Identification and characterization of the bacteriocin Carocin S3 from the multiple bacteriocin producing strain of Pectobacterium carotovorum subsp. carotovorum

Abstract

Background: Pectobacterium carotovorum subsp. carotovorum belongs to the Enterobacteriaceae family, which causes soft-rot disease in numerous plants worldwide resulting in significant economic losses. Results from our previous studies showed that the strain H-rif-8-6 produces low-molecular-weight bacteriocin (LMWB) Carocin S1. Interestingly, TH22-10, the caroS1K:Tn5 insertional mutant in H-rif-8-6, loses Carocin S1 producing ability, but still produces other LMWBs which the indicator strain SP33 can detect. The SP33 is one of the many strains that are sensitive toward the cytotoxic effects of Carocin S3K, but not Carocin S1. The result revealed that H-rif-8-6 is a multiple-bacteriocin producing strain.

Results: In this study, a 4.1-kb DNA fragment was isolated from the chromosomal DNA of Pcc strain, H-rif-8-6, by a DNA probe using the caroS1K gene as the template. DNA sequencing and analysis by GenBank revealed two complete open reading frames (ORFs), designated ORF1 and ORF2, which were identified within the sequence fragment. ORF1 and ORF2, similar to the identified carocin S2 genes, encode the killer (Carocin S3K) and the immunity (Carocin S3I) proteins, respectively, which were homologous to the colicin E3 gene. Carocin S3K and Carocin S3I were expressed, isolated, and purified in Escherichia coli BL21 after subcloning of the expression plasmid pGS3KI or pGSK3I. SDS-PAGE analysis showed that the relative masses of Carocin S3K and Carocin S3I were 95.6 kDa and 10.2 kDa, respectively. The results reveal that Carocin S3K has higher antimicrobial and specific antimicrobial activities for Pcc along with a nuclease activity than Carocin S3I. However, Carocin S3I inhibits the activity of Carocin S3K. Interestingly, a high concentration of Carocin S3I protein is also a DNA nuclease, and Carocin S3K also inhibits its activity.

Conclusion: This study showed that another type of bacteriocin was found in Pectobacterium carotovorum. This new type of bacteriocin, Carocin S3, has the killer protein, Carocin S3K, and the immunity protein, Carocin S3I.

Keywords: Carocin S3; Low-molecular-weight Bacteriocin; Pectobacterium carotovorum subsp. carotovorum.

Fig. 1

Flow diagram of the research process. To confirm the presence of another bacteriocin from Pcc, we initially subjected the TH22–10 (caroS1K:Tn5) to bacteriocin activity assay, RT-PCR, and western blotting. After confirming that the Pcc strain H-rif-8-6 produces another bacteriocin apart from the previously identified bacteriocin, Carocin S1, the genomic DNA from H-rif-8-6 was isolated and digested using several restriction enzymes and was assayed with the best restriction enzyme for creating DNA libraries as demonstrated by the Southern blotting experiment. Then, bacteriocin production was tested through the bacteriocin activity assay. After that, the DNA nucleotide sequence and the deduced amino acid sequence of other known bacteriocins were compared. Subsequently, subcloning for the novel bacteriocin, designated as Carocin S3, and transformation were performed. Recombinants were used to express the Carocin S3 proteins, Carocin S3K and Carocin S3I. Furthermore, bacteriocin expression and bacteriocin activity assays were performed. Also, protein purification and Carocin S3 antibiotic activity test were carried out. Finally, Electrospray Ionization Mass Spectrometry Molecular Weight Assay was done to determine the molecular weights of the killer protein, Carocin S3K, and immunity protein, Carocin S3I

Fig. 2

A Bacteriocin assays of Pectobacterium carotovorum subsp. carotovorum. Numbered strains show the wild type H-rif-8-6 (1) and the caroS1K:Tn5 insertion mutant, TH22–10 (2). Other unlabeled strains are Tn5 insertion mutants of progeny of H-rif-8-6 strain. The indicators were Pcc strain SP33 (A) and Ea1068 (B). B In vitro analysis of the DNase activity of rough protein extract from TH22–10. Samples were subjected to gel electrophoresis and labeled as follows: Lane M, the HindIII-digested λ DNA marker; 1, the equal quantities of EcoRI-digested genomic DNA; 2, the genomic DNA with protein extract from H-rif-8-6; 3, the genomic DNA with protein extract from TH22–10; 4, the genomic DNA only with reaction buffer; 5, the genomic DNA only. C Carocin S1 protein detection. Carocin S1K antiserum antibody (anti-CaroS1K) and Carocin S1I antiserum antibody (anti-CaroS1I) were detected from rabbits

Fig. 3

Carocin S3 protein analysis by 15% SDS-PAGE and comassie-blue stained. M, protein molecular weight marker; 1, BL31/pET32a (control); 2, BL21/pES3KI cells; 3, BL21/pES3KI protein extracts. The molecular weight of Carocin S3K is 95.6 kDa, and Carocin S3I is 10.2 kDa

Fig. 4

Survival of SP33 cells treated with Carocin S3. Aliquots of indicator SP33 cells were treated with increasing concentrations of Carocin S3K ( ) and Carocin S3K:Carocin S3I in a molar ratio of 1:1 ( ). The effect of trypsin on the Carocin S3K was also assayed ( ). The data are reported as means ± standard deviations

Fig. 5

Molecular weight analysis for Carocin S3I from Carocin S3 by ESI-MS

Fig. 6

SDS-PAGE analysis of purified protein. Shown are the Carocin S3K (a) and Carocin S3I (b). Samples were subjected to electrophoresis in 10% polyacrylamide gels, which were stained with Coomassie blue. These were labeled as follows: lane M, molecular weight standards (kDa); lane 1, cell lysate of E. coli BL21/pET32a; lanes 2 and 4, IPTG-induced cell lysates of BL21/pES3kI and BL21/pES3I, respectively; lanes 3 and 5, a purified protein obtained after elution. The arrowheads indicate the killing protein of Carocin S3K (a) and the immunity protein of Carocin S3I (b)

Fig. 7

In vitro hydrolysis of DNA and RNA by Carocin S3. a Analysis of the DNase activity of Carocin S3. The samples are labelled as M, marker; S, genomic DNA of Ea1068; 1, genomic DNA/ddw; 2, genomic DNA/buffer (contains Mg²⁺); 3, genomic DNA/BamHI + buffer; 4, genomic DNA/EcoRI + buffer; 5, genomic DNA/Carocin S3I (1 μM) + buffer; 6, genomic DNA/Carocin S3K(1 μM) + buffer; 7, genomic DNA/Carocin S3K(1 μM) + Carocin S3I(1 μM) + buffer. b 0.15 μg of genomic DNA per sample were incubated with different concentration of Carocin S3I. Lane S, contains 0.15 μM genomic DNA; lane K, 0.15 μM genomic DNA and 1 μM Carocin S3K; all other lanes contain 0.15 μM genomic DNA and different concentrations of Carocin S3I protein. All samples contained 10 mM Mg²⁺

Fig. 8

Region similarity of the putative domains of the Carocin S3 with those of related bacteriocins. The related ORFs are shown. Percentage values indicate the percent relatedness to the corresponding regions in Carocin S3. The length of each domain is proportional to the number of amino acids, and homologous domains are shaded similarly

Fig. 9

The DNA nuclease activity of CaroS3I and CaroS3K in the producer cell (a) and target cell (b)