Open Access

Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum

  • Yung-Chieh Chan1,
  • Jian-Li Wu1,
  • Huang-Pin Wu2,
  • Kuo-Ching Tzeng3 and
  • Duen-Yau Chuang1Email author
BMC Microbiology201111:99

DOI: 10.1186/1471-2180-11-99

Received: 21 September 2010

Accepted: 12 May 2011

Published: 12 May 2011

Abstract

Background

Most isolates of Pectobacterium carotovorum subsp. carotovorum (Pcc) produce bacteriocins. In this study, we have determined that Pcc strain F-rif-18 has a chromosomal gene encoding the low-molecular-weight bacteriocin, Carocin S2, and that this bacteriocin inhibits the growth of a closely related strain. Carocin S2 is inducible by ultraviolet radiation but not by mutagenic agents such as mitomycin C.

Results

A carocin S2-defective mutant, TF1-2, was obtained by Tn5 insertional mutagenesis using F-rif-18. A 5706-bp DNA fragment was detected by Southern blotting, selected from a genomic DNA library, and cloned to the vector, pMS2KI. Two adjacent complete open reading frames within pMS2KI were sequenced, characterized, and identified as caroS2K and caroS2I, which respectively encode the killing protein and immunity protein. Notably, carocin S2 could be expressed not only in the mutant TF1-2 but also in Escherichia coli DH5α after entry of the plasmid pMS2KI. Furthermore, the C-terminal domain of CaroS2K was homologous to the nuclease domains of colicin D and klebicin D. Moreover, SDS-PAGE analysis showed that the relative mass of CaroS2K was 85 kDa and that of CaroS2I was 10 kDa.

Conclusion

This study shown that another nuclease type of bacteriocin was found in Pectobacterium carotovorum. This new type of bacteriocin, Carocin S2, has the ribonuclease activity of CaroS2K and the immunity protein activity of CaroS2I.

Background

The phytopathogenic enterobacterium, Pectobacterium carotovorum subsp. carotovorum, is a phytoparasitic, Gram-negative, facultative anaerobic bacterium [1]. Pcc produces many extracellular pectic enzymes (pectate lyase, pectin lyase, exopolygalacturnoate lyase) and hydrolytic enzymes causing soft-rot disease, tissue maceration, and cell wall collapse [2, 3]. The only current strategy against soft-rot disease involves chemical agents that unavoidably contaminate the environment [4]. Kikumoto et al. have demonstrated that mixed bacteriocin-producing avirulent strains of Pcc show high efficacy against soft-rot disease of Chinese cabbage [5].

Bacteriocins are bactericidal, extracellular toxins, produced by both Gram-positive and Gram-negative bacteria [6, 7]. These proteinaceous molecules kill closely related bacteria. The susceptible cell is recognized by specific target receptors on the membrane, and the producer cell evades lethality by expressing a cognate immune protein. The colicin family produced by Escherichia coli is divided into DNase (colicins E2, E7, E8 and E9), RNase (colicins E3, E4 and E6), tRNase (colicins D and E5), and pore-forming colicins (colicins A, E1, Ia and Ib) [8]. Bacteriocins (especially nuclease bacteriocins) have a high amino acid sequence homology.

Natural bacteriocin molecules act via a number of mechanisms. For example, colicin E3 is a well-known ribonuclease that specifically cleaves 16S rRNA at the 3'-end of the coding sequence both in vivo and in vitro, which leads to the abolishment of protein synthesis resulting in death of the susceptible cell [912]. Previous reports indicate that colicin E3 consists of a killer protein with three domains (i.e., a translocation domain [T domain], receptor binding domain [R domain], and nuclease domain) and an immunity protein that retards antibiotic activity [13, 14]. The R domain recognizes a specific receptor, BtuB on the cell membrane and the T domain interacts with the TolB protein in the cell periplasm of the sensitive cell to facilitate entry of the killer domain through the cell membrane. In addition to the attack mechanism, the immunity mechanism has been extensively elucidated. Notably the immunity protein and the killer protein interact initially at very high affinity because of charge attraction, and are separated at the cell surface through energy generated from the proton motif force [1517].

In general, the C-terminal domain determines the type of bacteriocin. The C-terminal nuclease domains are not only interchangeable but also lack species specificity [18]. Strikingly, the tRNase type of bacteriocin may accelerate exhaustion of tRNA in the cytoplasmic pool and thereby impair protein synthesis in vivo. Ogawa et al. have demonstrated that particular tRNA molecules can be digested by colicin D as well as by colicin E5 [19, 20]. It has been suggested that phage-associated klebicin D is a tRNase type of bacteriocin based on similarity to the nuclease-like domain of colicin D [21].

Nguyen et al. reported production of a high-molecular-weight bacteriocin (carotovoricin Er) and Chuang et al. reported production of a low-molecular-weight bacteriocin (LMWB; carocin) by Pectobacterium[22, 23]. The former has a bulky antenna-like tail, inner core, and contractile cylindrical structure, and the carotovoricin-caused inhibition zone can be easily distinguished from that of carocin by its low diffusibility. Carocin S1 is a deoxyribonuclease type of LMWB (indicated by the letter S) and is secreted by Pcc strain 89-H-4. Additionally, export of Carocin S1 utilizes the type III secretion system in Pcc, which also controls the cell motility of the bacterium [24].

Pcc strain F-rif-18 is a spontaneous rifampin-resistant mutant of the wild-type 3F-3. Ultraviolet radiation can induce Pcc strain F-rif-18 to produce the LMWB Carocin S2. One of several sensitive cells, SP33, was selected as an indicator strain here. In the present study, the chromosomal bacteriocin gene, carocin S2, was introduced into an expression plasmid encoding two proteins, CaroS2K and CaroS2I. These proteins were purified and characterized and their primary activities of killing (CaroS2K) and immunity (CaroS2I) were investigated in vivo and in vitro.

