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Diverse Antibacterial Activity of Pectobacterium Carotovorum Subsp. Carotovorum Isolated in Korea

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Diverse Antibacterial Activity of Pectobacterium Carotovorum Subsp. Carotovorum Isolated in Korea J. Microbiol. Biotechnol. (2009), 19(1), 42–50 doi: 10.4014/jmb.0803.209 First published online 1 August 2008 Diverse Antibacterial Activity of Pectobacterium carotovorum subsp. carotovorum Isolated in Korea Roh, Eunjung1, Seungdon Lee1, Yonghoon Lee1, Dongsu Ra1, Jaehyuk Choi2, Eunpyo Moon3, and Sunggi Heu1* 1Division of Plant Pathology, National Institute of Agricultural Science and Technology, Rural Development Administration, Suwon 441-707, Korea 2Department of Molecular Science and Technology, Ajou University, Suwon 442-749, Korea 3Department of Biological Science, Ajou University, Suwon 442-749, Korea Received: March 17, 2008 / Accepted: June 17, 2008 Fifty-four Pectobacterium carotovorum subsp. carotovorum Archaebacteria [15, 16]. Given their often narrow range of strains isolated in Korea were characterized by a spectrum activity, it has been proposed that the primary role of of antibacterial activities against 7 indicator strains chosen bacteriocins is to mediate intraspecific, or population-level, to represent various regions and host plants. All P. interactions [15]. Bacteriocins include a diversity of proteins carotovorum subsp. carotovorum isolates tested could be in terms of size, microbial targets, mode of action, and immunity grouped into 4 classes depending on the pattern of antibacterial mechanism. Bacteriocin genes are encoded in both plasmids substance production. All tested strains had DNA fragment(s) and chromosomes [17]. Escherichia coli encodes its colicins homologous to the genes encoding carotovoricin and 21 of exclusively on plasmids [14]. The pyocins of Pseudomonas them had genes homologous to DNA invertase. Sixteen aeruginosa, which show high sequence similarity to colicins strains had genes homologous to the genes encoding carocin and others, as yet uncharacterized, are found exclusively S1. Several isolates produced antibacterial substances active on the chromosome [19]. Another close relative to the against strains in Brenneria, Pantoea, and Pectobacterium colicin family, the bacteriocins of Serratia marcescens, are genera that belonged formerly to the genus Erwinia. Strains found on both plasmids and chromosomes [5]. in Pseudomonas or Xanthomonas sp. were not sensitive to Many phytopathogenic bacteria produce proteinaceous the antibacterial substances produced by P. carotovorum bacteriocins. Only a few isolated and/or purified bacteriocins, subsp. carotovorum, except for X. albilineans that was including members of the Corynebacteria, Erwinia, sensitive to antibacterial substances produced by most strains Pseudomonas, and Xanthomonas, have been reported in P. carotovorum subsp. carotovorum and P. betavasculorum [1, 3, 7, 9]. The fraudulent adenine nucleotide antibiotic KACC10056. These results demonstrated the diverse patterns Agrocin 84, produced by Agrobacterium radiobacter strain of antibacterial substance production and the possibility of 84, is used as an effective biological control of the plant the existence of new antibacterial substance(s) produced by cancer, crown gall [8, 10]. The elucidation of the ecological P. carotovorum subsp. carotovorum isolated in Korea. significance of inhibitory substances as bacteriocins produced Keywords: Soft rot, bacteriocin, antibacterial substance, by crop pathogens is important to understanding the factors carotovoricin, carocin S1 that affect the population dynamics on plant surfaces. Therefore, the production of antibiotics by antagonists is important in the biological control of plant diseases [20]. Pectobacterium carotovorum subsp. carotovorum (Erwinia Bacteriocins are one of the most abundant and diverse carotovora subsp. carotovora) [4, 6] is a phytopathogenic classes of antibacterial substances. Bacteriocins are a enterobacterium responsible for the soft rot, blackleg, or subgroup of the antibacterial peptides that were originally stem rot of a number of economically important crops. defined as proteinaceous compounds that kill strains of the Bacterial soft rot is found all over the country and causes same or closely related species [21]. Their production serious diseases of crops in the field, in transit, and especially occurs across all major groups of the Eubacteria and the in storage, resulting in a greater total loss of produce than any other bacterial disease. Various aspects of the epidemiology *Corresponding author Phone: 82-31-290-0420; Fax: 82-31-290-0406; of the disease caused by this phytopathogen are understood, E-mail: [email protected] but there is no efficient method to control the global ANTIBACTERIAL ACTIVITY OF PECTOBACTERIUM CAROTOVORUM 43 disease. Up to now, two bacteriocins had been reported in P. DNA Techniques and PCR Amplification carotovorum subsp. carotovorum. One of them is carotovoricin Standard procedures were used in the isolation of total DNA and gel Er or CGE (CtvEr or CtvCGE), a high-molecular-weight