Journal of Bacteriology and Virology 2009. Vol. 39, No. 4 p.269 – 276 DOI 10.4167/jbv.2009.39.4.269 Original Article Bacteriocin from Purple Nonsulfur Phototrophic Bacteria, Rhodobacter capsulatus * * Sang-Seob Lee1, Tae Jung Oh2, Jaisoo Kim1 , Jong-Bae Kim3 and Hyun-Soon Lee4 1Department of Life Science, Kyonggi University, Suwon, Korea 2Genomictree, Inc., Daejon, Korea 3School of Life & Food Sciences, Handong Global University, Pohang, Korea 4Department of Biological Sciences, Sungkyunkwan University, Suwon, Korea To find whether productivity of bacteriocin is controlled between different species under unusual cultural conditions, we used Rhodobacter capsulatus ATCC 17016 as a producer and Rhodopseudomonas palustris ATCC 17003 as an indicator. Rhodobacter capsulatus was cultured under aerobic conditions in the dark in Lascelles medium containing 0.3% Triton X-100. As a result, bacteriocin productivity increased enormously. The optimal pH range of bacteriocin production was 6~7.8. Through partial purification of bacteriocin, the molecular weight was roughly estimated at 14 kDa. Plasmid had no influence on bacteriocin production by Rhodobacter capsulatus. Our findings indicate that culture conditions affect bacteriocin productivity between more distantly related species, and bacteriocin of Rhodobacter capsulatus is not encoded by a plasmid. Key Words: Bacteriocin, Rhodobacter capsulatus, Lascelles medium by lactic acid bacteria kill some pathogenic bacteria (e.g., INTRODUCTION Listeria) with high efficiency (2). Most research has been dedicated to lactic acid bacteria Many gram-positive bacteria and some gram-negative for combination of bacteriocin with antimicrobial agents, bacteria produce ribosomally-synthesized antimicrobial membrane permeability and LPS affinity (2, 10, 11). peptides or proteins, often termed bacteriocins (1~5). The However, bactericidal peptides are produced by several other important role of bacteriocins is to inhibit or kill bacteria classes of bacteria, such as strict and facultative anaerobes. that are usually closely related to the producer strain (6~8). Some facultative anaerobic purple nonsulfur bacteria such Bactericidal effects of bacteriocins have been recognized as as Rhodobacter capsulatus, Rhodobacter sphaeroides and an important subject in food fermentation and preservation. Rhodopseudomonas palustris have been found to produce Those produced by lactic acid bacteria have been largely bacteriocins (12~14). In this work, we isolated bacteriocin studied as potentially safe food-grade preservatives of from Rhodobacter capsulatus ATCC 17016 cultured in biological origin (9). The class IIa bacteriocins produced Lascelles medium to study bacteriocin production from purple nonsulfur phototrophic bacteria, because Lascelles Received: August 19, 2009/ Revised: September 22, 2009 Accepted: October 16, 2009 medium has long been recognized for bacteriocin production * Corresponding author: Jaisoo Kim. Department of Life Science, by purple nonsulfur phototrophic bacteria (12). But this Kyonggi University, Suwon 443-760, Korea. Phone: +82-31-249-9648, Fax: +82-31-249-9139 species didn't produce enough bacteriocin. Once Triton e-mail: [email protected] X-100 was added to Lascelles medium, much more **This work was in part supported by a grant from the Korea Research Foundation. bacteriocin was produced than that in Lascelles medium 269 270 S-S Lee, et al. alone. Under diversified nutrient conditions, the bacteriocin B. Braun, Allentown, PA, USA) and centrifuged (15,000 of Rhodobacter capsulatus ATCC 17016 was studied and its rpm) for 15 min at 4℃. The supernatant was separated into molecular weight was roughly estimated. Lantibacteriocins three fractions of molecular weight <103, 103~104, >104 (4, 15) and circular bacteriocins (16, 17) were encoded by Dalton using ultrafiltration membranes (Amicon TC-10, the responsive plasmids. However, Rhodobacter capsulatus Millipore, Billerica, MA, USA). Then they were lyophilized didn't show plasmid dependence. and tested for bacteriocin activity, for purification on FPLC system. MATERIALS AND METHODS Activity was determined according to Brock et al. (19) and Guest (12). Briefly, 4 wells were made in agar plates Strains and cultures (1.5%) and filled with mixtures of equal amount of 3% Bacterial strains used in this study were Rhodobacter agar media and bacteriocin samples dissolved in sterilized capsulatus ATCC 17016 as a producer strain and DW. After solidifying, the bacteriocin-seeded plates were Rhodopseudomonas palustris ATCC 17003 as an indicator overlaid with soft agar (0.5%) medium containing 2 ml of strain. Precultures were grown on a medium 27 (DSM, indicator strain (ca. 4.8 × 108 cells/ml). The overlaid plates Braunschweig, Germany) supplemented