FEMS Microbiology Letters 12 (1981) 327-331 327 Published by Elsevier Biomedical Press Poly-/3-hydroxybutyrate parasporal bodies in Bacillus thuringiensis Kenneth W. Nickerson *, William J. Zarnick and Vance C. Kramer School of Life Sciences, Universi(v of Nebraska, Lincoln, NE 68588, U.S.A. Received and accepted 14 August 1981 1. INTRODUCTION 2. MATERIALS AND METHODS The insecticidal bacterium Bacillus thuringiensis produces an intracellular proteinaceous crystal concomitant with sporulation that is toxic upon ingestion to the larvae of Lepidoptera insects. The entomocidal activity resides primarily in this parasporal crystal and extensive studies have been devoted to characterizing the crystal's protein and/or glycoprotein subunits [1-3]. However, de- spite the widespread use of B. thuringiensis as a commercial insecticide, we still have very little information on fundamental questions such as why B. thuringiensis forms a crystal in the first place. What physical or enzymatic factors are responsible for crystal formation? To this end, we thought it worthwhile to examine any other parasporal com- ponents produced by B. thuringiensis. In particu- lar, density gradient centrifugation in either re- nografin [4] or NaBr [5] results in the separation of some phase-dark granules at a density significantly less than that exhibited by the proteinaceous crystals. These small granules have been termed black dots [6]. The present paper reports the chem- ical composition of the black dots and discusses their influence on certain structural features [7,8] of the B. thuringiensis crystal. * Author to whom inquiries and reprint requests should be addressed. B. thuringiensis var. HD-1 [9] was grown on a glucose-yeast extract-salts medium [10] at 22°C on a New Brunswick Scientific G-52 gyrotory shaker set at 200 rev./min. Following sporulation the cells were allowed to autolyse and the crystals and black dots were purified by zonal gradient centri- fugation employing NaBr gradients [5]. The crystals were collected at 30-32% NaBr and the black dots at 23-25% NaBr; both preparations were then dialysed extensively (4 × 1 liter of dis- tilled water). Prior to chemical analysis, the black dots were repurified on NaBr, dialysed, and lyophilized. The quantitative elemental analysis of the black dots was performed by Galbraith Laboratories, Inc., P.O. Box 4187, Knoxville, TN 37921, U.S.A. 3. RESULTS 3.1. Scanning electron microscopy Fractionation of fully sporulated cultures of B. thuringiensis by density gradient centrifugation in NaBr [5] produced three separate bands: (i) spores, (ii), crystals,and (iii) black dots. Phase-contrast microscopy of this latter fraction revealed phase- dark granules considerably smaller than the crystals and spores. In order to better assess the dimensions of the black dots, we examined their morphology via scanning electron microscopy 0378-1097/81/0000-0000/$02.75 ~(: 1981 Federation of European Microbiological Socictics Downloaded from https://academic.oup.com/femsle/article-abstract/12/4/327/571625 by guest on 16 June 2020