J. Microbiol. Biotechnol. (2009), 19(1), 42–50 doi: 10.4014/jmb.0803.209 First published online 1 August 2008

Diverse Antibacterial Activity of carotovorum subsp. carotovorum Isolated in Korea

Roh, Eunjung1, Seungdon Lee1, Yonghoon Lee1, Dongsu Ra1, Jaehyuk Choi2, Eunpyo Moon3, and Sunggi Heu1*

1Division of 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 . 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 , Pantoea, and Pectobacterium colicin family, the bacteriocins of Serratia marcescens, are genera that belonged formerly to the genus . Strains found on both plasmids and chromosomes [5]. in Pseudomonas or Xanthomonas sp. were not sensitive to Many phytopathogenic 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 [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. The induced producer strains were killed subsp. carotovorum strains collected nationwide in Korea, by the exposure of bacterial cells to 700 µl of chloroform vapor for including Jeju Island, were tested against 7 P. carotovorum 10 min. After aeration for 20 min to remove traces of chloroform, the surface of the solid media was subsequently overlaid with 7 ml of 0.7% subsp. carotovorum isolates (Pcc1, 3, 21, 26, 27, 49, and soft agar containing 10 µl of an overnight culture of the indicator 56) representing each collection region. The antibacterial organism. The production of antibacterial substance was detected as activities to the indicator strains were diverse and the sizes growth-free inhibition zones (clear zones) around the spotted area of inhibition zones were varied depending on the producer after overnight incubation at 28oC [11]. or the indicator strains (Table 1 and Fig. 1). The productions 44 Roh et al.

Table 1. Antibacterial spectrum of Pectobacterium carotovorum subsp. carotovorum against P. carotovorum subsp. carotovorum. a Type Pcc1 Pcc3 Pcc21 Pcc26 Pcc27 Pcc49 Pcc56 Group strains LB MMCb UVc LB MMC UV LB MMC UV LB MMC UV LB MMC UV LB MMC UV LB MMC UV Ad Pcc3+ + +-- -++ +++ ++++++ +- -- (48.15%)' Pcc6+ + ++ + ++ + ++ + ++ + ++ + +-- - Pcc7+ + ++ + ++ + ++ + ++ + ++ + +-- - Pcc8+ + +-- -++ +++ ++++++ +-- - Pcc19++ ++ + ++ + ++ + ++ + ++ + +-- - Pcc20++ ++ + ++ + ++ + ++ + ++ + +-- - Pcc33++ ++ + ++ + ++ + ++ + ++ + +-- - Pcc40++ ++ + ++ + ++ + ++ + ++ + +-- - B Pcc24++ ++ + ++ + ++ + ++ + ++ + ++ + + (14.81%)' Pcc30++ ++ + ++ + ++ + ++ + ++ + ++ + + Pcc31++ ++ + ++ + ++ + ++ + ++ + ++ + + Pcc50++ ++ + ++ + ++ + ++ + ++ + ++ + + Pcc51++ ++ + ++ + ++ + ++ + ++ + ++ + + Pcc52++ ++ + ++ + ++++++++ ++ + ++ + + Pcc91++ ++ + ++ + ++ + ++ + ++ + ++ + + Pcc92++ ++ + ++ + ++ + ++ + ++ + ++ + + C Pcc2+ + ++ + ++ + ++ + ++ + ++ + +- +++ +++ (20.37%)' Pcc13 + ++ + + +++ +++ -- -++ +---++ +- ++ Pcc14 + ++ + + +++ +++ -- -++ +---++ +- ++ Pcc15 + ++ + + +++ +++ -- -++++ ---++ +- ++ Pcc17 + + + -- -++ +++ ++++++ +- ++ Pcc21 ++ +++ +++ ++ +++ +++ -- -+ +++ + + +++ ++ + +++ ++ ++ +++ +++ Pcc22 -- ++ + ++ + + + ++ + +-- --++ Pcc25 + + +++ ++ +++ +++ + + + + +++ ++ + + + + - ++ + +++ +++ Pcc27++ ++ + ++ + ++ + +----- + - ++ Pcc29 ++ ++ +++ +++ +++ +++ ++ +++ +++ + + + ++ + +++ + +++ ++ + +++ +++ Pcc47 -- --++- + + +++ +++ +++ --+ -- + -- + D Pcc36 -- --++- ++- ++- ++- + -- ++ (16.67%)' Pcc37 -- --++- ++- ++- ++- + -- + - Pcc39 -- --++- ++- ++- ++- + -- + - Pcc41 -- --++- ++- ++- ++- + -- ++ Pcc42 -- --++- ++- ++- ++- + -- ++ Pcc43 -- --++- ++- ++- ++- + -- ++ Pcc44 -- --++- ++- ++- ++- + -- ++ Pcc45 -- --++- ++- ++- ++- + -- ++ Pcc93 -- --+ ------aPcc, Pectobacterium carotovorum subsp. carotovorum. bMMC, Producer strains were incubated in LB medium containing 0.5 µg/ml mitomycin C. cUV, Producer strains were treated with UV light for ten minutes. dTotal 26 strains belonged to group A. This table shows 8 typical strains. Other strains that belonged to group A are Pcc1, 4, 11, 12, 18, 23, 32, 34, 35, 49, 56, 65, 89, 90, 94, 96, 102, and 103. +; 0-0.5 cm in diameter of inhibition zone, ++ 0.5-1 cm in diameter of inhibition zone; +++ 1-2 cm in diameter of inhibition zone of antibacterial substances in several strains were induced typing of the 54 P. carotovorum subsp. carotovorum strains or the size of inhibition zone was increased by treatment of could identify four groups on the basis of their bacteriocin mitomycin C or UV light. Since the productions of carocin production and bacteriocin sensitivity profiles. Most tested and carotovoricin were induced by the UV treatment and strains (48.15%) belonged to group A. Groups B, C, and D the mitomycin C treatments, respectively, the induction of contained 14.81%, 20.37%, and 16.67% of tested strains, the antibiotic production was also tested [3, 13]. Bacteriocin respectively. The production of antibacterial substances by ANTIBACTERIAL ACTIVITY OF PECTOBACTERIUM CAROTOVORUM 45

Fig. 1. Antibacterial activity of P. ca rot ov or u m subsp. carotovorum against P. c aro to vo r um subsp. carotovorum Pcc3. Producer strains were incubated on LB media (A), LB media containing mitomycin C (B), and LB media followed by UV treatment (C), respectively. strains belonging to groups A and B was not induced by is responsible for the antibacterial activity of P. carotovorum mitomycin C or UV light treatments (Table 1). Small clear subsp. carotovorum strains isolated in Korea, the presence zones around bacterial colonies were visible on rich media (LB) of genes related to carotovoricin Er or carocin S1 was tested containing mitomycin C or UV treatment. The only difference by PCR. Since P. carotovorum subsp. betavasculorum between groups A and B was the activity against indicator KACC10056 was able to inhibit the growth of P. carotovorum strain Pcc56. All the other strains except strains in Group subsp. carotovorum strains, P. carotovorum subsp. A inhibited the growth of the indicator strain Pcc56. The betavasculorum KACC10056 was also included. production of antibacterial substances of strains in group C The carotovoricin Er gene cluster consists of a lysis cassette and group D was induced by mitomycin C or UV light of major and minor structural protein genes. Holin, lytic treatments. Strains in group D were not able to produce murein transglycosylase, and putative Rz lysis protein are antibacterial substances in LB medium. These strains were components of the phage-type lysis cassette [22]. tail sheet, able to show weak antibacterial activity only after treatment tail and tail fiber are the structural components of with mitomycin C or UV light. However, the strains belonged carotovoricin