Cloning and expression of the Erwinia carotovora subsp. carotovora gene encoding the low-molecular-weight bacteriocin carocin S1

Abstract

The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp. carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp. carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (-312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.

FIG. 1.

Bacteriocin activities of Tn5 insertion mutants of E. carotovora subsp. carotovora strains: 1, E. coli 1830/pBJ4JI (containing Tn5); 2, H-rif-8-6 (parent); and 3, TH22-10 (insertion mutant). The unlabeled strains are all Tn5 insertion mutants of the H-rif-8-6 parental strain. The indicator was Ea1068.

FIG. 2.

Transcription analysis of the carocin S1 gene. (A) Northern hybridization analysis of caroS1K and caroS1I. Total RNAs (20 μg) from H-rif-8-2, TH22-10, TH22-10/pAYL4, Ea1068, and Ea1068/pAYL4 cells incubated in BSM at 28°C for 24 h were subjected to Northern blot analysis. E. carotovora subsp. carotovora strain Ea1068B was used for the bacteriocin activity test. (B) Carocin S1 expression after water, UV, glucose, and lactose stimulation. The producer strains were 89-H-4 and Ea1068/pAYL4, and the indicator strain was Ea1068.

FIG. 3.

DNase activity of carocin S1 against genomic DNA from E. carotovora subsp. carotovora Ea1068. (A) Analysis of the killing activity of purified carocin S1. Carocin S1 was purified from 89-H-4 (1), H-rif-8-6 (2), TH22-10/pAYL4 (3), and Ea1068/pAYL4 (4) strains and then added to the indicator plates to test its killing activity. The indicator strains were Ea1068/pAYL4 in the left plate and Ea1068 in the right plate. (B) The reaction mixture (50 μl) containing 1 μg of DNA and 20 μl of sample solution in 10 mM Tris, pH 7.5, 4 mM MnCl₂ was incubated at 28°C for 2 h. Samples 1 to 5 were collected from Ea1068, and samples 6 to 10 were collected from Ea1068/pAYL4. Lanes 1 and 6 were from the 11th fraction tubes, lanes 2 and 7 from the 12th fraction tubes, lanes 3 and 8 from the 13th fraction tubes, lanes 4 and 9 from the 14th fraction tubes, and lanes 5 and 10 from the 15th fraction tubes. Lane 11 was genomic DNA isolated from Ea1068. Lane 12 was the positive control (DNA mixed with EcoRI and buffer), and lane 13 was the negative control (DNA and buffer). Samples were treated and analyzed by agarose gel electrophoresis.

FIG. 4.

Alignment of the amino acid sequences of carocin S1, pyocin S3A, and pyocin AP41. (A) Killer protein. (B) Immunity protein. The numbers refer to the positions of the amino acid residues in the sequence of each protein. The putative structural domains (I to IV) of carocin S1K are indicated.