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

Abstract

BackgroundMost 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.ResultsA 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.ConclusionThis 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.

Bacteriocins and their position in the next wave of conventional antibiotics

“…carotovorum, has been shown to have a ribonuclease mode of action against P. carotovorum SP33 [61]. Bacillus clausii strain GM17 and is found to be both heat-and pH-stable (between pH 3 and pH 9).…”

Ribosomally encoded antibacterial proteins and peptides fromPseudomonas

“…Their carboxy-terminal domain shows significant similarity to the cytotoxic domain of E. coli colicin D (Cascales et al, 2007), Klebsiella oxytoca klebicin D (Chavan et al, 2005), and Pe. carotovorum carocin S2 (Chan et al, 2011). Colicin D cleaves anticodon loops of at least three of the four tRNA Arg , in contrast to the tRNAse from colicin E5 (same type as present in pyocin S4) that prefers tRNA Asn , tRNA Asp , tRNA His , and tRNA Tyr (Papadakos et al, 2012).…”

Engineering bacteriocin‐mediated resistance against the plant pathogen Pseudomonas syringae

“…(which causes the greening and blight of rice and banana; Pham et al , ; Ghequire et al , ); Ralstonia solanacearum (which causes potato brown rot and bacterial wilt of tomato; Huerta et al , ) and Pectobacterium and Dickeya spp. (which cause potato soft rot and blackleg; Roh et al , ; Chan et al , ; Grinter et al , ; Czajkowski et al , ) .…”