Results

Isolation of Transposon Insertion Mutants

Conjugation between F-rif-18 and E. coli 1830 resulted in ~3,500 colonies after selection on Modified Drigalski's agar medium containing rifampin and kanamycin. In bacteriocin assay, the size of the inhibition zone around each isolate was compared with that of F-rif-18. Mutant colonies were identified by smaller inhibition zones. This evidence of mutation suggested that transposon Tn5 had been inserted into LMW bacteriocin-related genes. The strain TF1-2, a putative insertion mutant, would no longer produce LMW bacteriocin (Figure 1).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-11-99/MediaObjects/12866_2010_Article_1397_Fig1_HTML.jpg
Figure 1

Bacteriocin assays of Tn 5 insertion mutants of Pcc strains. Strain number: 1, 3F3 (wild type); 2, 1830 (E. coli); 3, F-rif-18 (parent); 4, TF1-1 and 5, TF1-2 (insertion mutant). Other unlabelled strains are Tn5 insertion mutants of F-rif-18 strain. The indicator is Pcc strain SP33.

To ascertain whether Tn5 was actually introduced into the genomic DNA of putative isolates, the nptII gene of isolates was amplified using two primers P3 and P4 [23]. Southern blot technology showed that Tn5 had been inserted (Additional file 1, Figure S1).

Identification of Tn5-inserted DNA Structures

To identify Tn5-interrupted genes, genomic DNA from TF1-2 was amplified with TAIL-PCR using an array of specific primers (Additional file 1, Figure S8). A 2621-bp DNA fragment, including two open reading frames (ORFs), was identified as the sequence containing the bacteriocin structural gene. This gene was designated the carocin S2 gene. To characterize the carocin S2 gene, the TF1-2 probe was designed to hybridize in Southern blots with a Bam HI-digested DNA fragment from the genomic library of F-rif-18 (Figure 2A). A 5706-bp Bam HI-digested DNA fragment (Figure 2B), harboring two complete ORFs of carocin S2, was cloned into the plasmid pMCL210 (Additional file 1, Figure S2). The carocin-producing plasmid was designated as pMS2KI. The amplicon, comprising the predicted ORF2 of caroS2I, was subcloned into the pGEM-T easy vector, resulting in the plasmid pGS2I (Additional file 1, Figure S5).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-11-99/MediaObjects/12866_2010_Article_1397_Fig2_HTML.jpg
Figure 2

DNA library screening and scheme of carocin S2 gene. (A) The TF1-2 probe was used to screen DNA fragments from the genomic DNA library of F-rif-18. The DNA was digested with various restriction enzymes as follows: 1. Hpy188I; 2. HindIII; 3 HpaI; 4. EcoRV; 5. EcoRI; 6. ClaI; 7. BsaAI; 8. BglII; 9. BamHI; 10. AhdI; M. DNA leader marker; C. The TF1-2 probe DNA. The arrowhead indicates the 5.7-kb carocin S2 fragment. (B) Shown is the 5.7-kb segment of DNA containing the carocin S2. The location of TF1-2 probe and part amplicon of cDNA of caroS2K and caroS2I were shown.

Transcriptional analysis and in vivo expression of carocin S2 gene

To determine whether the carocin S2 gene is transcribed in a series of recombinant strains, reverse transcription-PCR was used to estimate RNA level. Two sets of intergenic primers were designed to amplify parts of transcripts from caroS2K or caroS2I, respectively (Figure 2B). Amplification of parts of 16S ribosomal RNA transcripts indicated that RNA in these bacterial cells is expressed at normal levels (Figure 3).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-11-99/MediaObjects/12866_2010_Article_1397_Fig3_HTML.jpg
Figure 3

Reverse Transcription PCR of RNA. Shown are cDNA from the following strains: Lanes 1, F-rif-18; 2, TF1-2; 3, TF1-2/pMS2KI, 4, DH5α; 5, DH5α/pMS2KI.; 6, SP33; 7, SP33/pGS2I. The amplicons of caroS2K and caroS2I are 925 bp and 259 bp, respectively. The corresponding amplicons of 16S rRNA from the examined strains (lower panel). All samples were loaded equally.

The presence of the 925-bp amplicon revealed that caroS2K was being transcribed in the cell (panel caroS2K in Figure 3). The TF1-2 strain, which is a Tn5 insertional mutant, could not transcribe caroS2K (lane 2), but the ability of TF1-2 to transcribe caroS2K was restored by introduction of pMS2KI (lane 3). It was apparent that the amount of caroS2K expression was dependent on the number of copies of plasmid pMS2KI (compare lane 1 to lane 3). Additionally, carocin S2 can be expressed in E. coli strain DH5α by introduction of pMS2KI (lane 4 and lane 5).

The presence of a 259-bp amplicon showed that caroS2I was transcribed constitutively (panel caroS2I in Figure 3). The caroS2I gene was transcribed unexpectedly in mutant strain TF1-2 even though the plasmid pMS2KI was introduced (lane 3). This demonstrated that caroS2I is expressed constitutively regardless of whether the gene caros2K is transcribed. Possibly an individual promoter for caroS2I gene is located behind the Tn5 insertion site in the caroS2K gene. CaroS2I transcripts were detected in strain SP33 with plasmid pGS2I (lanes 6 and 7). Although both the SP33 strains (with or without pGEM T-easy) were susceptible to Carocin S2, SP33/pGS2I appeared to grow in the presence of CaroS2K (Figure 4B).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-11-99/MediaObjects/12866_2010_Article_1397_Fig4_HTML.jpg
Figure 4

Recovery and immunity activity of carocin S2. (A) Antibacterial activity of carocin S2 from different strains. The indicator was Pcc strain SP33. Strain number: 1, F-rif-18; 2, TF1-2; 3, TF1-2/pMS2KI; 4, DH5α/pMS2KI; 5, DH5α. (B) Assay for caroS2I. The colony and inoculated strains were F-rif-18. The indicator strains were: 1, SP33; 2, SP33/pGEM-T easy; 3, SP33/pGS2I.

To prove that pMS2KI contained the gene for Carocin S2, pMS2KI was introduced into TF1-2 and E. coli DH5α. Both TF1-2/pMS2KI and DH5α/pMS2KI had ability to express the activity of Carocin S2 (Figure 4A). The size of inhibition zone around strain TF1-2/pMS2KI was equal to that around DH5α/pMS2KI but still smaller than that around the wild-type strain F-rif-18. On the other hand, the quantity of transcripts expressed in vivo and in vitrodid not usually correspond.