electrophoresis [18]. The native plasmids were isolated from P. bacteriocin that contains a lysis cassette of a major and minor carotovorum subsp. carotovorum using the Axygen midi kit (Axygen, structural protein gene cluster located in chromosomal DNA U.S.A.). Electrophoresis was conducted on 1% agarose gels in Tris acetate buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA [12]. The complete nucleotide sequences of CtvEr genes have [pH 8.0]), using a constant voltage of 100 V for 1 h. PCR amplification been determined [22]. Sequence comparison showed high of known structural genes of carocin S1 [3], carotovoricin Er [13], homology between carotovoricin and phage proteins. The and carotovoricin CGE [22] was performed with the specific primers killing spectra of the two carotovoricins CtvEr and CtvCGE listed in Table 2. PCR was performed on a DNA thermal cycler are different depending on the C-terminal region of the tail PTC-100 (MJ Research Inc. Watertown, U.S.A.) using the Maxime fiber proteins. The other bacteriocin is a low-molecular- PCR PreMix i-StarTaq (Intron Biotechnology, Seongnam, Korea) in weight bacteriocin, carocin S1, which consists of a killing a final volume of 20 µl containing 50 nM of each primer and 1 µg protein and an immunity protein. Production of carocin S1 of total DNA or 50 ng of plasmid DNA. The cycles used were 95oC is induced by glucose and lactose [3]. The carocin S1 gene for 5 min for the first cycle, 95oC for 1 min, 55oC (for the primers of o is homologous to the pyocin S3 and pyocin AP41 genes of carocin S1) or 57 C (for the primers of tail, tail fiber, DNA invertase) o o Pseudomonas aeruginosa. These genes encode proteins with or 58 C (for the primers of tail core) for 1 min, and 72 C for 1 min for the next 30 cycles; 72oC for 5 min was used for the last cycle. nuclease activity [3]. In this study, we collected 54 strains of P. carotovorum Southern Hybridization subsp. carotovorum in various regions of Korea and tested Genomic DNA of 54 P. carotovorum subsp. carotovorum strains their antibacterial activities. We could detect the existence were isolated and transferred to Hybond N+ nylon membranes of either one of two already known bacteriocin genes in (Amersham, Buckingham shire, U.K.). The genomic DNA isolated was most isolates. However, some isolates showed a different digested by BamHI restriction enzyme. Genomic DNA fragments spectrum of antimicrobial activity and induction mechanism, were separated by electrophoresis in 0.8% agarose gel. The gel was suggesting the possibility of the existence of new kinds of dipped in 250 mM HCl for 10 min. For denaturation, the gel was bacteriocins produced by P. carotovorum subsp. carotovorum dipped twice in denaturation solution (0.5 M NaOH, 1.5 M NaCl) for isolated in Korea. 30 min. After washing with distilled water, the gel was neutralized with neutralization solution (1 M Tris-HCl [pH 7.4], 1.5 M NaCl) for 20 min. DNA was transferred to nylon membranes by the capillary blot procedure. The transferred DNA was cross-linked on the membrane MATERIALS AND METHODS with UV for 3 min. The membrane was prehybridized with hybridization o solution (0.5 M NaPO4, 1 mM EDTA, 1% BAS, 7% SDS) at 65 C Bacterial Strains and Media for 2 h. The carocin S1 PCR product of Pcc25 used as probes was A total of 54 strains of P. carotovorum subsp. carotovorum were labeled by a 32P extension labeling procedure with the Rediprime II used in this study. The strains were isolated from various host plants DNA Labeling System (Amersham, U.K.). Hybridization was performed and locations in Korea. The identity of the strains was confirmed by with fresh hybridization solution containing the denatured labeled the Biolog GN microplate system (Biolog Inc, U.S.A.). Bacterial DNA probe at 65oC for 20 h. Hybridized membrane was washed with strains were cultured in LB medium (10 g of tryptone, 5 g of yeast washing solution I (5×SSC, 0.1% SDS) at 65oC for 30 min, followed extract, and 10 g of NaCl in 1 l, pH 7.2) at 28oC. Soft agar was by washing solution II (1×SSC, 0.1% SDS) at 65oC for 30 min, and prepared by adding 0.7% agar to broth medium. Rifampicin was finally by washing solution III (0.5×SSC, 0.1% SDS) at 65oC for 30 min. used at 100 µg/ml concentrations The membrane was combined with an Image plate (Fujifilm, Japan), incubated at -70oC for 24 h, and developed. Detection of Antibacterial Substances Antibacterial activity was detected by the spot-on-lawn method for screening of inhibitory activity against P. carotovorum subsp. RESULTS carotovorum and various bacteria. A spot was made on solid LB media with a bacterial strain to be tested as producer, and the spotted Detection of Diverse Antibacterial Activities producer strains were incubated for 12 h at 28oC. For the UV induction, To determine the antibacterial activity of P. carotovorum producer strains were exposed to UV light for 15 min. For mitomycin C subsp. carotovorum isolated in Korea, the spot-on-lawn (MMC) induction, MMC was added to LB media at a final assay on agar plate was carried out. Total 54 P. carotovorum concentration of 0.5 µg/ml.
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