The Effects Of PA0861 Gene In Pseudomonas Aeruginosa PAO1

Subjective: To investigate the biological function of PA01 Pseudomonas aeruginosa GGDEF-EAL domain protein PA0861, then to analyze the relationship c-di-GMP and absorption of iron in order to have deep insight into the related gene of chronic disease.Methods: Getting transposons PA0861 mutant strains and wild-type strain PAO1 from Manoil. Firstly, we conduct the polymerase chain reaction(Polymerase Chain Reaction PCR) method to verify the mutant site of gene PA0861.Then the changes of biofilm formation were performed by phenotypic analysis. Subsequently, PA0861 mutant strains and wild-type strain PAO1 were performed as micro sequence(Microarray) analysis to obtain gene expression differeces of PAO1 and PA0861. Microarray data analysis is the first to get through Capitalbio Company GO gene expression data, mutant PA0861 and wild-type PAO1 were performed by comparative analysis of the different gene expression, and then apply KEGG and DAVID to analyze its role in the channel. We performed semi-quantitative reverse transcription polymerase chain reaction to verify differences of PA0861 mutant strains and wild-type PAO1 by the molecular level in the regulation of siderophore PAO1. In addition, we conducted to verify the differences between the two strains at the gene expression level by thin layer chromatography and spectrophotometer. Thereby, we can know that how it affects the absorption of iron and the regulation of c-di-GMP to adjust Pseudomonas aeruginosa biological processes.Results:1. PCR and agarose gel electrophoresis results showed that the template and primer of A2 and C2 is PA0861 and Lac Z148, it is confirmed that the mutant strain PA0861,PA0861 gene has been inactivated.2. PA0861 mutant strains and wild-type phenotype PAO1 were performed comparative analysis by crystal violet staining biofilm quantitative experiments, two strains were grown at4,8,12 hours in LB liquid medium, mutant PA0861 formed more biofilms at each time point than the wild-type PAO1, there was statistically significant(P<0.05).3. We used the expression profile of Pseudomonas aeruginosa to perform Microarray analysis, showed that the mutant PA0861 and wild type PAO1 2782 transcripts were changed after filtration analysis, in which there are approximately 2,400 genes were upregulated and 382 genes were down at least twice fold. Because upregulated genes were much more than down-regulated genes, we corrected this standard increasing up to 6-fold and lowering to 2.5-fold, and false discovery rate FDR was less than 5%, to test the validity of Wilcoxon Rank-Sum was at 0.05(P<0.05). As a result, compared with wild type PAO1, the mutant PA0861 was 319 up-regulated genes and 198 down-regulated genes.(1) Comprehensive understanding genes of expression difference in the mutant PA0861 and wild type PAO1 by GO analysis, it was found that 517 changing transcripts in the regulation of biological function in both groups, there are 215 with significant difference, accounting for 42% of biological processes regulating Pseudomonas aeruginosa.(2) When we use WEGO to analyze biological function of mutant PA0861, found that there are 26 genes about "pigmention". Which was consistent with microarray analysis.(3) We analyze the channel with KEGG, in the case of P<0.05, in addition to involving nucleic acid synthesis, cell cycle, energy production, etc., the most important is that five genes regarding siderophore biosynthesis in the mutant PA086 are also activated.4. P. aeruginosa has two main siderophore including Pseudomonas hormone(Pyoverdine) and Pseudomonas aeruginosa chelating Ferritin(Pyochelin). We used the KEGG channel analysis, we found a message that there was five gene concerning siderophore biosynthesis were activated in the mutant PA0861. The results of such analysis are consistent with the microarray results. For the regulation of gene Pyoverdine generated genes situate on PAO1 genome, there are about 120 kb from gene A2383 to PA2452, we selected 11 genes from 70 gene regulating Pyoverdine, representing up-regulation of two-fold to 19-fold, and then useGAPDH as internal control and conducted the analysis by RT-PCR, the gene expression of PA2392, PA2394, PA2395, PA2398, PA2403, PA2443, Pvd S increase, but PA2388 and PA2418 is down, while there is no statistical significance of PA2444 between two types in 6 and 18 hours. There is only 11 genes concerning regulation for Pyochelin. In mutant PA0861, all genes are upregulated between 3.86-14 folds. Overall most genes is up-regulated, and only a few number of a reduction or no change, which are consistent with our analysis of the gene chip.5. We conducted semi-quantitative comparative analysis for Pyochelin by TLC method, and used salicylic acid as a standard, in addition, served Roc R gene of PDR activity and Wsp R gene of DGC activity as controls, the results showed that mutant PA0861 generated more Pyochelin than in that of PA01, which was similar to ROCR synthesis. 6.Pyoverdine of PA0861 mutant strains and the wild type PAO1 was determined by quantitative analysis on a spectrophotometer, the content of Pyoverdine in mutant PA0861 was higher that of PAO1 in 6 hours and 18 hours and were statistically significant(P<0.05), suggesting that the genes regulating c-di-GMP metabolism genes can regulate siderophore biosynthesis. Thus, it can also affect the biological function of Pseudomonas aeruginosa.ConclusionIn a recent study, We have already known that PA0861 gene is c-di-GMP regulatory factors, and influence biofilm formation. In order to get through PA0861 gene molecular mechanisms to regulate iron absorption, thus to control pseudomonas aeruginosa associated biological functions. We have found that its absence induce control gene product including siderophore Pyoverdine and Pyochelin have been upregulated by analyzing the level of transcription, suggesting that PA0861 have hindered the formation of Pseudomonas aeruginosa siderophore, which is the antagonism to form biofilms, which is consistent with previous analysis of our biological phenotype. So, the conclusion is that genes regulate Pseudomonas aeruginosa PA0861 absorption of iron, providing more inspiration for thebiological function of c-di-GMP genes in Pseudomonas aeruginosa.