Evaluation of Two-Dimensional Polyacrylamide Gel Electrophoresis of Acidic Proteins of Ribosome Preparations for Identifying Plant Pathogenic Soft-Rotting Bacteria

Moline, H. E., Johnson, K. S., and Anderson, J. D. 1983. Evaluation of two-dimensional polyacrylamide gel electrophoresis of acidic proteins of ribosome preparations for identifying plant pathogenic soft-rotting bacteria. Phytopathology 73:224-227. Two-dimensional polyacrylamide gel electrophoretic separation of proteins ribosomal-enriched protein of Erwinia spp. were in those of the acidic was successfully used for identifying cultures of plant pathogenic bacteria, proteins in the 20-50 Kdalton molecular weight range. Although several Profiles of soluble proteins were very complex; however, profiles of protein clusters were similar on all strains of E. carotovora and E. ribosomal protein-enriched fractions contained only 5-10 major proteins atroseptica examined, significantly different clusters were observed and could be used to distinguish strains of Erwinia carotovora, E. between these strains and those of E. chrysanthemi. atroseptica, and E. chrysanthemi. Most significant differences in profiles of The soft-rotting bacteria are a heterogeneous group of related JA 14 rotor in a refrigerated Beckman J-21 C preparative centrifuge genera (9) that include the Erwinia species. Numerous investigators at 0 C. The pelleted cells were washed in 0. 15 M NaCl, centrifuged have attempted to improve the taxonomic characterization of these as above, and resuspended in 10 ml of the following extraction genera (7,9,12); however, a good deal of disagreement remains as to medium: 0.5 M RNAse-free sucrose (Bethesda Research their status. A simple method of distinguishing these soft-rot Laboratories [BRL]), 5 mM tris, 5 mM MgCI2, 6 mM 2bacteria based on separation of cellular proteins would be of great mercaptoethanol, and 5% ribonuclease inhibitor (BRL). All help in characterizing strains of Erwinia. extractions were carried out on ice at 0 C. Cells were disrupted by The O'Farrell (15) method for obtaining high-resolution, twosonication with a Sorvall sonicator for 3 X 30 sec at setting 5. dimensional (2-D) electrophoretic separation of soluble proteins in Cellular debris was removed by centrifuging at 10,000 g for 30 min. polyacrylamide gels and staining them with silver (14) is potentially Desoxycholic acid was added to the supernatant at a final valuable for identifying bacteria isolated from diseased plant concentration of 0.5% to solubilize membrane fragments. tissues. A single 2-D profile, which may reveal as many as 100 Ribosomes were pelleted by centrifuging at 100,000 g for 90 min silver-stained proteins, may be as effective as the series of 50 or with a model 40 rotor in a model L Spinco ultracentrifuge at 0 C. more conventional biochemical tests currently needed for Pelleted ribosome-enriched fractions were resuspended in the identifying these bacteria (3,6,12). However, these profiles of extraction media and clarified by centrifugation at 10,000 g for 30 soluble proteins are quite complex, whereas profiles of the acidic min. The ultraviolet absorption spectrum (260-235 nm ratio) of a ribosomal-enriched proteins contain only a few proteins and are portion of the ribosome-enriched fraction was determined from a much easier to analyze. Since ribosomal proteins are structural sample resuspended in lysis buffer (in which Cleland's reagent proteins they do not vary with the environment as may be the case replaced 2-mercaptoethanol as the antioxidant) using a Beckman with other soluble proteins (11). The purpose of this study was to model 35 spectrophotometer. compare the ribosomal-enriched protein profiles of a number of Extraction and solubilization of protein sample. Samples of soft-rotting Erwinia strains (Table 1) using the O'Farrell (15) 2-D electrophoretic technique. TABLE 1. List of Erwinia spp. and strains included in this study of biochemical taxonomy MATERIALS AND METHODS biochemicaltaxonomy Erwinia spp. Growth of cells and extraction of ribosomes. Ribosomes were and strains' Host of origin Source extracted from bacterial cells according to the procedures of E. carotovora Tissieres et al (20) and Schaad (17) with the following C7 Iris rhizome W. L. Smith, Jr. Collection, modifications. A 24to 48-hr-old bacterial liquid culture was used Beltsville, MD to seed 150 ml of sterile Difco nutrient broth in 250-ml Erylenmeyer C7s Iris rhizome flasks. Suspensions seeded with E. carotovora and E. atroseptica E31 Iris leaf were shaken at 20 C for 18-24 hr and those seeded with E. E. atroseptica chrysanthemi were shaken at 37 C for 24 hr (Table 1). E18 Potato tuber W. L. Smith, Jr. Collection, Cells were harvested by centrifugation at 10,000 g for 15 min in a Beltsville, MD E25 Potato stalk The publication costs of this article were defrayed in part by page charge payment. This E. chrysanthemi article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. § M 80-I Sweet potato root Salisbury, MD; H. E. Moline, 1734 solely to indicate this fact. 1980 This article is in the public domain and not copyrightable. It may be freely AN7 Sweet potato root Experiment, GA; reprinted with customary crediting of the source. The American N.W. Schaad Phytopathological Society, 1983. a As identified by Burkholder and Smith, 1949.