Structural plasmid evolution as a result of coupled recombinations at bom and cer sites.

We have studied the recombination of plasmids bearing bom and cer sites. The bom ( basis of mobilization) site is required for conjugative transfer, while the cer ( Col E1 resolution) site is involved in the resolution of plasmid multimers, which increases plasmid stability. We constructed a pair of parent plasmids in such a way as to allow us select clones containing recombinant plasmids directly. Clone selection was based on the McrA sensitivity of recipient host DNA modified by M. Ecl18kI, which is encoded by one of the parent plasmids. The recombinant plasmid contains segments originating from both parental DNAs, which are bounded by bom and cer sites. Its structure is in accordance with our previously proposed model for recombination mediated by bom and cer sequences. The frequency of recombinant plasmid formation coincided with the frequency of recombination at the bom site. We also show that bom-mediated recombination in trans, unlike in cis, is independent of other genetic determinants on the conjugative plasmids.

Characterization of pECL18 and pKPN2: a proposed pathway for the evolution of two plasmids that carry identical genes for a Type II restriction-modification system.

The primary structures of the plasmids pECL18 (5571 bp) and pKPN2 (4196 bp) from Escherichia coli and Klebsiella pneumoniae, respectively, which carry genes for a Type II restriction-modification system (RMS2) with the specificity 5'-CCNGG-3', were determined in order to elucidate the structural relationship between them. The data suggest a possible role for recombination events at bom (basis of mobility) regions and the sites of resolution of multimer plasmid forms (so-called cer sequences) in the structural evolution of multicopy plasmids. Analysis of the sequences of pECL18 and pKPN2 showed that the genes for RM* Ecl18kI and RM* Kpn2kI, and the sequences of the rep (replication) regions in the two plasmids, are almost identical. In both plasmids, these regions are localized between the bom regions and the cer sites. The rest of the pECL18 sequence is almost identical to that of the mob (mobilization) region of ColE1, and the corresponding segment of pKPN2 is almost identical to part of pHS-2 from Shigella flexneri. The difference in primary structures results in different mobilization properties of pECL18 and pKPN2. The complete sequences of pECL18, pKPN2 and the pairwise comparison of the sequences of pECL18, pKPN2, ColE1 and pHS-2 suggest that plasmids may exchange DNA units via site-specific recombination events at bom and cer sites. In the course of BLASTN database searches using the cer sites of pECL18 and pKPN2 as queries, we found twenty cer sites of natural plasmids. Alignment of these sequences reveals that they fall into two classes. The plasmids in each group possess related segments between their cer and bom sites.

Effects of Cryptococcus humicola killer toxin upon Cryptococcus terreus envelope: combined fluorometric and microscopic studies.

Killer toxin (microcin) produced by Cryptococcus humicola 9-6 induced interaction of the fluorogenic dyes, ethidium bromide, propidium iodide, and hemimagnesium 8-anilino-1-naphtalenesulfonate, with the sensitive strain of Cryptococcus terreus VKM Y-2253. The toxin also made the cells susceptible to cetyltrimethylammonium bromide and leaky for K+. When excited at 360 nm, cell-bound ethidium (propidium) fluorescence was enhanced by 8-anilino-1-naphtalensulfonate, and cell-bound 8-anilino-1-naphtalensulfonate fluorescence was quenched by ethidium (propidium), indicating energy transfer from 8-anilino-1-naphtalensulfonate to ethidium (propidium). These results suggest that at least a portion of the probe molecules had the same binding site, possibly the cytoplasmic membrane. The parameters of kinetics of microcin action were evaluated fluorometrically. They were found to be identical for all probes and depended on microcin concentration. The fluorescence increment of ethidium and 8-anilino-1-naphtalensulfonate upon binding to microcin-treated cells correlated with the fraction of stainable cells and viability.