Research design and methodology Essay

Despite the fact that the complete genome of the organism was already sequenced, the specific genes coding for the needed enzymes to form pores in the host cell were still unidentified. With this lack of information, this study is formulated and designed. Culturing of B. bacteriovorus HD100 on prey dependent and prey independent set-ups: Predatory (HD) cultures of B. bacteriovorus HD100 will be grown on E. coli in Ca2_-HEPES buffer at 30Â °C, with shaking at 200 rpm (8). Escherichia coli ML35 and E. coli W7-M5 (10) will be used as the prey throughout the experiments. Escherichia coli ML35 will be cultured in nutrient broth (Difco Laboratories), and E. coli W7-M5, a lysine and DAP auxotroph, will be cultured in nutrient broth supplemented with 0. 2 mM lysine and 0. 1 mM DAP at 37Â °C with shaking at 200 rpm. Prey-independent HI strains will be plated on rich peptone-yeast extract (PY) medium (8). Synchronous cultures: Synchronous cultures will be used for performing various experiments as described below. Briefly, fresh bdellovibrios will be added to prey cells in HM buffer (3 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-1 mM CaCl. LQ. One mM of MgCl2 will be adjusted to pH 7. 6 using NaOH (10). The organisms will be grown until a final concentration of 1010 bdellovibrios per ml and 5 x 109 E. coli per ml is reached. For proper aeration, volumes will be kept to ? 20% of the flask’s volume and incubated at 30Â °C with shaking at 400 rpm. Synchronous cultures will be examined at intervals for attachment and penetration with a Nikon model L-Ke microscope (Nippon Kogaku Inc. ) equipped with phase-contrast optics and a Nikon model AF camera. Time course Microarray analysis. Time course Microarray analysis will be performed to identify the genes to be expressed during the entry phase, specifically during pore formation on the host cell membrane of B. bacterovorus H100. Microarray slides of B. bacteriovorus H100 will be ordered from Advanced Throughput, Inc Services. Total cellular RNA will be extracted from B. bacteriovorus H100 cells at entry phase using the RNeasy mid kit (Qiagen). The RNA of the organism will also be extracted during the other stages of infection. This will serve as a reference for comparison of the genes expressed and not expressed at the desired stage. Complementary DNA synthesis, fragmentation, labeling, hybridization, staining and washing will be performed according to the Affymetrix B. bacteriovorus H100 GeneChip array expression analysis protocol (Affymetrix). Briefly, cDNA will be synthesized from RNA using Superscript II (Invitrogen) according to the manufacturer’s instructions. RNA will be removed by alkaline treatment and subsequent neutralization. Complementary DNA will be purified with QIAquick PCR purification columns (Qiagen). Purified cDNA will be fragmented by DNase I (Amersham) at 37Â °C for 10 min followed by end labeling with biotinddUTP, using an Enzo BioArray terminal labeling kit (Affymetrix), at 37Â °C for 60 min. Hybridization will be performed in an Affymetrix GeneChip hybridization Oven 640. Washing and staining will be performed using an Affymetrix Fluidics Station 400. Arrays will be scanned with an Agilent GeneArray Scanner G2500A. GeneChip scans will be initially analyzed using the Affymetrix Microarray Suite 5. 1 software, from which PivotData tables will be exported. Raw data from the PivotData Tables will be analyzed in GeneSpring software version 6 (Silicon Genetics), using the parameters suggested by Silicon Genetics for analysis of Affymetrix Microarrays. Real-time PCR: Real-time PCR using the Applied Biosystems 7500 Real-time PCR system will be performed to confirm microarray results. RNA will be extracted from B. bacteriovorus H100 at initial phases of predatory life cycle up to entry phase as described above. RNA will be reverse transcribed into cDNA and simultaneously labelled using the iScript One-step RT-PCR kit with SYBR Green (Biorad). RT-PCR reactions will also be performed to amplify cDNA of housekeeping genes (identified from micro array studies) for normalization of fluorescence values. Identifying the specific hydrolytic enzymes of B. bacteriovorus which are involved in pore formation on host cell membrane. Many experiments showed that B. bacteriovorus H100 releases hydrolytic enzymes during predatory life cycle. According to Thomashow and Ritterberg, glycanases and lipopolysaccharideases are required for pore formation in the prey’s peptidoglycan and LPS layers respectively. The glycanase and/or peptidase could be responsible for weakening the peptidoglycan layer of the prey and thereby responsible for permitting conversion of the substrate cell to a spherical shape (10). Tudor et al. proposed another model for penetration. According to them peptidase is responsible for pore formation but not glycanase (11). Specific enzymes involved in pore formation are not known. The genes identified from the time course micro array technique will be mutated as described previously using suicide vector pSSK10. Resulting mutants will be complemented by using vector pMMB206 (8). Mutants will be analysed for the specific enzymes (using 2D-gel electrophoresis) and their actions on host cell i. e, as a glycanase, LPSase or peptidase will be observed by radio labelling experiments (10). Wild-type B. bacteriovorus H100 and complemented strains will be used as controls. Radio labeling experiments: Escherichia. coli W7-M5, auxotroph for lysine and DAP and cannot metabolize glucosamine, will be radiolabelled as described previously (9,10). Peptide portion of E.coli W7-M5 peptidoglycan will be labelled with [3H] DAP and the lipopolysaccharides and glycan portions of the peptidoglycan will be labeled with [3H]glucosamine. Various mutants and wild-type strains will be tested for predation using this radiolabelled strain. Solubilisation of glucosamine and DAP from labelled prey peptidoglycan will be measured as described previously (11). Briefly, samples taken at intervals will be precipitated with an equal volume of cold 10% trichloroacetic acid for 30 min followed by centrifugation. Resulting supernatants will be assayed for soluble radioactivity in a scintillation counter (Rackbeta II). Two-dimensional gel electrophoresis: The hydrolytic enzymes released by B. bacteriovorus H100 during its predatory life cycle will be analyzed by performing two-dimensional gel electrophoresis. Sample preparation for 2D-gel electrophoresis: Escherichia coli ML35 cells will be challenged with B. bacteriovorus H100 wild-type as well as the mutant strain. Culture fluid will be drawn from synchronous cultures during attachment and entry phases of B. bacteriovorus H100. Culture fluid will be centrifuged to discard any cell debris. Proteins in the supernatant will be precipitated using cold acetone. The precipitated proteins will be separated by centrifugation. The precipitated pellet will be air dried and will be dissolved in rehydration solution (8M urea, 2% CHAPS {3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 18 mM DTT, 0. 5% IPG buffer pH range 4-7; Amersham Biosciences), plus a trace of bromophenol blue. Sample protein concentrations will be determined using the BCA protein assay (Pierce). Resulting protein pellet will be subjected to 2D-gel electrophoresis.