Er. The DNA invertase gene in carotovoricin to group C were able to produce antibacterial substances in Er is known to be involved in host range specificity [13]. LB medium without any treatment but the production was PCR primers were designed based on the published sequences greatly induced by mitomycin C or UV light treatment. Taken of the carotovoricin Er structural gene cluster as shown in together, these data suggest the possibility that P. carotovorum Table 2. The homologous DNA fragments of holin and tail subsp. carotovorum strains isolated in Korea were able to core were detected in the total DNA of all 54 tested strains. produce several different types of antibacterial substances. The key components for the bacterial lysis are transglycosylase and Rz lysis protein. Some strains did not have transglycosylase Identification of Carotovoricin and Carocin Structural but did have Rz lysis protein. Therefore, they did have all Gene Homologs lysis-related components. Most of the strains belonging to Two bacteriocins, carotovoricin Er and carocin S1, had groups A, B, C, and D showed homologous DNA been reported to be produced by P. carotovorum subsp. fragments for all components of the carotovoricin Er gene carotovorum [3, 22]. In order to identify which bacteriocin cluster except DNA invertase. The DNA invertase fragment

Table 2. Specific primers for PCR detection of carocin and carotovoricin structural genes. Product Tm Target gene Forward primer Reverse primer (bp) (oC) Carocin S1 ERB1-F CCT CAA CAA gTg gTA ATg gC ERB1-R gAA TgT AAT CCC gAA AAT CC 616 55 Holin ERB2-F TAT Cgg gCT gAT TTC ACC gC ERB2-R CAA ACg gCT gAC ACA ATC Ag 240 58 Lytic murein transglycolaseERB3-F TgC gAC ACC TCC TCA TCA Cg ERB3-R CTT ATC ACg CTg TAA CCA gC 437 59 Putative Rz lysis protein ERB4-F gTC TgT TTC TCA ATA gCC Tg ERB4-R CTg AAA gTT gTA ATT Cgg Cg 528 55 Tail sheat ERB5-F gAA gTT gAA ACC ggT gCT Cg ERB5-R CTC CAg ATC gAC TTT ggC AC 858 59 Tail core ERB6-F gAT TAT gCA ggA gCA CAA gg ERB6-R CAC CgT TCA TCA CCT gCT Tg 326 58 Tail ERB7-F CTg CgT TAA Cgg AAg AAC TC ERB7-R CAC CgT Tgg CTT CAT AAA CC 744 57 Tail fiber ERB9-F ATg gCg AAT TTg TCA gAg AA ERB9-R ggC gCA TAT TgC gTA TgC gg 212 57 DNA invertase ERB10-F ACg CgT gTC TAC TAC CgA TC ERB10-R CCT gTT gCC AAC ATT gAT TC 456 57 46 Roh et al. was detected in 21 out of 54 P. carotovorum subsp. carotovorum strains. Interestingly, all P. carotovorum subsp. carotovorum strains, except the Pcc93 strain that belonged to group D, contained the DNA invertase gene, but the influence of this gene on host range is not clear. Fig. 2. Southern hybridization analysis using total genomic These data showed that all the P. carotovorum subsp. DNA from 54 isolates of P. carotovorum subsp. carotovorum. carotovorum strains tested carried carotovoricin genes and Genomic DNA was digested with BamHI, fractionated on a 0.9% agarose gel, were able to produce carotovoricin. Moreover, all genes in and transferred to a Nylon membrane. The Nylon membrane was hybridized the carotovoricin gene cluster, except Rz lysis protein, were with 32P-labeled carocin S1 PCR product. Numbers 7, 8, 13, 14, 15, 20, 25, 27, 29, and 47 indicate P. carotovorum subsp. carotovorum strains. detected in P. betavasculorum KACC10056. Several strains

