| Objective:Our previous study showed that there were 7 strains harboring pks island in 213 strains of Escherichia coli (E. coli.) which did not belong to intestinal pathogenic E. coli. And two new islands, Insert1 and Insert2, were found to appear in the pks island of 3 E. coli. strains containing the same molecular typing. The sizes of Insert1 and Insert2 were around 13 kb and 90 kb, respectively, evaluated by PCR, sequencing and Southern blot. The results of gene function prediction showed that Insert1 mainly coded proteins related to the synthesis, transport and regulation of yersiniabactin, while Insert2 might be associated with the regulation of the pentose phosphate pathway and drug resistance. In this study, in order to preliminary probe on the gene function of island, we studied the biochemical properties and cytotoxicity of the bacterium which was deleted the Inserts from using strain 81 containing Insert1 and Insert2.Methods:Two insert fragments (Insert1 and Insert2) in the pks island of strain 81 were deleted with one step deletion method-λRed homologous recombination system. Successful deletion was confirmed by PCR and sequencing. Bacterial Biochemistry Identification Instrument (Walk Away 40 SI, Siemens) was used to test the biochemical properties, sensitivity to antibiotics, growth rate and resistance to acid and alkali of the 81 wild strain and the mutant strain. Cytotoxicity experiments were done with Hela cells. Hela cells in 80% confluency were infected with bacteria for 2 hours in 100:1 (bacterium:cell) before they were treated with gentamycin (200μg/mL) for another 2, 3 or 5 hrs. Changes in cell number and morphology were observed under a microscope after Giemsa staining. The bacterial cytotoxicity was determined by measuring lactate dehydrogenase test (LDH) release after incubation with gentamycin for 8 hr.Statistical analysis was done with SPSS 14.0 software. Data was presented as mean±SD. Differences among various groups was evaluated by one way analysis of variance. And LSD was used to compare two means. A value of P≤0.05 was considered statistically significant.Results:1) Seventeen possible deletion mutants were screened by PCR. Among them, two strains were deletion mutants identified by sequencing. However, no mutant strain with Insert1 deletion was obtained.2) There were differences only in resistance to kanamycin but in biochemical identification, resistance to acid and alkali, resistance to antibiotics, and growth rate between strain 81 with Insert2 and strain 17 without Insert2. The results of sensitivity to antibiotics showed that strain 17, but not strain 81, was resistant to kanamycin. This indicated that the exogenous kanamycin- resistant gene was successfully integrated into bacterial chromosome through homologous recombination.3) Compared to strain 17, pre-infection of Hela cells with strain 81 induced a larger number of detached cells from culture flasks, larger swelled cell bodies and neucli.4) The results of LDH showed that the cell death rates of strain 17 ((13.2±9.5) %) was significantly lower than that of strain 81 ((25.1±6.2) %) (P<0.05). It suggested that bacterial cytotoxicity was reduced with the deletion of Insert2 from the bacterial genome.Conclusion:1) The new island, Insert2, was successfully deleted from the pks island of E. coli. by one step deletion.2) There was no difference in the biochemical properties no matter whether the Insert2 was deleted (strain 17) or not (strain 81). 3) The new island, Insert2, might contain unknown cytotoxin genes which were not predicted by gene function prediction. |
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