Deduction of the amino acid sequence of Carocin S2

The carocin S2 gene consists of two ORFs (Additional file 1, Figure S7): one containing the 2352-bp caroS2K gene and the other containing the 273-bp caroS2I gene. The stop codon (TGA) of caroS2K overlaps the first start codon of caroS2I by 4-bp (ATGA). The amino sequences were deduced from the nucleotide sequence of the carocin S2 gene using DNASIS-Mac software (HITACHI, Japan) and compared to other analogous proteins using the BLAST and FASTA search tools.

ORF1 was found to encode a 783-amino acid protein with a high degree of homology to Pcc21 carocin D, Escherichia coli colicin D and Klebsiella oxytoca klebicin D (Figure 5); ORF2 was found to encode a 90-amino acid protein that shows homology to the immunity proteins of colicin D and klebicin D (Figure 5). Thus, caroS2K produces an antibiotic with a deduced molecular mass of 85 kDa. CaroS2I (a 10-kDa protein of 90 amino acids) was shown to confer resistance to CaroS2K. It is particularly noteworthy that the homology between CaroS2K and Colicin D and Klebicin D is at the C-terminal end of these proteins where the catalytic center of a ribonuclease is located. According to the FASTA program, the amino acid segment between Asp677 and the C-terminus of CaroS2K shares almost 60% similarity with the minimal tRNase domain of colicin D and klebicin D (Figure 5). Since the colicin D and klebicin D are well-known tRNase family of bacteriocins, suggests that Carocin S2 might therefore be a ribonuclease.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-11-99/MediaObjects/12866_2010_Article_1397_Fig5_HTML.jpg
Figure 5

Region similarity of the putative domains of carocin S2 with those of related bacteriocins. The related ORFs are shown. Percentage values indicate the percent relatedness to the corresponding regions in carocin S2. The length of each domain is proportional to the number of amino acids. Homologous domains are shaded similarly. Domain I is homologous with the N-terminal T domain of colicin E3 [27]. Domain II resembles the receptor binding domains of other bacteriocins, but has no significant homology to other sequences in the database [8, 30]. Domain III and ORF2 of carocin S2 are highly homologous to colicin D and klebicin D.

Purification and characterization of Carocin S2

E. coli BL21 (DE3) recombinants, which were transformed with pES2KI or pES2I, were used to express CaroS2K protein or CaroS2I protein individually. Coomassie blue stained SDS-PAGE gels of purified Carocin S2 are shown in Figure 6. The band corresponding to CaroS2K was purified. The gel indicates a relative mass (Mr) of about 85 kDa (Figure 6A), enrichment of the purified CaroS2K (arrowhead), and disappearance of other bands. Purification of CaroS2I by the same procedure resulted in a more intense band in the region of Mr 10 kDa (arrowhead; Figure 6B).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-11-99/MediaObjects/12866_2010_Article_1397_Fig6_HTML.jpg
Figure 6

SDS-PAGE analysis of purified protein. Shown are the CaroS2K (A) and CaroS2I (B). Samples were subjected to electrophoresis in 10% polyacrylamide gels, which were stained with Coomassie blue. Lane M, molecular weight standards (kDa); lane 1, cell lysate of E. coli BL21/pET32a; lane 4, cell lysate of BL21/pET30b; lanes 2 and 5, IPTG-induced cell lysates of BL21/pES2kI and BL21/pES2I, respectively; lanes 3 and 6, purified protein obtained after elution. The arrowheads indicate the killing protein of carocin S2K (A) and the immunity protein of carocin S2I (B).

The purified CaroS2K involved in the growth inhibition of the susceptible indicator strain SP33 was then characterized. The number of viable cells decreased with increasing concentration of CaroS2K (Figure 7). Almost all cells were dead at the initial concentration of 4 μg ml-1, indicating that about 90% of indicator strains are killed at this concentration. However, the activity of CaroS2K was inhibited by trypsin, but not inhibited by CaroS2I.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-11-99/MediaObjects/12866_2010_Article_1397_Fig7_HTML.jpg
Figure 7

Survival of SP33 cells treated with Carocin S2. Aliquots of indicator SP33 cells were treated with increasing concentrations of CaroS2K () and CaroS2K:CaroS2I in molar ratio of 1:1 (▲). The effect of trypsin on the CaroS2K was also assayed (■). The data are reported as means ± standard deviations.

Carocin S2 has ribonuclease activity

In order to confirm the role of carocin S2 as a ribonuclease type bacteriocin, we set up a RNA degradation assay. Northern blots of 5'-32P-labeled total RNA extract treated with increasing concentrations of CaroS2K (Figure 8B) showed a markedly lower intensity of labeled RNA fragments compared to untreated extracted RNA (Figure 8B, lane 1), suggesting that CaroS2K has ribonuclease activity.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-11-99/MediaObjects/12866_2010_Article_1397_Fig8_HTML.jpg
Figure 8

In vitro hydrolysis of DNA and RNA by Carocin S2. (A) Analysis of the DNase activity of carocin S2. Lane M, the HindIII-digested λ DNA marker; lane 1, genomic DNA only; lanes 2 and 3, genomic DNA treated or untreated with carocin S2 in buffer, respectively; lane 4, equal quantity of EcoRI-digested genomic DNA. The 5'-labeled total RNA (B) and 3'-labeled total RNA (C) (1 μg of RNA per sample) were incubated without (lane 1) or with 1 μg (lane 2), 100 ng (lane 3), 10 ng (lane 4), or 1 ng (lane 5) of Carocin S2 and the result was assessed by autoradiography. The arrowhead indicates that the RNA segment digested from ribosome. Equal amounts of Carocin S2I and Carocin S2K mixed before RNA digestion (lane 6).

