铜绿假单胞菌PA0861突变菌株表达谱检测分析

Analysis of the gene expression profile to reveal biological functions of the gene PA0861 from the PAO1 strain of Pseudomonas aeruginosa

目的分析铜绿假单胞菌野生型菌株PAO1及PA0861突变菌株基因表达谱,探讨铜绿假单胞菌中GGDEFEAL基因PA0861的生物学功能。方法以野生型铜绿假单胞菌PAO1及PA0861基因转座子插入失活的突变体菌株为材料,利用结晶紫染色方法进行细菌生物膜的表型分析;利用Microarray技术分析其基因表达谱的变化;采用半定量RT-PCR验证Microarray结果。结果 PA0861突变株生物膜的合成量在4、8、12h均高于PAO1(P<0.05),在12h时增加了近1倍;利用Microarray技术筛选出差异表达基因517个,其中319个上调,198个下调。生物分子功能注释和Pathway分类显示这些差异表达基因除涉及代谢过程中基本的物质变化和能量转换外,突变体PA0861中涉及铁载体生物合成基因的表达增高。半定量RT-PCR验结果一致。结论铜绿假单胞菌突株PA0861基因表达谱发生改变,其中铁载体生物合成基因高表达,提示c-di-GMP可能通过对铁载体的调控影响细菌生物膜的形成。

Objective To analyze the gene expression profile of the wild strain PAO1 and the mutant strain PA0861 of Pseudomonas aeruginosa to examine the biological functions of the GGDEF-EAL gene PA0861. Methods Transposons were inserted into inactivated PAO1 and PA0861 strains of P. aeruginosa. Biofilm formation was analyzed using crystal violet staining. Gene expression profiles were compared using a microarray. A semi-quantitative reverse transcription pol- ymerase chain reaction was used to verify the microarray results. Results Biofilm formation by the PA086t mutant strain was significantly （P^0.05） greater than that by the PAO1 wild-type strain at 4, 8, and 12 h and double at 12 h. Five hundred and seventeen genes that were differentially expressed were screened using a microarray. Of those genes, 319 were up regulated and 198 were down-regulated. A molecule annotation system and Pathway classification indicated that these differentially expressed genes were involved in basic material changes and energy conversion in the metabolic process. The PA0861 mutant strain had increased expression of genes involved in siderophore biosynthesis Semi-quantita- tive RT-PCR. Conclusion The gene expression profile of the PA0861 mutant strain of P. aeruginosa changed. The strain had increased expression of genes involved in siderophore biosynthesis, suggesting that c-di-GMP may affect the formation of bacterial biofilm through the regulation of siderophores.