A plate containing 96 mutants

was randomly selected from

A plate containing 96 mutants

was randomly selected from the mutant library and total DNA was extracted as described above. DNA samples were cleaved with the restriction enzyme Eco RI and separated in a 1% agarose gel in TBE buffer for 12 h at 35 V. At the end of this process, the gel was stained with ethidium bromide and the image was documented. The DNA was transferred from the gel to a Hybond N+ nylon membrane, following the manufacturer’s instructions www.selleckchem.com/products/blz945.html (Amersham Biosciences). Transposon Tn5 DNA (100 ng) was labeled using an AlkPhos Direct RPN 3680 labeling kit and probe signals were detected with a Gene Images CDP-Star RPN 3510 kit (Amersham Biosciences), according to the manufacturer’s instructions. The membrane was finally exposed to X-ray film, stored at room temperature for 1 h and developed using the GBX kit (Kodak). The film was analyzed under a white light transilluminator. Two independent hybridizations

were carried out to confirm results. The same mutants were independently multiplied and the process was fully repeated. Determination of Xanthomonas citri subsp. citri growth curves in planta Eight mutants with altered virulence (02H02, 03C01, 06H10, 11D09, 18C05, 18D06, 11D03, 10H02) and a wild-type strain (isolate 306) were chosen for determination of growth curves in planta. These mutants carry knock-out versions of ORFs XAC0410, XAC1266, see more XAC0789, XAC4040, XAC0340, XAC3673, XAC1201 and XAC0095, respectively, created by transposon aminophylline insertion. Mutant and wild-type strains were multiplied in TSA culture medium as above described. After growth, an aliquot of each was transferred to 1.5 mL microcentrifuge tubes containing

1 mL of sterile distilled water. After complete dissolution of the cell pellet, the concentration was adjusted to an OD of 0.1 at 600 nm then diluted to OD 0.01 (approximately 104 CFU/mL). Using a syringe, an orange leaf was infiltrated with each bacterial suspension. Quantitative analyses were performed 0, 2, 4, 6, 8 and 10 days after inoculation. The number of cells per leaf area was measured in three disks of 1 cm2 from each inoculated leaf. With a pestle, leaf disks were ground in 1 mL of double-distilled sterile water. Serial dilutions of 10-1 to 10-7 were prepared and 10 μL of each dilution was used to inoculate TSA culture medium containing kanamycin (except for the wild type) using a microculture technique [54]. Plates were kept at 28°C for 2 days, and isolated colonies (cells) were counted. The experiment was repeated independently three times. Gene expression analysis detected through nucleic acid hybridization using cDNA probes Bacterial cells were grown in a plate for 72 h under the above conditions. To obtain RNA from cells growing in the culture media, suspension of Xcc 306 cells was adjusted for OD 0.3 at 600 nm, and 1 mL was inoculated in 50 mL liquid NA medium, then inoculated for 48 h in a shaker (200 rpm) at 28°C.

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