Surprisingly the RNA segments were larger when the RNA was 3'-32P-labeled compared with 5'-32P-labeling (Figures 8B and 8C). As the concentrations of 23S RNA and 16S RNA decrease on the addition of increasing concentrations of CaroS2K, it is assumed that more ribosomal RNA is degraded leaving material that is ostensibly the ribosome. When excess concentrations of caroS2K (i.e 1 μg) are added then most of the ribosomal RNA is degraded leading to a destabilization and subsequent degradation of the ribosome (Figure 8C, lane 2). We hence consider that CaroS2K (in sufficient amount) would degrade the ribosome. CaroS2I inhibits the killing activity of CaroS2K because a mixture of equal quantities of CaroS2K and CaroS2I prevented digestion of RNA segments by CaroS2K (Figure 8C, lane 6).

Subsequently, treatment of the genomic DNA of the indicator strain SP33 with the purified CaroS2K protein had no effect on deoxyribonuclease activity, as compared to the pattern of EcoRI-digested genomic DNA (Figure 8A and Additional file 1, Figure S4).

Nucleotide sequence accession number

The Genbank accession number of the sequence of the carocin S2 gene is HM475143.

Discussion

In this study, a chromosome-borne gene encoding bacteriocin, carocin S2, in Pcc strain 3F3 was shown to possess ribonuclease activity. According to Bradley's classification, Carocin S2 is a low-molecular-weight bacteriocin [25]. Two genes, caroS2K and caroS2I, encode the 85-kDa and 10-kDa components, respectively, of Carocin S2. The substrate and gene structure of carocin S2 were unlike those of other bacteriocins from Pcc.

On the basis of sequence analysis, carocin S2 comprises these two overlapping ORFs, caroS2K and caroS2I (Additional file 1, Figure S7). A putative Shine-Dalgarno sequence 5'-AUGGA-3', which has also been seen in the DNA sequence of carocin S1, is located upstream (-9 bp to -13 bp) of the start codon AUG, suggesting that it could be a ribosome binding site for caroS2K [23]. Comparison of the upstream sequences of both caroS2K and caroS2I has shown that the two consensus sequences, 5'-TATAAAAA-3' (-34 bp to -41 bp) and 5'-GAAGT-3' (-61 bp to -65 bp), are both upstream from the start codon. Presumably, 5'-TATAAAAA-3' is the -10 promoter and 5'-GAAGT-3' is the -35 promoter for the carocin S2 gene, even though they differ from those of E. coli[26].

A putative -10 promoter is 33 bp upstream from the initiator ATG of the caroS2K gene, in which the SD sequence is embedded, while the -35 promoter is 19 bp upstream of the -10 promoter region. The putative promoter of the -35 box of caroS2I is located similarly near the -10 box, but the -10 box is just 24 bp upstream of the start codon where no SD sequence is apparent. Although those hypothesized promoters are located within the caroS2K structural gene, transcripts of caroS2I are routinely produced (Figure 3). This suggests that caroS2I RNA expression may be regulated posttranscriptionally, in spite of close neighboring genes downstream of the gene caroS2K; that is, core promoter elements may influence the expression of caroS2I gene.

In the present study, we attempted to separate CaroS2K from CaroS2I attached to (His)6-tag using a Nickel column (pEH2KI; Additional file 1, Figure S5), but a small amount of CaroS2I (Mr ~10 kDa) was observed in SDS-PAGE gels (Figure 6, bottom in lane 3), which had little influence on the activity of CaroS2K as the purified protein still had transient killing activity. Additionally, the activity of the Carocin S2 complex at 4℃ was long-lasting indicating good stability.

The C-terminal amino acid sequence of Carocin S2 had higher homology to those of colicin D and klebicin D, which are produced by E. coli and Klebsiella oxytoca, respectively, than to the amino acid sequence of carocin S1 from the same species (Additional file 1, Figure S6B).

The amino acid sequence of CaroS2K has three putative domains. Domain I (the N-terminal 314-residue sequence ending in Pro314) is regarded as the translocation domain and is homologous to the translocation domains of carocin D and colicin E3 (Figure 5). It is assumed to direct the cytotoxic domain to the periplasmic space [27, 28]. Additionally, the putative TonB box (a sequence recognition motif DTMTV) was found in the N-terminal domain of CarocinS2, which is thought to participate in bacteriocin translocation [8]. Thus, we suggested that Carocin S2 could be a TonB-dependent bacteriocin.

Domain III (extending from Asp677 to the carboxyl terminus) is the killer domain. Particularly noteworthy is the resemblance of the killer domain to the tRNase domain of colicin D and klebicin D (Figure 5), and thus we suggested that carocin S2 might have tRNase activity [2931]. Domain II extends 141 residues from Ilu315 to Val455 and is hypothesized to be the binding site that recognizes specific receptors on cell membranes. Additionally, domain III has no significant homology to carocin D, suggesting that carocin S2 and carocin D have different functions [28].

Finally, we showed that total RNA (whether labeled with radioactive phosphate at the 5'- or at the 3'- end) is sensitive to Carocin S2. Carocin S2 degraded 5'-labeled total RNA but not 5'-labeled CaroS2K-free RNA (Figure 8B), and the amount of degradation was not dose-dependent (arrowhead). However, the appearance of segments of unknown origin paralleled partial degradation of 23S and 16S rRNA (Figure 8C). These results suggest that the site of excision (either conformational or sequential) is close to the 5'-terminus of rRNA. Notably, the decrease in the amount of rRNA depended on the amount of Carocin S2 protein present, with complete degradation occurring in the presence of excess Carocin S2. Ogawa et al. reported that RNase type of bacteriocins, colicin E3 and colicin E5, catalyze the hydrolysis of the shorter RNAs from 16S rRNA [19, 32]. Moreover, colicin E5 was found to hydrolyze tRNA in vitro. Furthermore, it was previously reported that colicin E3 cleaved 16S rRNA completely, and even 30S rRNA [11, 33]. In our study, carocin S2 acted as an RNase that hydrolyzes rRNA (both 23S and 16S) in vitro. In terms of enzymatic function, Carocin S2 may act as an endo- and exo-ribonuclease simultaneously. Moreover, CaroS2I significantly inhibited nuclease activity in vitro but not in vivo (Figures 7, Figure 8 andAdditional file 1, Figure S3). We speculated that immunity protein CaroS2I might not be able to cross the cell membrane, as previously described [14]. Although our in vitro experiment showed that carocin S2 was a ribonuclease, further investigation is needed to clarify its function in cells.

