biosynthesis ; genetics ; Cloning, Molecular ; Colicins ; biosynthesis ; genetics ; Escherichia coli ; drug effects ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

NB-Cl gene is a potential class IIa bacteriocin gene. To obtain its soluble expression, NB-C1 was fused with the green fluorescent protein (GFP) gene and a recombinant expression vector plVEX 2.4d-GFP-NB-C1 was constructed, which was transformed into Escherichia coli BL21(DE3) pLysS. The expressed fusion protein GFP-NB-CI was purified by Ni-NTA affinity chromatography and the bioactivity was examined using Listeria monocytogenes as the indicator bacteria. The results showed that the expressed fusion protein GFP-NB-C1 was soluble and the final concentration of the purified fusion protein was 36.1 mg/L E. coli culture and had the purity above 95%. The antimicrobial assay of GFP-NB-C1 was analyzed and showed its high activity against Listeria monocytogenes.
Amino Acid Sequence ; Anti-Bacterial Agents ; pharmacology ; Bacteriocins ; biosynthesis ; genetics ; Chromatography, Affinity ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Listeria monocytogenes ; drug effects ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

Cholesterol-dependent cytolysins (CDC) are pore forming toxins. A prototype of the CDC family members is perfringolysin O (PFO), which directly binds to the cell membrane enriched in cholesterol, causing cell lysis. However, an exception of this general observation is intermedilysin (ILY) of Streptococcus intermedius, which requires human CD59 as a receptor in addition to cholesterol for its hemolytic activity. A possible explanation of this functional difference is the conformational variation between the C-terminal domains of the two toxins, particularly in the highly conserved undecapeptide termed tryptophan rich motif. Here, we present the crystal structure of suilysin, a CDC toxin from the infectious swine pathogen Streptococcus suis. Like PFO, suilysin does not require a host receptor for hemolytic activity; yet the crystal structure of suilysin exhibits a similar conformation in the tryptophan rich motif to ILY. This observation suggests that the current view of the structure-function relationship between CDC proteins and membrane association is far from complete.
Amino Acid Sequence ; Animals ; Bacterial Toxins ; chemistry ; Bacteriocins ; chemistry ; Cholesterol ; chemistry ; Crystallography, X-Ray ; Cytotoxins ; chemistry ; Hemolysin Proteins ; chemistry ; genetics ; Molecular Sequence Data ; Point Mutation ; Protein Structure, Tertiary ; Sequence Alignment ; Streptococcus suis ; metabolism ; Swine