Identification and Characterization of the Bacteriocin Carocin S3 from the Multiple Bacteriocin Producing Strain of Pectobacterium carotovorum subsp. carotovorum

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.

Computational approach and functional analysis of the Pectobacterium carotovorum subsp. carotovorum low-molecular weight bacteriocin Carocin S2

Pectobacterium carotovorum subsp. Carotovorum 3F3 is a gram-negative phyto-parasitic enterobacterium. This strain is a producer of Carocin S2 bacteriocin, which comprises of two proteins of different sizes. Carocin S2K (killer protein) which is responsible for antibiotic resistance and Carocin S2I (immunity protein) which inhibits the antibiotic activity.The present study aimed to predict the structure and functional properties of Carocin S2. Computational approaches utilizing various bioinformatic tools predicted that Carocin S2 is a putative membrane protein having the N-terminal at the extracellular side and the central domain at the coiled-coil region. Carocin S2 was predicted to have three domains, the translocation domains, receptor binding domain and the killer domain. Moreover, the killer domain was calculated to have the catalytic cleft. The in-vivo assays confirmed that for Carocin S2K, bound immunity protein was not a pre-requisite for cell attachment or translocation. The site-directed mutagenesis experiment led us to hypothesized the hydrolysis mechanism of Carocin S2.The predicted structure of Carocin S2K provided a system of understanding on the biochemical and structural function which led to the mechanism of Carocin S2. It revealed that the role of immunity protein to Carocin S2 is not a pre-requisite for translocation pathway. Furthermore, this research led to hypothesized a hydrolytic mechanism of Carocin S2 to target the tRNA.

Identification and Characterization of the Bacteriocin Carocin S3 from the Multiple Bacteriocin Producing Strain of Pectobacterium carotovorum subsp. carotovorum

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.

Identification and Characterization of the Bacteriocin Carocin S3 from the Multiple Bacteriocin Producing Strain of Pectobacterium carotovorum subsp. carotovorum

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 can be detected by using the indicator strain SP33. 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, high concentration of Carocin S3I protein is also a DNA nuclease, and its activity is also inhibited by Carocin S3K.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.

Identification and Characterization of the Bacteriocin Carocin S3 from the Multiple Bacteriocin Producing Strain of Pectobacterium Carotovorum Subsp. Carotovorum

BackgroundPectobacterium 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 can be detected by using the indicator strain SP33. The SP33 is one of the many strains that are sensitive toward the cytotoxic effects of CaroS3K, but not Carocin S1. The result revealed that H-rif-8-6 is a multiple-bacteriocin producing strain.ResultsIn this study, a 4.1-kb DNA fragment was isolated 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 (CaroS3K) and the immunity (CaroS3I) proteins, respectively, which were homologous to the colicin E3 gene. CaroS3K and CaroS3I 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 CaroS3K and CaroS3I were 95 kDa and 10 kDa, respectively. The results reveal that CaroS3K has higher antimicrobial and specific antimicrobial activities for Pcc along with a nuclease activity than CaroS3I. However, CaroS3I inhibits the activity of CaroS3K. Interestingly, high concentration of CaroS3I protein is also a DNA nuclease, and its activity is also inhibited by CaroS3K.ConclusionThis study showed that another type of bacteriocin was found in Pectobacterium carotovorum. This new type of bacteriocin, Carocin S3, has the killer protein, CaroS3K, and the immunity protein, CaroS3I.