One of the other Tn5 insertional mutants, TF1-1, which disrupted the coding sequence of the fliC gene, was found to halt expression of Carocin S2 (Figure 1), indicating that Carocin S2 can also be secreted via the type III secretion system [24]. The role of carocin S2 as an RNase in the cytoplasm is to prevent protein synthesis by cleaving either 23S rRNA or 16S rRNA. The role of the immunity protein, CaroS2I, is usually to stop the damage caused by CaroS2K in the cytoplasm. More details of the actual mechanism of carocin S2 remain to be elucidated.

Conclusion

As shown herein, the novel bacteriocin, Carocin S2, was characterized as a ribonuclease. It is the first bacteriocin with ribonuclease activity to be found in Pectobacterium strains. We suggested that Carocin S2 kills the indicator cell by exhausting its supply of some kinds of RNA, leading to inactivation of protein biosynthesis. It will be of interest to study the proteomics of Carocin S2 and its mechanism of action in the future.

Methods

Bacterial strains, media, and growth conditions

Bacterial strains and plasmids used in the study are listed in Table 1. Isolates of Pcc were grown at 28°C in Luria-Bertani (LB) medium or IFO-802 medium. The IFO-802 medium was supplemented with 1% polypeptin, 0.2% yeast extract, 0.1% MgSO4 (pH 7.0), and 1.5% agar. Isolates of Pcc were distinguished from Escherichia coli by their ability to grow on Modified Drigalski's agar medium [34]. Antibiotics (final concentration, 100 μg ml-1 of media) were added when necessary.
Table 1

Bacteria and plasmids used in the study

Strain or plasmid

Description

Source

Escherichia coli

  

1830

pro¯ met¯ Kan r Nmr, containing transposon Tn5 on the suicidal plasmid pBJ4JI

[44]

DH5α

supE44ΔlacU169(Φ80lacZΔM15) hsdR17recA1 gyrA96thi-1relA1

[39]

BL21(DE3)

hsdS gal(λcIts857 ind1 Sam7 nin5 lac UV5-T7 gene 1)

[45]

Pectobacterium carotovorum subsp. carotovorum

  

3F-3

Pcc, wild-type

Laboratory stock

F-rif-18

3F3, Rifr, wild-type

This study

TF1-1

F-rif-18, fliC::Tn5, Rifr, Kanr

This study

TF 1-2

F-rif-18, CarocinS2::Tn5, Rifr, Kanr

This study

SP33

Pcc, wild-type

Laboratory stock

Plasmid

  

pMCL210

p15A, Cmlr, Low copy number

[46]

pGEM T-Easy

Ampr; lacZ cloning vector

Promega

pET32a

Ampr; expression vector with the N-terminal His-tag

Novagen

pET30b

Kanr; expression vector with the C-terminal His-tag

Novagen

pMS2KI

5.7-kb BamHI DNA fragment harboring carocin S2 gene from 3F3 genome, cloned into pMCL210

This study

pEN2K*

caroS2K subcloned into pET32a

This study

pES2KI

Derived from pEN2K; deleted series of Tag element in front of expressed caroS2K

This study

pEH2KI*

Derived from pES2KI; adding (His)6-Tag adjacent to caroS2I

This study

pGS2I

caroS2I and its putative promoter from pMS2KI, subcloned into pGEM T-easy

This study

pECS2I*

caroS2I subcloned into pET30b, but the expressed fusion CaroS2I has no activity

This study

pES2I

Derived form pECS2I, the (His)6-Tag element was deleted

This study

Kanr: Kanamycin; Cmlr: Chloramphenicol; Rifr: Rifampicin; Ampr: Ampicillin.

*: See Additional file 1, Figure S5.

Bacterial conjugation

Overnight cultures of Pcc (recipient) and E. coli (donor) were mixed and spread onto 0.22-μm membrane filters placed on LB agar media and incubated overnight at 28°C [23]. The progeny after conjugation were appropriately diluted and cultivated on Modified Drigalski's medium (with ampicillin and kanamycin [100 μg ml-1]) overnight at 28°C. All isolates were placed on IFO-802 medium and tested for bacteriocins. Bacteriocin was assayed using the double-layer method, and Pcc SP33 was used as indicator strain [35]. The cells were incubated for 12 hours to form colonies, exposed to ultraviolet irradiation, incubated again for 12 hours, treated with chloroform to kill the cells, and then covered with soft agar containing indicator cells. The bacteriocin production was indicated by a zone of inhibition of indicator-cell (SP33) growth around the colony.

Genetic-engineering technique

The procedures of plasmid preparation, genomic DNA isolation, and DNA manipulation were performed as described by Sambrook et al. [36]. Oligonucleotide DNA primers were synthesized by MD Bio Inc. (Taipei, Taiwan). The PCR was amplified with Go-Taq DNA polymerase (Promega, USA). The thermal asymmetric interlaced PCR (TAIL-PCR) was performed as previously described [37].

Plasmids were introduced into Pcc strains using electroporation (1.25 kV/cm, 200 Ω, 25 μF) [38]. For heat-shock transformation, the competent cells of E. coli were prepared according to the method of Hanahan [39].

Exponentially growing cells (OD595 of about 6.0) were harvested for RNA preparation. Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA was resuspended in diethylpyrocarbonate (DEPC)-treated water. The concentration of RNA was determined by OD260 absorption, and RNA was analyzed by electrophoresis on 1.5% formaldehyde-morpholinepropanesulfonic-agarose gel.

Reverse transcription-PCR (RT-PCR) was carried out with AMV Reverse Transcriptase (Promega Inc., Taiwan) according to manufacturer's instructions. RNA (1 μg) was subjected to RT-PCR containing CaroS2_re_1 used as a reverse primer in first-strand cDNA synthesis. The RT mixtures were diluted and used as templates in a PCR reaction with two primers CaroS2_re_1 and CaroS2_for_1 (Additional file 1, Table S1).

