Transfer of Bacillus lentimorbus andAlmost complete 165 rRNA gene sequences were generated for the type strains of the obligate insect pathogens Bacillus lentimorbus and Bacillus popilliae and a second strain of Bacillus popilliae (NRRL B-4081) received as 'Bacillus popilliae var. melolonthae I . A phylogenetic tree was constructed which grouped these strains into a well defined subcluster within the genus Paenibacillus. Bacillus popilliae NRRL B-4081 occupied an intermediate position between the type strains of Bacillus lentimonbus and Bacillus popilliae but with a marked clustering to the latter. The phylogenetic assignment of these strains to Paenibacillus is in contrast to earlier studies which placed these bacteria in the genus Bacillus, close to Bacillus subtilis. Indeed, the rRNA sequences generated in this study share less than 88% similarity to the deposited sequences for Bacillus popilliae ATCC 14706T and Bacillus lentimorbus ATCC 14707T. The results obtained by using different tree algorithms, bootstrap analysis, branch lengths and verification by signature nucleotide analysis supported the reclassification of these species in the genus Paenibacillus as Paenibacillus /en timorbus comb. nov. and Paenibacillus popilliae comb. nov.

The origin of high-level vancomycin resistance in enterococci is unknown. Biopesticidal powders containing spores of Bacillus popilliae, which is vancomycin-resistant, have been used for >50 years in the United States for suppression of Japanese beetle populations. Using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci, an amplicon in B. popilliae was identified and sequenced. The putative ligase gene in B. popilliae had 76.8% and 68.4%-68.9% nucleotide identity to the sequences of the vanA and vanB genes, respectively. There was 75.3% and 69.3%-69.9% identity between the translation of the putative ligase gene in B. popilliae and the translation of the vanA and vanB genes, respectively. We have identified a gene resembling vanA and vanB in B. popilliae. The gene in B. popilliae may have been a precursor to or have had an ancestral gene in common with vancomycin resistance genes in enterococci.

Bacillus popilliae and Bacillus lentimorbus, causative agents of milky disease in Japanese beetle and related scarab larvae, have hitherto been differentiated based upon a small number of phenotypic characteristics, but they have not previously been examined at the molecular level. In this study 34 isolates of these bacteria were examined for DNA similarity and by random amplified polymorphic DNA (RAPD) analysis. Two distinct but related similarity groups were identified: the f i r s t contained strains of B. popilliae and the second contained strains of B. lentimorbus. Two strains distinct from but related t o B. popilliae may represent a subspecies. Some strains received as B. popilliae were found to be most closely related to B. lentimorbus and some received as B. lentimorbus were found to be most closely related t o B. popilliae. RAPD analysis confirmed the DNA similarity results. Paraspore formation, previously believed to be a characteristic unique to B. popilliae, was found to occur among a sub-group of B. lentimorbus strains. Growth in media supplemented with 2% NaCl was found to be a somewhat less reliable characteristic in distinguishing the species than vancomycin resistance, the latter being present only in B. popilliae.

A hydrogen peroxide‐resistant mutant of the catalase‐negative microaerophile, Spirillum volutans, constitutively expresses a 21·5 kDa protein that is undetectable and non‐inducible in the wild‐type cells. Part of the gene that encodes the protein was cloned using amino acid sequence data obtained by both mass spectrometry and NH2‐terminal sequencing. The deduced 158 amino acid polypeptide shows high relatedness to rubrerythrin and nigerythrin previously described in the anaerobes Clostridium perfringens and Desulfovibrio vulgaris. The protein also shows high similarity to putative rubrerythrin proteins found in the anaerobic archeons Archaeoglobus fulgidus, Methanococcus jannaschii and Methanobacterium thermoautotrophicum. This is the first report of this type of protein in an organism that must respire with oxygen. It seems likely that the novel combination of methodologies used in this study could be applied to the rapid cloning of other genes in bacteria for which no genomic library yet exists.

Bacillus sphaericus strains isolated on the basis of pathogenicity for mosquito larvae and strains isolated on the basis of a reaction with a B. sphaericus DNA homology group IIA 16s rRNA probe were analyzed for DNA similarity. All of the pathogens belonged to homology group IIA, but this group also contained nonpathogens. It appears inappropriate to designate this homology group a species based solely upon pathogenicity.Aerobic bacilli that form spherical endospores are common in soil and water and are usually classified as Bacillus sphaericus. There are few useful phenotypic tests for identification of these bacteria. Spore morphology combined with negative reactions in tests for fermentation products and extracellular enzymes have been the basis for taxonomic placement. The species was found to be comprised of at least five distinct homology groups, each sufficiently separated from the others to merit species status (5). Representative strains of the homology groups have also been examined by rRNA gene restriction fragment length polymorphisms analyses (ribotyping), and these analyses confirmed that there are distinct groups within the B. sphaericus complex (2). Recently, randomly amplified polymorphic DNA analysis has also clearly distinguished the groups originally identified by DNA similarity analysis (9). These five groups have not been designated separate species because of the lack of readily utilizable phenotypic tests to distinguish them.In the original study of Krych et al. (9, group I1 was divided into two subgroups based on levels of DNA similarity and DNA heteroduplex stability. It was of considerable interest that all of the isolates in group IIA were pathogenic for mosquito larvae. No mosquito pathogens were found in any other group. These bacteria are pathogenic because they produce one or more of four toxins, a binary toxin composed of two distinct proteins and three additional toxins designated Mtx, Mtx2,. Strains that produce the binary toxin are highly toxic (50% lethal concentrations, around lo2 to lo3 cells ml-'), and strains that produce only toxins Mtx, Mtx2, and Mtx3 have low toxicity (50% lethal concentrations, about lo5 to lo7 cells ml-'). It appeared that the group IIA mosquito pathogens might be designated a separate species. However, only seven pathogenic isolates were available at the time of the original DNA similarity study. Now, many more pathogenic isolates from many geographic locations are available, and although they have been referred to as group IIA strains on the basis of ribotyping data, DNA similarity studies have never actually been performed with them. In this paper we report DNA similarity results for a large number of strains from diverse geographic locations.Bacteria and DNA isolation. The strains of B. sphaericus used in this study are listed in Table 1. The bacteria were grown in NY broth (Difco nutrient broth supplemented with 0.05% yeast extract) at 30°C with shaking at 150 rpm. Cells were recovered by centrifugation, suspended in 20 ml of pH 8.0 buffer (10 ...