• Korean Journal of Microbiology
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• v.28 no.3
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• pp.219-228
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• 1990

A kanamycin resistance($Km^r$) gene was studied for its transfer in natural freshwater environments by using the natural bacterial isolate(M1) of DK1 and the DKC601 strain, $Km^r$ plasmid of which was genetically engineered from the NI strain. The transfer frequency ofthe $Km^r$ gene and rearrangement of the $Km^r$ plasmid were compared between the gnetically engineered microorganism(GEM) and the NI parental strain by conjugation with the same recipient strain. The transfer frequency of the $Km^r$ gene was about $9.1\times 10^{-12}-1.8\times 10^{-11}$ in both the GEM and NI strains at 5 to $10^{\circ}C$, but the frequency of the NI was about 10 times higher than that of the GEM at 20 to $30^{\circ}C$. The $Km^r$ plasmid in the transconjugants obtained by conjugation of the NI with the MY1 strain as a ricipient showed alot of rearrangement, but the $Km^r$ plasmid transferred from the GEM was stable without alteration of its size. When the MT2 strain was used as a recipient, however, such a rearrangement of the $Km^r$ plamid was observed in the transconjugants obtained from the GEM as well as the NI strain. In those transconjugants obtained from different mating pairs and water environments, the plasmid were appeared to decrease in their number as the period of conjugation time was prolonged, but only the $Km^r$ plasmid transferred from the GEM kept having its size of 52kb. Therefore, the $Km^r$ gene was transferred at the same rate from the GEM and NI strains in natural freshwater environment, but the gene of the GEM strain was more stable than the NIduring conjugation and the $Km^r$ plasmid was rearranged by changing the recipient strain for conjugation in any water environments.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.286-291
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• 1993

The $Km^{r}$ gene and plasmid of natural isolate and genetically modified microorganisms (GMM) rearranged by conjugation in water environments were comparatively analyzed by agarose gel electrophoresis and Southern analysis. The transfer rates of the $Km^{r}$ gene from GMM strains were generally 100 times higher than thosc of natural iso]ate(DKI) under laboratory cnvironments, but their transfer rate was not much different in Moosimcheon River water. The conjugants obtained in LB(Luria-Bertani broth) and FW(filtered river water) water under laboratory conditions showed same number of the plasmids. but the sizes of the plasmids were changed. The $Km^{r}$ gene in the conjugants was found in the same position as the pDKJO] $Km^{r}$ plasmid. In case of the GMM strains as donor. the large plasmids of 180 kb appeared in conjugants obtained in LB and FW water. Especially, the $Km^{r}$ gene in the donor of DKC600 was found to be inserted into chromosome of the conjugant obtained in FW water. However. in the conjugants obtained from DKl and DKB 701 in Moosimcheon River water, the plasmids were rearranged by 4 and 8. respectively, and all of them showed hybridization by the $Km^{r}$ probe. But the small plasmids of the recipient disappeared in the conjugant from DKC600 as donor, and the rearranged plasm ids and chromosome in the conjugants were observed to be hybridized with the $Km^{r}$ probe. Therefore, rearrangement of $Km^{r}$ gene and plasmids by conjugation was found to be afTected diversely by cellular characteristics as well as by environmental factors.

• Microbiology and Biotechnology Letters
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• v.20 no.4
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• pp.483-490
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• 1992

In order to understand the transfer behavior of a particular gene in water environments, kanamycin resistance ($Km^r$) gene was tracked by Southern hybridization with DNA probe in its conjugal transfer. A $Km^r$ natural bacterial isolate and genetically modified microorganisms (GMMs) constructed from the isolate were used as donor for conjugal transfer of the $Km^r$ gene. The transfer frequencies of the $Km^r$ gene from GMM strains were generally 10 to 100 times higher than those from the natural isolate. The conjugants obtained from GMM strains in LB broth had more plasmids newly appeared, and particularly the conjugants in A Wand FW waters revealed more rearrangement in their plasmids as a function of conjugation time. When plasmids of the conjugants obtained in LB broth were Southern hybridized with DNA probe of the $Km^r$ gene, the $Km^r$ plasmids in the conjugants were detected at the same position of the plasmids in donor cells, in spite of the fact that the plasmids were highly rearranged in conjugant cells. But the $Km^r$ plasmids in the donor of DKI and DKC601, and DKC600 were not identified in the conjugants obtained after 50 h conjugation in AW and after 30 h in AW, respectively. The size of the $Km^r$ plasmids showing hybridization signal were a little changed in the conjugants obtained in A Wand FW waters. Therefore, the method of Southern hybridization with DNA probe was proved to be very specific and useful for tracking of particular genes in water environments.

• Korean Journal of Microbiology
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• v.30 no.4
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• pp.322-331
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• 1992

In order to understand the transfer and behavior of R gene in water environments. the Kmr gene in the genetically modified microorganisms(GMMs) w,is studied by conjugation. The plasmid variously rearranged in the conjugants were comparatively analyzied by agarosc gel electrophoresis and the specific Km' genes in the gel were tletected with DNA probe. The Kmr genes of the GMM strains(DKC600 and DKC601) were transferred at higher rate than those of natural isola~e(DKI)b, ut the ratc was a little diflurent depending upon the recipient strains. Rearrangement of the plasmids appeared morc drastic in GMM strains than in IIKI as donor. The transfer frequencies of the Km' genes in LR broth were remarkably higher than in the water of AW and FW without regards to the strains. In LA breth. the frequencies of Kmr genes were higher at 25'C-30$^{\circ}$C than at 10$^{\circ}$C and at pH - 7 than pH 9, but temperature and pH of the FW did n,,t affect to the frequency. And the conjugants from GMM strains in FW did not showed any plasmids. except tor 43 kb plasmiil. As results of Southern analysis of the plasmid, variously rearranged in eonjugant cells obtained in LB broth, the Kmr genes were detected at the same position of Km' plasrnids of the donor cell(DK1 and GMM strains). But Km' plasmid disappeared in the conjugants obtained in F'W and their chronlosomes showed strong signal of hybridization. The Kmr plasmid of DKl in the conjugants obtained in FW water was transferred and maintained its size, but the Kmr plasinids of the GMM strains were all integrated into chromosome. Therefore, the Kmr plasmids of DKI anit GMM strains in LH were intactly transferred and other plasmitls were variously rearranged. but Km' gene of DKC600 in FW water was integrated into the chromosorn: without regards to the temperature and pH of the water.