Table 3. The presence of carotovoricin structural genes and carocin S1 gene in P. ca rot ov or u m subsp. carotovorum isolated in Korea. Carocin Target ORFs of known and suggested genes of carotovoricin Er coding region Type Group Carocin Trans- Rz lysis Tail Tail Tail DNA strains Holin Tail S1 glycosylase protein sheat core fiber invertase A Pcc3 - ++ +++-- + (48.15%) Pcc6 - ++ +++++- Pcc7++ + ++++++ Pcc8++ + ++++++ Pcc19 - + + ++++++ Pcc20 + + + + + + + + - Pcc33 - ++ +++++- Pcc40 - ++ +++++- B Pcc24 - + + ++++++ (14.81%) Pcc30 - ++ - ++++ + Pcc31 - + + ++++++ Pcc50 - ++ +++++- Pcc51 - ++ +++++- Pcc52 - + - + - +++ - Pcc91 - ++ - ++++ - Pcc92 - ++ - ++++ - C Pcc2 - ++ - ++++ - (20.37%) Pcc13 + + + + + + + + - Pcc14 + + + + + + + + - Pcc15 + + + + + + + + - Pcc17 - ++ +++++- Pcc21 -- +++++++ Pcc22 - + + ++++++ Pcc25 + + + + + + + + - Pcc27 + + + + + + + + - Pcc29 + + + + + + + + + Pcc47 + + + + + + + + - D Pcc36 - + + ++++++ (16.67%) Pcc37 - + + ++++++ Pcc39 - + + ++++++ Pcc41 - + - +++- ++ Pcc42 - ++ +++- ++ Pcc43 - ++ +++- ++ Pcc44 - ++ - ++- ++ Pcc45 - ++ +++- ++ Pcc93 - ++ ++++-- P. betavasculorum ++ + - ++++ + All PCR reactions were repeated three times. ANTIBACTERIAL ACTIVITY OF PECTOBACTERIUM CAROTOVORUM 47 without homologous genes of tail formation or DNA invertase still produced active carotovoricin activity, and therefore tail formation or DNA invertase genes may not have any effect on the carotovoricin activity. Based on the published sequences (Accession No. AF205141) of the carocin S1 structural gene [3], primers, ERB1-F and ERB1-R, which are complementary to the sequences proximal to the 5' ends of the carocin S1 structural gene (CaroS1K), were synthesized and used for PCR amplification. The presence of the carosin S1 structural gene or its homologs in Korean isolates was tested with Southern hybridization analysis of chromosomal DNA, and positive signals were detected in 16 of 54 tested strains (29.6%), as Fig. 3. Agarose gel electrophoresis of native plasmid DNA shown on Fig. 2. P. carotovorum subsp. betavasculorum isolated from P. carotovorum subsp. carotovorum strains. Plasmid profiles of P. ca rot ov or um subsp. carotovorum digested with KACC10056 also showed a DNA fragment homologous BamHI/HindIII. The plasmids were separated by 1% agarose gel to carocin S1 (Table 3). Only the strains that belong to electrophoresis. Lanes 2 to 7, plasmid DNA isolated from Pcc 2, 24, 35, 49, groups A and C carried the DNA homologous to the carocin 56, 65 respectively; lane 1, 1-kb DNA size marker. S1 gene, and none of the strains that belong to group B and group D showed any homologous DNA bands at all. of the bacteriocins produced by P. carotovorum subsp. Strains that belonged to group D did not have any carotovorum strains were studied. Since the production of antibacterial activity against all indicator strains in LB medium most antibacterial substances produced by P. carotovorum The production of antibacterial substances was only induced subsp. carotovorum strains was induced by mitomycin C, by mitomycin C or UV treatment but the activity was very all tests were carried out with strains grown in LB medium low. moreover, none of the strains that belong