with trace element were incubated in anaerobic jars (CO2 + H2 gas pack, BBL, SL-6 under anaerobic photosynthetic conditions illuminated Becton Dickinson, Cockeysville, MD, USA) under light at with incandescent lamp (ca. 3000 Lux) at 28℃. Cells were 28 ± 2℃ for 5 days. If the bacteriocin activity were routinely prepared in 10 ml screw cap tubes completely present, growth inhibition zones could be observed around filled with media at these conditions and used as a standard the wells containing the indicator strains. Activity units per strain. milliliter were defined according to the method of Jorger Strains of producer and indicator were cultivated in the and Klaehammer (20). medium described by Lascelles (18) according to procedures pH and absorbance estimation by Guest (12) instead of medium 27 (DSM). Lascelles medium was supplemented with Triton X-100 (0.3%) for Studies on the effects of growth medium pH on the producer, Rhodobacter capsulatus ATCC 17016. The bacteriocin productivity were followed by the method of pH of all media was adjusted to pH 6.8. Jorger and Klenhammer (20). Producer strain Rhodobacter Aerobic cultures in the dark were carried out with 5,000 capsulatus, with initial culture medium pH adjusted to 5.0, ml bottles filled with 300 ml Lascelles medium supple- 6.0, 6.8 and 8.0 with 5 N NaOH was cultured aerobically mented with Triton X-100 (0.3%). These were inoculated by shaking (50 rpm) in the liquid in the dark. Every 24 hrs, with 5 ml of the standard strain (OD600 1.5) and shaken at cultures of Rhodobacter capsulatus were measured for pH 50 rpm and 28 ± 2℃ for 4 days. (Fisher 230A, Fisher Scientific, Pittsburgh, PA, USA) and absorbance at 660 nm using spectrophotometer (spectronic Bacteriocin detection and activity assay 20, Bausch & Lomb, Inc., Rochester, NY, USA). To produce the bacteriocin, the producer strains were After inoculation, Rhodobacter capsulatus was aerobically aerobically cultured in the dark conditions described as cultured in the dark for 4 days. Two grams of cell mass above. Cells were collected by centrifugation for 10 min at were dissolved in 2 ml DW, disrupted with ultrasonic 4℃ (8,000 rpm, Sorvall Gs-3 rotor, RC-5B, DuPont, St. system (3 mm probe, 80 Hz for 1 min, 3 times), and mixed Louis, MI, USA). Cell pellets were washed twice with DW equally with 3% agar medium. Then bacteriocin activity was and finally re-suspended in DW (1 g/ml, wet weight). Cells determined as mentioned above according to the method of were disrupted by ultrasonic system under ice base for 21 Brock et al. (19) and Guest (12). min (Ultrasonic 19 mm probe, 230 Hz, 3 min × 7 times, Bacteriocin from Purple Nonsulfur Phototrophic Bacteria 271 The materials for estimation of molecular weight of Bacteriocin productivity under chloramphenicol bacteriocin under SDS-PAGE and silver staining were tris treatment (BRL, Grand Island, NY, USA), acrylamide (BRL), bis- It was reported that bacteriocin production was coded by acrylamide (BRL), SDS (Sigma, ST. Louis, MO, USA), plasmid DNA (21~25). In order to increase bacteriocin β-mercaptoethanol (LKB, Bromma, Sweden), bromophenol production, plasmid DNA was amplified with the method blue (Sigma), TEMED (Sigma), glycin (Sigma), silver of Yasuyoshi et al. (24). After shaking aerobically in the nitrate (Sigma), nitrocellulose filter (Hoefer, San Fransisco, dark, it was cultured for 48 hrs in a chloramphenicol- CA, USA), and an electrophoresis unit & transfer unit containing medium (concentration of 50 μg/ml) and the (Hoefer). Molecular weight of bacteriocin was estimated activity was tested. by western blotting and silver staining. Plasmid isolation Immunization and confirmation test of antibody generation Plasmid DNA was isolated from nonsulfur phototrophic bacteria by alkaline lysis (26). For the generation of rabbit polyclonal antibody to bacteriocin, the following reagents were required: Freund's Electrophoresis adjuvant (complete & incomplete: Gibco BRL, Grand Island, Plasmid separation was conducted using 0.9% agarose NY, USA), maxi mix (Thermolyne Co., Dubuque, IA, USA). gel electrophoresis, which was modified from the methods The equipments for identification of bacteriocin antigen by of Maniatis et al. (26), Bearden (27) and Kado and Liu (28). an ELISA were bovine serum albumin (Sigma), plate λDNA was cut with Hind lll for measuring plasmid DNA shaker (Dynatech Laboratories, Inc., Chantilly, VA, USA), size, development was conducted under 60 V for 4.5 hrs, goat-anti-rabbit-IgG-HRP (Molecular Probes, Eugene, OR, and the gel was photographed by konix pan Tri-x film on USA), OPD (Sigma), and ELISA autoreader (Pharmacia, UV-transilluminator (245 nm). Uppsala, Sweden). Bacteriocin (100 μg) dissolved in PBS (phosphate- Plasmid curing test buffered saline)