Novel variants of Bacillus thuringiensis were isolated from the phylloplane of deciduous and conifer trees as well as of other plants. These isolates displayed a range of toxicity towards Trichoplusia ni. Immunoblot and toxin protein analysis indicate that these strains included representatives of the three principal B. thuringiensis pathotypes active against larvae of the orders Lepidoptera, Diptera, and Coleoptera. We propose that B. thuringiensis be considered part of the common leaf microflora of many plants.

The entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus produces two types of intracellular inclusion bodies during in vitro culture. Large cigar-shaped inclusions (designated type 1) and smaller ovoid inclusions (designated type 2) were purified from cell lysates, using differential centrifugation in discontinuous glycerol gradients and isopycnic density gradient centrifugation in sodium diatrizoate. The inclusions, composed almost exclusively of protein, are readily soluble at high and low pH values and in the presence of cation chelators such as EDTA, anionic detergents (sodium dodecyl sulfate), or protein denaturants (urea, NaBr). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inclusions revealed a single 26-kilodalton protein (IP-1) in type 1 inclusions and a 22-kilodalton protein (IP-2) in type 2 inclusions. Analysis of these proteins by isoelectric focusing in the presence of 8 M urea showed that IP-1 is acidic and IP-2 is neutral. Furthermore, each protein occurred in multiple forms differing slightly in isoelectric point. Other variations in peptides released by trypsin digestion, immunological properties, and amino acid composition revealed significant structural differences between IP-1 and IP-2. Kinetic studies using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting procedures showed that inclusion protein synthesis occurs only during the second half of exponential culture growth. Synthesis of inclusion proteins and their aggregation to form inclusions occurred concurrently. Possible functions for these abundant proteins are discussed.Xenorhabdus species are entomopathogenic bacteria symbiotically associated with insect parasitic nematodes of the families Heterorhabditidae and Steinernematidae (1,3,30). The bacterial symbionts are asporogenous gram-negative rods, which are carried monoxenically in the intestine of nonfeeding infective-stage nematodes. On penetration of an insect host, the bacteria are released into the hemocoel. Colonization by the bacteria kills the host and establishes a suitable environment for reproduction of the nematodes by providing nutrients and inhibiting the growth of other microorganisms (1,24).When cultured in vitro, Xenorhabdus bacteria occur in two forms designated primary and secondary, which can be distinguished according to colony morphology and pigmentation on various bacteriological media, or on the basis of antibiotic production (1, 2). Both forms are pathogenic to insects, but only the primary form is isolated from infective nematodes (1). Xenorhabdus spp. will grow in a wide variety of artificial media which are rendered suitable for nematode reproduction, thus providing the basis for economical mass production of nematodes. Our nematophilus, in contrast to X. luminescens in which both primary and secondary forms produced inclusions (10). Intracellular inclusion bodies have also been described in several ultrastructural studies of Xenorhabdus spp. (4,7,15,18).In this repor...

The carbohydrate content of purified Bacillus

Entomopathogenic bacteria of the genus Xenorhabdus produce crystalline inclusion bodies during in vitro culture. When cultured in liquid media, inclusions were present in primary forms but not secondary forms of X. nematophilus. In contrast, both primary and secondary forms of X. luminescens produced inclusion bodies during liquid culture. Two morphologically distinct forms of inclusion bodies were found in X. nematophilus subsp. nematophilus strain All. They were proteinaceous, and one of the proteins (IP-1) was present in seven strains of X. nematophihrs subsp. nematophilus, but not in strains of X. nematophilus subsp. bovienii, X. nematophilus subsp. poinarii, X. nematophilus subsp. beddingii or X. luminescens. This protein may be useful as a taxonomic indicator. Plasmids were isolated from seven of ten strains of Xenorhabdus. They varied in size from 12 to 3.6 kb, and were present in both primary and secondary forms of X. nematophilus subsp. nematophilus and X. nematophilus subsp. bovienii.

The immunological relationships among the proteins of the mosquito larvicidal toxin produced by Bacillus thuringiensis subsp. israelensis have been investigated by using polyclonal antisera specific for the 28-, 70-, and 135-kilodalton proteins. Each of these proteins was immunologically distinct. There was no cross-reaction among the three proteins and the two non-homologous antisera. Treatment of toxin proteins with larval gut enzymes for 20 h identified protease-resistant domains at approximately 65, 38, and 22 kilodaltons. Similar domains were generated by treatment with trypsin and chymotrypsin. Our immunological and kinetic data indicate that the 28-kilodalton protein is degraded successively to protein bands at 26, 25, 23, and 22 kilodaltons, the 70-kilodalton protein is degraded to a protein at 38 kilodaltons, and the 135-kilodalton protein is degraded successively to protein bands at 94, 72, and, probably, 65 kilodaltons. Solubilized toxin possesses two biological activities, larvicidal and general cytolytic (hemolytic). We used nondenaturing gel electrophoresis to show that the hemolytic activity resides in the 28-kilodalton protein. However, higher-molecular-weight proteins are required to achieve the level of toxicity observed in intact toxin.