A 2621-bp BamHI-HindIII digested DNA fragment, including the caroS2K and caroS2I genes, was amplified from pMS2KI with primers of CarocinS2K_for2 and CarocinS2I_rev2 (Additional file 1, Table S1) and subcloned into pET32a to give the plasmid pEN2K (Additional file 1, Figure S5). The pES2KI was obtained by excision of the Tag element between the rbs (ribosome binding site) and start code (for CaroS2K) in pEN2K using the SLIM method as previously described [40, 41]. The 5IHT32a2KI_forT, 5IHTGT2KI_forS, 5IHT32a3KI_revT, and 5IHT32a4KI_revS primers were used. A 273-bp fragment of the caroS2I gene was amplified by PCR and ligated into the NdeI and XhoI site of pET30b to form the plasmid pEC2I. Similarly, the plasmid pES2I was obtained by deleting the (His)6-tag of pEC2I (carried out as described above with primers of X21_forT, X21_forS, X21_revT and X21_revS). Subsequently, pES2KI and pES2I were introduced into E. coli BL21 (DE3) cells, respectively.

Restriction DNA library screening and Southern blots

Southern blots were performed according to the DIG Application Manual (Roche, USA). A 543-bp DNA fragment (TF1-2 probe) was amplified with TF1-2P and TF1-2A2 primers (Additional file 1, Table S1), subcloned into pGEM-T Easy vector (Promega Inc., USA), and labeled using a Random Primed DNA Labeling Kit (Roche Diagnostics, USA).

The genomic DNA of the wild-type strain F-rif-18 was digested with various restriction endonucleases, with sites located outside the putative open reading frame. Samples were electrophoresed and analyzed with Southern blotting. After detection using the TF1-2 probe, the DNA from positive gel slices was purified and cloned into pMCL210 to give the carocin-producing plasmid pMS2KI. The pMS2KI construct was isolated and detected as above with the TF1-2 probe.

Protein purification

The transformant cells of BL21, harboring pES2KI or pES2I, were grown in 500 ml to an OD595 of 0.4. The cells were induced with isopropyl-β-D-thiogalactopyranoside (IPTG; final concentration, 0.1 mM; at 25°C for 12 h). Subsequently, the cells were pelleted and the pellets were sonicated (10 cycles of 9 s with 9-s intervals). BL21/pES2KI pellets were subjected to ammonium sulfate precipitation (30-40%), resuspended in buffer A (30 mM NaCl and 20 mM Tris-Cl, pH 8.0), and applied to a Fractogel column (Merck, USA). The fraction was eluted by a NaCl gradient (30 mM-1.4 M). After purification through a P-100 size-exclusion column (BioRad, USA), the CaroS2K fractions were pooled and concentrated using an Amicon centriprep-50 column (Millipore, USA) and dissolved in buffer A. BL21/pES2I pellets were precipitated by ammonium sulfate (70-100%) and resuspended in buffer A. CaroS2I purification involved a similar chromatographic procedure using the Amicon centriprep-3 column (Millipore, USA). The concentration of protein was determined by the Bradford assay (Amresco, USA).

In vitro determination of Carocin S2 activity

Total RNA was treated with calf intestinal alkaline phosphatase (Promega, USA) at 55°C for 30 min as recommended by the manufacturer. The reaction was arrested by adding 5 mM nitrilotriacetic acid, and RNA was extracted with equal volumes of phenol/chloroform. An aliquot of phosphatase-treated RNA was 5'-32P-labeled at 37°C for 30 min by incubation with a mixture of [γ-32P]ATP, T4 polynucleotide kinase (Promega Inc, USA), and reaction buffer in nuclease-free water [42]. [5'-32P]Cytidine 3',5'-bisphosphate (pCp) and T4 RNA ligase (Promega, USA) were used for 3'-labeling of RNA [43]. Subsequently, the mixture was purified by MicroSpin G-25 columns (GE Healthcare, USA). The purified labeled RNA was divided into aliquots and incubated without or with Carocin S2 at 28°C for 60 min, respectively. To measure its activity, CaroS2I was pre-mixed with an equal amount of CaroS2K. The mixtures were subjected to electrophoresis on a 9% polyacrylamide gel (19:1) containing 7M urea, 50 mM Tris, 50 mM boric acid, and 1 mM EDTA, pH 8.3. All samples were electrophoresed at 15℃ by PROTEIN II xi (BioRad, USA).

To confirm DNase activity, 1 μg of genomic DNA from SP33 in solution containing buffer A was incubated with or without Carocin S2 at 28°C for 90 min. An equal quantity of genomic DNA was digested with EcoRI at 28°C for 90 min. Samples were then subjected to electrophoresis on 1% agarose gel.

Antibiotic activity of Carocin S2

Overnight cultures of SP33 were diluted (1:100) with LB medium and grown at 28°C to a density of approximately 105 CFU ml-1. The activity of increasing concentrations of Carocin S2 on cells in suspension incubated at 28°C for 60 min was assessed. CaroS2I was pre-mixed with an equal molar ratio of CaroS2K. All reaction mixtures were spread onto LB agar plates and incubated at 28°C for 16 h. The experiment was performed three times. Colonies growing on a series of plates were respectively counted.

Computer analysis of sequence data

Sequencing of the DNA fragments was carried out using an ABI automated DNA sequencer 373S. The nucleotide sequence data were compiled by DNASIS-Mac software (Hitachi, Japan). Amino acid sequences were compared using international BLAST and FASTA servers. Also, the putative domains of Carocin S2 were predicted using the PSI/PHI-BLAST.

Declarations

Acknowledgements

The support of this work by grants from the National Science Council (grants NSC-97-2313-B-005-027-MY3) of Taiwan (R.O.C.) is gratefully acknowledged.

