Research On Antibiotics Resistance-related Gene Of Xenorhabdus Nematophila Var. Pekingensis

Xenorhabdus nematophila var. pekingensis is a bacterium symbiotically associated with Steinernema carpocapose, an insect pathogenic nematodes which was firstly collected from soil in suburb of Beijing, China. Previous reports in our experiment have revealed that high insecticidal, antimicrobial, and anti-tumour activities are conferred by the metabolites of this strain, among which a variety of antibiotics-active substances show attractive prospect for development and application. So it is important to do some researches concerning its antibiotics through approaches of biology, genetics and molecular biology. This paper was mainly focused on the effects of different plasmid transformation techniques on the physiological and biochemistry properties of this bacterium, and cloning, functional identification and localization of its antibiotics- related gene fragment. The major results were as follows:1 The antibiotics resistance of X. nematophila var. pekingensis was analyzed. The results indicated that the strain was sensitive to chloramphenicol, neomycin and kanamycin, while possessed various degrees of resistance against streptomycin, penicillin, and ampicillin. The result was laid the foundation for the plasmid selection in following transformation techniques and the basis of molecular biological research on antibiotics- related gene from this strain.2 With several transformation approaches to prepare competent cells of Form I of X. nematophila var. pekingensis, the effects of these gene manipulation methods on physiological and biochemistry properties of the strain were investigated systematically. It was proved that various methods caused different effects on bacterial metabolism, and conjugation was the most appropriate approach for genetic manipulation. Contrarily, after electroporation, the strain became resistant to kanamycin, the color of colony on NBTA dark red, the V-P, SAC, GLU, and SOR metabolism on API strip were changed, the pattern of growth curve was influenced, and inhibitory activity against Bacillus subtilis was decreased. And the influence of CaCl₂-mediated chemical transformation was similar to electroporation, especially for some properties such as kanamycin resistance, dye absorption, inhibitory activity. Besides, the catalase activity was altered from negative to positive reaction. Comparatively, there is no observable change between the metabolism pathways of cell undergoing conjugation procedure and those of wild type strain.3 Through three-parent-mating of conjugation procedure, plasmids pSZ21 and pLA2917 were transferred into Form I cell of X. nematophila var. pekingensis, respectively. However, the introduction of exogenous plasmid affected some metabolism pathways and straits greatly. Compared with wild type strain, both pSZ21 and pLA2917 transconjugatants were altered in some aspects: the color of colonies was dark red on NBTA medium, the growth rate promoted rapidly, some physiological and biochemistry properties affected accordingly, and the antibiotics- producing ability decreased sharply.4 An antibiotics-related gene xbl fragment was firstly cloned from X. nematophila var. pekingensis with the accession number of DQ631811. It was showed that 15-731 base of xbl gene fragment is highly homologous to the counterpart sequence of X. nematophila ATCC19061 (99% identity). Also the...