| Phosphatidylcholine (PC), a component of the membrane phospholipids, forms bacterial membrane system with other phospholipids, membrane proteins and other ingredients together, maintaining physiological metabolism of bacterial cells and exercising specific biological functions. Variation of PC content in the membrane phospholipids can result in certain change of the physiological and biochemical characteristics of bacterial membrane, which directly or indirectly affects biological function of whole cell. In this study, we focused on studying whether PC affects the secretion of the harpin HrpZ secreted specifically by the type Ⅲ secretion system, and the major research results are presented as follows:1. Identification of the open reading frame (ORF) of Pseudomonas.sp593pcs gene. Soil bacterium Pseudomonas.sp593isolated previously in our laboratory, and its pcs gene was cloned. However, the cloned pcs gene contains two Orbs. The small ORF (696) has been found to be located in the large ORF (897bp), and the initial code of the small ORF (pcs2) is the67th code of the large ORF (pcsl) and the stop codon is the same. To understand which ORF really encodes phosphatidylcholine synthase (Pcs), two Orbs were respectively inserted into the expression vector pET23a and the cloning vector pMD-T, and then separately transformed into E. coli BL21(DE3) plysS. Positive transformants were incubated in LB plus0.5%choline. Thin layer chromatography (TLC) showed (6) both pET23a//pcs1and pET23a/pcs2transformants were able to synthesize PC, and (7) only pMD-T/pcsl transformants synthesized PC but pMD-T/pcs2transformant did not. It is understood that two Orbs inserted into pET23a were able to express Pcs because a strong promoter in the pET23a vector can initiate transcription of downstream DNA sequence. The cloning vector pMD-T has no promoter for the transcription of DNA insert.2. Impact of PC on incretion of HrpZ. At first, the gene hrpZ, cloned in previous study from Pseudomonas syringae pv. syringae Van Hall, was inserted into pET23a vector and transformed into E. coli BL21(DE3) plysS. The expressed product of the hrpZ gene was purified by using Ni-affinity chromatography, and then injected into New Zealand rabbits to produce anti-HrpZ rabbit polyclonal antiserum. Meanwhile, the recombinant vector pUC19-ptac-hrpz-Tetr constructed in this study was transformed into Pseudomonas.sp593 and Pseudomonas.sp593pcs-strains using pulse-electrical DNA transformation technique to get two transformants, Pseudomonas.sp593pcs-/pUC19-ptac-hrpz-Tetr and Pseudomonas.sp593/pUC19-ptac-hrpz-Tetr. Both transformants were cultivated in the secretion medium, and then the extracellular and intracellular cytoplasmic proteins were prepared. The HrpZ protein band in the cytoplasmic proteins of both two transformants was detected by Western blotting. Band intensity for both cytoplasmic proteins was almost identical. This result indicates that PC has not affect HrpZ expression inside bacterial cells. In contrast, the HrpZ band was only detected in the extracellular protein of Pseudomonas.sp593wild type transformant, other than pcs-mutant transformant. This result shows that PC in bacterial membrane is essential for HrpZ secretion.3. Pseudomonas.sp593/pUC19-ptac-hrpz-Tetr induces hypersensitive reaction (HR) of tobacco and soybean leaves. Pseudomonas.sp593lacks hrpz gene. The wild type strain does not infect tobacco or soybeans, and also does not induce any hypersensitive reaction. The wild type transformant Pseudomonas.sp593/pUC19-ptac-hrpz-Tetr and the pcs-mutant transformant were inoculated into tobacco and soybean leaves. The wild type transformant induced HR plots observed obviously in both tobacco and soybeans leaves postinnoculation3days. However, the pcs-mutant transformant did not elicit any visible HR. Bacterial number in inoculation area was count. Bacterial number for the wild type transformant showed2.5fold more than that for the pcs-mutant transformant. Those results demonstrate that:(1) PC indeed affects the secretion of HrpZ;(2) Introduction of single hrpZ gene into virulent soil bacterial strain effectively changes the phenotype of Pseudomonas.sp593from HR-to HR+... |
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