Authors’ Affiliations

(1)
Department of Chemistry, National Chung-Hsing University
(2)
Division of Pulmonary Medicine, Department of Internal Medicine, Chang Gung Memorial Hospital
(3)
Department of plant pathology, National Chung-Hsing University

References

  1. Pe'rombelon MCM: Potato diseases caused by soft-rot erwinias: an overview of pathogenesis. The role of pectic enzymes in plant pathogenesis. Plant Pathol. 2002, 51: 1-12. 10.1046/j.0032-0862.2001.Short title.doc.x.View ArticleGoogle Scholar
  2. Collmer A, Keen NT: The role of pectic enzymes in plant pathogenesis. Annu Rev Phytopathol. 1986, 24: 383-409. 10.1146/annurev.py.24.090186.002123.View ArticleGoogle Scholar
  3. Barras F, Van Gijsegem F, Chatterjee AK: Extracellular enzymes and pathogenesis of soft-rot Erwinia. Annu Rev Phytopathol. 1994, 32: 201-234. 10.1146/annurev.py.32.090194.001221.View ArticleGoogle Scholar
  4. Eckert JW, Ogawa JM: The Chemical Control of Postharvest Diseases: Deciduous Fruits, Berries, Vegetables and Root/Tuber Crops. Annu Rev Phytopathol. 1988, 26: 433-469. 10.1146/annurev.py.26.090188.002245.View ArticleGoogle Scholar
  5. Kikumoto T, Kyeremeh AG, Chuang DY, Gunji Y, Takahara Y, Ehara Y: Biological Control of Soft Rot of Chinese Cabbage Using Single and Mixed Treatments of Bacteriocin-producing Avirulent Mutants of Erwinia carotovora subsp. carotovora. J Gen Plant Pathol. 2000, 66: 264-268. 10.1007/PL00012957.View ArticleGoogle Scholar
  6. Jack RW, Tagg JR, Ray B: Bacteriocins of Gram-Positive Bacteria. Microbiol Rev. 1995, 59: 171-200.PubMedPubMed CentralGoogle Scholar
  7. Daw MA, Falkiner FR: Bacteriocins: Nature, Function and Structure. Micron. 1996, 27: 467-479. 10.1016/S0968-4328(96)00028-5.PubMedView ArticleGoogle Scholar
  8. Cascales E, Buchanan SK, Duche D, Kleanthous C, Lloube's R, Postle K, Riley M, Slatin S, Cavard D: Colicin Biology. Microbiol Mol Biol Rev. 2007, 71: 158-229. 10.1128/MMBR.00036-06.PubMedPubMed CentralView ArticleGoogle Scholar
  9. Boon T: Inactivation of Ribosomes In Vitro by Colicin E3. Proc Natl Acad Sci USA. 1971, 68: 2421-2425. 10.1073/pnas.68.10.2421.PubMedPubMed CentralView ArticleGoogle Scholar
  10. Mosbahi K, Walker D, James R, Moore GR, Kleanthous C: Global structural rearrangement of the cell penetrating ribonuclease colicin E3 on interaction with phospholipid membranes. Protein Sci. 2006, 15: 620-627. 10.1110/ps.051890306.PubMedPubMed CentralView ArticleGoogle Scholar
  11. Senior BW, Holland IB: Effect of colicin E3 upon the 30S ribosomal subunit of Escherichia coli. Proc Natl Acad Sci USA. 1971, 68: 959-963. 10.1073/pnas.68.5.959.PubMedPubMed CentralView ArticleGoogle Scholar
  12. Zarivach R, Ben-Zeev E, Wu N, Auerbach T, Bashan A, Jakes K, Dickman K, Kosmidis A, Schluenzen F, Yonath A, Eisenstein M, Shoham M: On the interaction of colicin E3 with the ribosome. Biochimie. 2002, 84: 447-454. 10.1016/S0300-9084(02)01449-9.PubMedView ArticleGoogle Scholar
  13. Lancaster LE, Savelsbergh A, Kleanthous C, Wintermeyer W, Rodnina MV: Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes. Mol Microbiol. 2008, 69: 390-401. 10.1111/j.1365-2958.2008.06283.x.PubMedPubMed CentralView ArticleGoogle Scholar
  14. Soelaiman S, Jakes K, Wu N, Li C, Shoham M: Crystal structure of colicin E3: implications for cell entry and ribosome inactivation. Mol Cell. 2001, 8: 1053-1062. 10.1016/S1097-2765(01)00396-3.PubMedView ArticleGoogle Scholar
  15. Jakes KS, Zinder ND: Highly purified colicin E3 contains immunity protein. Proc Natl Acad Sci USA. 1974, 71: 3380-3384. 10.1073/pnas.71.9.3380.PubMedPubMed CentralView ArticleGoogle Scholar
  16. Jakes K, Zinder ND, Boon T: Purification and properties of colicin E3 immunity protein. J Biol Chem. 1974, 249: 438-444.PubMedGoogle Scholar
  17. Vankemmelbeke M, Zhang Y, Moore GR, Kleanthous C, Penfold CN, James R: Energy-dependent immunity protein release during tol-dependent nuclease colicin translocation. J Biol Chem. 2009, 284: 18932-18941. 10.1074/jbc.M806149200.PubMedPubMed CentralView ArticleGoogle Scholar
  18. Kageyama M, Kobayashi M, Sano Y, Masaki H: Construction and characterization of pyocin-colicin chimeric proteins. J Bacteriol. 1996, 178: 103-110.PubMedPubMed CentralGoogle Scholar
  19. Ogawa T, Tomita K, Ueda T, Watanabe K, Uozumi T, Masaki H: A cytotoxic ribonuclease targeting specific transfer RNA anticodons. Science. 1999, 283: 2097-2100. 10.1126/science.283.5410.2097.PubMedView ArticleGoogle Scholar
  20. Tomita K, Ogawa T, Uozumi T, Watanabe K, Masaki H: A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops. Proc Natl Acad Sci USA. 2000, 97: 8278-8283. 10.1073/pnas.140213797.PubMedPubMed CentralView ArticleGoogle Scholar
  21. de Zamaroczy M, Mora L, Lecuyer A, Géli V, Buckingham RH: Cleavage of Colicin D Is Necessary for Cell Killing and Requires the Inner Membrane Peptidase LepB. Mol Cell. 2001, 8: 159-168. 10.1016/S1097-2765(01)00276-3.PubMedView ArticleGoogle Scholar
  22. Nguyen AH, Tomita T, Hirota M, Sato T, Kamio Y: A simple purification method and morphology and component analyses for carotovoricin Er, a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er. Biosci Biotechnol Biochem. 1999, 63: 1360-1369. 10.1271/bbb.63.1360.PubMedView ArticleGoogle Scholar
