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 Trichostatin A cost 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 Rucaparib manufacturer 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 Selleckchem Venetoclax 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 . 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 .