to group D containing mitomycin C. Total 29 different plant pathogens, had homologous bands with carocin S1. All these data representing 7 genera of Brenneria, Pantoea, Pectobacterium, suggested that the antibacterial substance produced by the Erwinia, Xanthomonas, Pseudomonas, and Burkholderia, strains that belong to group D might be a carotovoricin were tested for the sensitivity against the antibacterial only. Seven out of 11 strains in group C carried the carocin substances produced by P. carotovorum subsp. carotovorum S1 homologous gene and the production of antibacterial isolates (Table 4). Brenneria, Pantoea, Pectobacterium, activity in those strains was greatly induced by UV light as and Erwinia all previously belonged to Erwinia species in carocin S1. However, 4 strains in group C did not have [2, 4, 6]. Antibacterial substances produced by P. carotovorum any carocin S1 homologous gene, though they were able to subsp. carotovorum strains were active against strains in P. produce an antibacterial substance that was induced greatly carotovorum subsp. Specifically, two subsp. atrosepticum by the UV light or mitomycin C treatments. These data strains were very sensitive to the antibacterial substances indicate that these 4 strains might produce an unknown produced by P. carotovorum subsp. carotovorum strains. antibacterial substance(s) other than carocin S1 in addition None of the tested P. carotovorum subsp. carotovorum to carotovoricin. strains were able to produce antibacterial substances against three different P. chrysanthemi strains. Isolation of Native Plasmids In addition to strains in Enterobacteriaceae, other To characterize the correlation between antibacterial activity phytopathogenic bacteria were tested. We tested X. albilineans, and plasmid content in the P. carotovorum subsp. carotovorum X. axonopodis pvs. aurantifolia, citri, diffenbachia, glycines, isolates, the native plasmids were isolated from the P. X. campestris pvs. campestris, vesicatoria, X. oryzae pv. oryzae, carotovorum subsp. carotovorum strains. Among the 54 Pseudomonas cichorii, P. putida, and Burkholderia glumae, strains, 6 strains carried different sizes of native plasmids but none of them were sensitive to the antibacterial substances (Fig. 3). It was not clear whether strains Pcc2 or Pcc24 carry produced by the P. carotovorum subsp. carotovorum more than 2 plasmids. Four strains carrying plasmids (Pcc35, isolates except Xanthomonas albilineans (data not shown). 49, 56, and 65) were in group A, and strains Pcc24 and Pcc2 Xanthomonas albilineans was sensitive to the antibacterial were in group B and group C, respectively. Therefore, the substance produced by all tested P. carotovorum subsp. presence of plasmids in the P. carotovorum subsp. carotovorum carotovorum strains. strains had no relation with bacteriocin production. Response to Antibacterial Substances Produced by Antibacterial Activity Against Other Bacterial Species Other Bacterial Species Because most reported bacteriocins inhibit the growth of Since the genus Pectobacterium once belonged to the genus only closely related bacteria, the spectra of antibacterial activity Erwinia, the activity of the antibacterial substances produced 48 Roh et al.

Table 4. Antibacterial spectrum of P. carotovorum subsp. carotovorum against various bacteria (MMC)a. Indicator strains ) um ) ) r c3 a a lo c ) ) ic ic m u P a e t t ru sc r a m p m p o a m fe bi 5 u se u se l v u m ri a 8 ic o ic o cu ta or u o e s 0 pt tr pt tr s e v er d ia a 10 e a e a a b o if o b w s . s . v . ot r . a . C ro sp ro sp ta sp r ) o sp as sp 3 C at b at b be b ca ra od b w b 0 5 0 06 8 A . u . u . u . o . u . u 5 0 3 0 2 4 4 5 K p s p s p s p v p s p s 5 4 8 1 5 0 05 05 i s ra s ra s ra s to s ra s ra 3 4 4 C Type 0 0 0 0 rt ub o ub o ub o ub o ub o ub o L L L C Group 0 1 9 53 1 1 9 3 a s v s v s v s r s v s v S S S strains 1 C 4 0 C C 5 8 to to to ca to to i i i A C C 0 0 0 0 ew m o m o m o m . m o m o m m m K C A 0 1 C C 1 1 st ru r ru r ru r ru p ru r ru r he he he i A K 1 C A A C C . o ca o ca o ca o bs o ca o ca t t t d K s ) C C K K C C p v v v v u v v an i) an i) an i) e s ) n s C s s bs to ia to ia to ia to s to ia to ia s m s m s m ip n s e n A A n ) n ) A A u o in o in o in o a o in o in y e y e y e r ) e n ci e K a a a a K ) K ) s ) r r r r r r r r h r h r h p i u e a ci K a er l er l s is s is i i ca rw ca rw ca rw ca o ca rw ca rw ch t ch t ch t cy d fl lu if a is ) n o o ti t ti t t rt v an an an e ri if r if ic is ci m ic m ic a a a a r a m (E m (E m (E m to m (E m (E m s m s m s m ip g r b r l ic i lo b lo b n n n n a w u 8 u 0 u 6 u o u 6 u 1 u y u y u y u r i ig u b a l rs g er g er a na a na w e ri 7 ri 8 ri 5 ri r ri 8 ri 6 ri r ri r ri r ri p n n r ru s sa e g h g h n a n a te st te 4 te 4 te 0 te ca te 4 te 0 te ch te ch te ch te cy ia a ia a ia a p a a a a a a a a s a c 0 c 0 c 0 c a c 0 c 0 c a c a c a c a r i r i r i ia a i a i a i a i a i a 1 a 1 a 1 a i a 1 a 1 a i a i a i a i ne in ne in ne in n oe in oe in oe in oe in oe in b C b C b C b in b C b C b in b in b in b in n w n w n w i t w t w t w t w t w to C to C to C to w to C to C to w to w to w to w re r re r re r rw an r an r an r an r an r ec A ec A ec A ec r ec A ec A ec r ec r ec r ec r B (E B (E B (E E P (E P (E P (E P (E P (E P K P K P K P (E P K P K P (E P (E P (E P (E A Pcc3 ------(48.15%) Pcc6 - + ------+++- + ------Pcc7 - + ------+++- + ------Pcc8 ------Pcc19 --+ ------+++- + ------Pcc20 - + ------++------Pcc33 - ++- + ----++- + ------Pcc40 - ++- + ----++++------B Pcc24 + ------+ ------(14.81%) Pcc30 + + ---+++----+++++---- Pcc31 + + ---+++----+++++---- Pcc50 ------+ ------Pcc51 ------+ ------Pcc52 ------+ ------Pcc91 + + + --+ ---++++------Pcc92 + + + --+ ---++++------C Pcc2 ------+++ + ------(20.37%) Pcc13 ------++- +++ ------Pcc14 ------++- +++ ------Pcc15 ------++- +++ ------Pcc17 ------Pcc21 ------++ +++ - +++ +++ ----- Pcc22 ------+ ------Pcc25 ------++ +++ - +++ ------Pcc27 ------++----- Pcc29 ------++ +++ - +++ ------Pcc47 ------++----- D Pcc36 ------++ + - + ------(16.67%) Pcc37 ------++ + - + ------Pcc39 ------+ - + ------Pcc41 ------+ ------Pcc42 ------++ + - + ------Pcc43 ------++ + - + ------Pcc44 ------++ + - + ------Pcc45 ------++ + - + ------Pcc93 ------++--+ ----- aMMC, Experiments were conducted in LB medium containing 0.5 µg/ml mitomycin C. +, 0-0.5 cm in diameter of inhibition zone; ++, 0.5-1 cm in diameter of inhibition zone; +++, 1-2 cm in diameter of inhibition zone ANTIBACTERIAL ACTIVITY OF PECTOBACTERIUM CAROTOVORUM 49

Table 5. Antibacterial spectrum of various bacteria against P. carotovorum subsp. carotovorum (MMC)a. Indicator strains Producer strains (Synonym) Pcc1 Pcc3 Pcc21 Pcc26 Pcc27 Pcc49 Pcc56 Brenneria nigrifluens KACC10052 (Erwinia nigrifluens ) ------Brenneria rubrifaciens KACC10048 (Erwinia rubrifaciens ) ------ KACC10049 (Erwinia salicis) ------Erwinia persicina KACC10053 ------Pantoea agglomerans KACC10054 (Erwinia herbicola) ------Pantoea agglomerans KACC10055 (Erwinia herbicola) ------Pantoea ananatis KACC10059 (Erwinia ananatis) +++ +++ +++ +++ +++ ++ +++ Pantoea ananatis KACC10083 (Erwinia ananatis) ------Pantoea stewartii subsp. stewartii KACC10085 (Erwinia stewartii) ------Pectobacterium carotovorum subsp. atrosepticum KACC10478 ------(Erwinia carotovora subsp. atroseptica) Pectobacterium carotovorum subsp. atrosepticum KACC10480 ------(Erwinia carotovora subsp. atroseptica) Pectobacterium carotovorum subsp. betavasculorum KACC10056 - ++++++- ++ (Erwinia carotovora subsp. betavasculorum) Pectobacterium carotovorum subsp. carotovorum Pcc3 + - ++++- (Erwinia carotovora subsp. carotovora) Pectobacterium carotovorum subsp. odoriferum KACC10486 ++ + - ++- (Erwinia carotovora subsp. odorifera) Pectobacterium carotovorum subsp. wasabiae KACC10061 +++ +++ +++ +++ +++ ¯ +++ (Erwinia carotovora subsp. wasabiae) Pectobacterium chrysanthemi SL3550 (Erwinia chrysanthemi) ------Pectobacterium chrysanthemi SL4405 (Erwinia chrysanthemi) ------Pectobacterium chrysanthemi SL4830 (Erwinia chrysanthemi) ------Pectobacterium cypripedii KACC10063 (Erwinia cypripedii) ------aMMC, Experiments were conducted in LB medium containing 0.5 µg/ml mitomycin C by P. carotovorum subsp. carotovorum strains was tested against of those antibiotics varied depending on the producers or several phytopathogenic bacteria that formerly belonged to the indicators. The induction kinetics of the antibacterial the genus Erwinia. Most producer strains were not able to substance production by mitomycin C treatment or UV light inhibit the growth of P. carotovorum subsp. carotovorum was different depending on the strain. The P. carotovorum strains grown in LB medium except P. betavasculorum subsp. carotovorum isolates tested in this study could be KACC10056 and P. carotovorum subsp. carotovorum Pcc3. grouped into 4 classes based on the antibacterial activity However, Pantoea ananatis KACC10059, P. carotovorum patterns. These data suggested that the isolates of P. carotovorum subsp. odoriferum KACC10486, and P. carotovorum subsp. subsp. carotovorum studied here might carry more than 4 wasabiae KACC10061 produced an antibacterial substance different kinds of antibacterial substances active against P. against most P. carotovorum subsp. carotovorum strains carotovorum subsp. carotovorum. Two already known grown in LB medium containing mitomycin C. None of antibacterial substances of P. carotovorum subsp. carotovorum the strains that belonged to Brenneria sp. was able to inhibit are carocin S1, a low-molecular-weight bacteriocin, and P. carotovorum subsp. carotovorum strains (Table 5). carotovoricin, a high-molecular-weight bacteriocin. These bacteriocins inhibited the growth of P. carotovorum subsp. carotovorum. All Korean strains were able to produce DISCUSSION carotovoricin. Strains belong to group D might carry carotovoricin only. Almost that 30% of tested Korean strains P. carotovorum subsp. carotovorum is a phytopathogenic carried DNA homologous to carocin S1 and these strains were bacterium responsible for the soft-rot disease of many in groups A to C. However, we were not able to group strains plant species. We screened the antibacterial spectra of 54 that might carry only carocin S1. strains of P. carotovorum subsp. carotovorum isolated in Most Korean strains were able to produce antibacterial Korea using seven indicator strains chosen to represent substance(s) against P. carotovorum subsp. atrosepticum. various regions and host plants. Several strains in group A and B produced antibacterial Korean isolates showed diverse antibacterial activity substance(s) against strains in Brenneria, Pantoea, and other against P. carotovorum subsp. carotovorum. The sensitivity Pectobacterium genera. Those responses were not correlated 50 Roh et al. with the antibacterial activity of carotovoricin or carocin produced by most Serratia marcescens biotypes. Res. Microbiol. S1. 146: 477-483. When 19 strains of phytopathogenic bacteria formerly 6. Hauben, L., E. R. Moore, L. Vauterin, M. Steenackers, J. belonging to the genus Erwinia were tested for the Mergaert, L. Verdonck, and J. Swings. 1998. Phylogenetic production of antibacterial substance(s) against P. carotovorum position of phytopathogens within the Enterobacteriaceae. Syst. subsp. carotovorum, Pantoea ananatis KACC10059 produced Appl. Microbiol. 21: 384-397. 7. Heu, S., J. Oh, Y. Kang, S. Ryu, S. K. Cho, Y. Cho, and M. Cho. antibacterial substance against all tested P. carotovorum 2001. gly Gene cloning and expression and purification of subsp. carotovorum strains isolated in Korea. However, glycinecin A, a bacteriocin produced by Xanthomonas campestris KACC10083 was not able to produce antibacterial substance pv. glycines 8ra. Appl. Environ. Microbiol. 67: 4105-4110. against any P. carotovorum subsp. carotovorum strains. 8. Kerr, A. and M. E. Tate. 1984. Agrocins and the biological control Therefore, the production of antibacterial substance was of crown gall. Microbiol. Sci. 1: 1-4. very specific for strain KACC10059. There are 5 subspecies 9. Lavermicocca, P., S. L. Lonigro, F. Valerio, A. Evidente, and A. of Pectobacterium carotovorum; subspecies atrosepticum, Visconti. 2002. Reduction of olive knot disease by a bacteriocin betavasculorum, carotovorum, odoriferum, and wasabiae. from Pseudomonas syringae pv. ciccaronei. Appl. Environ. Among those 5 subspecies, only strains in subsp. atrosepticum Microbiol. 68: 1403-1407. were not able to produce any antibacterial substance(s) 10. McClure, N. C., A. R. Ahmadi, and B. G. Clare. 1998. Construction of a range of derivatives of the biological control strain against P. carotovorum subsp. carotovorum. However, all Agrobacterium rhizogenes K84: A study of factors involved in P. carotovorum subsp. carotovorum strains studied here were biological control of crown gall disease. Appl. Environ. able to produce antibacterial substance(s) against strains in Microbiol. 64: 3977-3982. subsp. atrosepticum. These data supported the close relationship 11. Morgan, S., R. P. Ross, and C. Hill. 1995. Bacteriolytic activity within P. carotovorum subspecies. caused by the presence of a novel lactococcal plasmid encoding Our results demonstrate that a putative new antibacterial lactococcins A, B, and M. Appl. Environ. Microbiol. 61: 2995- substance(s) in addition to carotovoricin and carocin S1 3001. may be present in P. carotovorum subsp. carotovorum 12. Nguyen, A. H., T. Tomita, M. Hirota, T. Sato, and Y. Kamio. 1999. isolated in Korea. Isolation of a gene encoding such new A simple purification method and morphology and component bacteriocin is now under way. analyses for carotovoricin Er, a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er. Biosci. Biotechnol. Biochem. 63: 1360-1369. 13. Nguyen, H. A., T. Tomita, M. Hirota, J. Kaneko, T. Hayashi, Acknowledgment and Y. Kamio. 2001. 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