  23. Chuang DY, Chien YC, Wu HP: Cloning and Expression of the Erwinia carotovora subsp. carotovora Gene Encoding the Low-Molecular-Weight Bacteriocin Carocin S1. J Bacteriol. 2007, 189: 620-626. 10.1128/JB.01090-06.PubMedPubMed CentralView ArticleGoogle Scholar
  24. Chan YC, Wu HP, Chuang DY: Extracellular secretion of Carocin S1 in Pectobacterium carotovorum subsp. carotovorum occurs via the type III secretion system integral to the bacterial flagellum. BMC Microbiol. 2009, 9: 181-10.1186/1471-2180-9-181.PubMedPubMed CentralView ArticleGoogle Scholar
  25. Bradley DE: Ultrastructure of bacteriophage and bacteriocins. Bacteriol Rev. 1967, 31: 230-314.PubMedPubMed CentralGoogle Scholar
  26. Ross W, Gosink KK, Salomon J, Igarashi K, Zou C, Ishihama A, Severinov K, Gourse RL: A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science. 1993, 262: 1407-1413. 10.1126/science.8248780.PubMedView ArticleGoogle Scholar
  27. Sharma O, Cramer WA: Minimum length requirement of the flexible N-terminal translocation subdomain of colicin E3. J Bacteriol. 2007, 189: 363-368. 10.1128/JB.01344-06.PubMedPubMed CentralView ArticleGoogle Scholar
  28. Roh E, Park TH, Kim MI, Lee S, Ryu S, Oh CS, Rhee S, Kim DH, Park BS, Heu S: Characterization of a new bacteriocin, Carocin D, from Pectobacterium carotovorum subsp. carotovorum Pcc21. Appl Environ Microbiol. 2010, 76: 7541-7549. 10.1128/AEM.03103-09.PubMedPubMed CentralView ArticleGoogle Scholar
  29. Chavan M, Rafi H, Wertz J, Goldstone C, Riley MA: Phage associated bacteriocins reveal a novel mechanism for bacteriocin diversification in Klebsiella. J Mol Evol. 2005, 60: 546-556. 10.1007/s00239-004-0263-9.PubMedView ArticleGoogle Scholar
  30. de Zamaroczy M, Buckingham RH: Importation of nuclease colicins into E coli cells: endoproteolytic cleavage and its prevention by the immunity protein. Biochimie. 2002, 84: 423-432. 10.1016/S0300-9084(02)01426-8.PubMedView ArticleGoogle Scholar
  31. Mora L, Klepsch M, Buckingham RH, Heurgué-Hamard V, Kervestin S, de Zamaroczy M: Dual roles of the central domain of colicin D tRNase in TonB-mediated import and in immunity. J Biol Chem. 2008, 283: 4993-5003.PubMedView ArticleGoogle Scholar
  32. Hirao I, Harada Y, Nojima T, Osawa Y, Masaki H, Yokoyama S: In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA. Biochemistry. 2004, 43: 3214-3221. 10.1021/bi0356146.PubMedView ArticleGoogle Scholar
  33. Ohno S, Imahori K: Colicin E3 is an endonuclease. J Biochem. 1978, 84: 1637-1640.PubMedGoogle Scholar
  34. Sano Y, Kobayashi M, Kageyama M: Functional domains of S-type pyocins deduced from chimeric molecules. J Bacteriol. 1993, 175: 6179-6185.PubMedPubMed CentralGoogle Scholar
  35. Fredericq P: Colicins. Annu Rev Microbiol. 1957, 11: 7-22. 10.1146/annurev.mi.11.100157.000255.PubMedView ArticleGoogle Scholar
  36. Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual. 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2Google Scholar
  37. Liu YG, Whittier RF: Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics. 1995, 25: 674-681. 10.1016/0888-7543(95)80010-J.PubMedView ArticleGoogle Scholar
  38. Metzger M, Bellemann P, Schwartz T, Geider K: Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells. Nucleic Acids Res. 1992, 20: 2265-2270. 10.1093/nar/20.9.2265.PubMedPubMed CentralView ArticleGoogle Scholar
  39. Hanahan D: Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983, 166: 557-580. 10.1016/S0022-2836(83)80284-8.PubMedView ArticleGoogle Scholar
  40. Liu H, Naismith JH: An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 2008, 8: 91-10.1186/1472-6750-8-91.PubMedPubMed CentralView ArticleGoogle Scholar
  41. Garinot-Schneider C, Pommer AJ, Moore GR, Kleanthous C, James R: Identification of putative active-site residues in the DNase domain of colicin E9 by random mutagenesis. J Mol Biol. 1996, 260: 731-742. 10.1006/jmbi.1996.0433.PubMedView ArticleGoogle Scholar
  42. Silberklang M, Gillum AM, RajBhandary UL: The use of nuclease P1 in sequence analysis of end group labeled RNA. Nucleic Acids Res. 1977, 4: 4091-4108. 10.1093/nar/4.12.4091.PubMedPubMed CentralView ArticleGoogle Scholar
  43. Bruce AG, Uhlenbeck OC: Reactions at the termini of tRNA with T4 RNA ligase. Nucleic Acids Res. 1978, 5: 3665-77. 10.1093/nar/5.10.3665.PubMedPubMed CentralView ArticleGoogle Scholar
  44. Gantotti BV, Kindle KL, Beer SV: Transfer of the drug-resistance transposon Tn5 to Erwinia herbicola and the induction of the insertion Mutation. Curr Microbiol. 1981, 6: 417-425.View ArticleGoogle Scholar
  45. Wood WB: Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. J Mol Biol. 1966, 16: 118-133. 10.1016/S0022-2836(66)80267-X.PubMedView ArticleGoogle Scholar
  46. Nakano Y, Yoshida Y, Yamashita Y, Koga T: Construction of a series of pACYC-derived plasmid vectors. Gene. 1995, 162: 157-158. 10.1016/0378-1119(95)00320-6.PubMedView ArticleGoogle Scholar

Copyright

© Chan et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement