Rapid Identification of E. coli Bacteriophages using Mass Spectrometry

Author(s): Serafim V*, Ring C, Pantoja L, Shah H, Shah A

 The current increasing interest within the application of mass spectrometry, especially MALDI-TOF MS, to identification of bacteria and fungi involves a requirement to utilise this technology for identification of other infectious agents like viruses. The aim of this study was to develop a rapid and reliable mass spectrometrybased proteomic method for identification of Escherichia coli phages. The approach was supported rapid insolution tryptic digestion of suspensions of plaque-purified bacteriophage followed by mass spectral analysis. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography – tandem mass spectrometry (LC-MS) were wont to analyse the tryptic digests. Processing of tandem mass spectrometry data and interpretation of results were achieved using Mascot software and therefore the Swiss-Prot database. Five bacteriophage species (Enterobacteria phages P2, T4, T5, T7 and Lambda) isolated in E. coli cultures were identified. The viral proteins were identified from a posh mixture of host bacterial proteins. additionally , employing a single ion monitoring method, a Lambda prophage derived protein was also identified.The data obtained demonstrate that LC-MS/MS are often used for accurate identification of E.coli- specific bacteriophages in both lytic and lysogenic cycles. Pepper (Capsicum annuum L.) is an economically important vegetable and spice crop. In our laboratory we've established a regeneration and transformation protocol for the sweet red pepper type 'Florinis' and for 2 pepper hybrids PO1 and C using hypocotyl explants. the speed of plant regeneration was found to depend upon the kinds of explants cultured and therefore the media used. In our regeneration protocol shoot bud initiation is simpler on MS media supplemented with IAA and BAP and shoot bud development is promoted with addition of GA 3. Rooted shoots are successfully established in soil. so as to realize the transformation of pepper we applied two different methods, using Agrobacterium and therefore the particle gun. Following the primary method fertile transgenic pepper plants were regenerated from hypocotyl explants that were co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a plasmid that contains the gus reporter gene and therefore the nptII selection gene or a plasmid with the Cu/Zn SOD gene of tomato, that's expressed in chloroplasts. Transgenic pepper plants were developed, verified and characterized but the share of transformed plants obtained using Agrobacterium is quite small which is why we've applied as alternative the biolistics method. consistent with the tactic pepper hypocotyls as explants were bombarded by the hand gene gun of Bio-Rad.

PDF