Messenger ribonucleic acid content of Bacillus amyloliquefaciens throughout its growth cycle compared with Bacillus subtilis 168

The messenger RNA contents of Bacillus amyloliquefaciens and B. subtilis 168, grown in a 1% maltose-0.5% casein hydrolysate complex medium, were determined throughout their growth cycles by a hybridization technique. In both cases there was a level equal to about 3% of the total cellular RNA during the exponential phase. In B. subtilis this level was maintained into the stationary phase. By contrast, in B. amyloliquefaciens the proportion of messenger RNA increased after the end of exponential growth levelling off in the stationary phase at a value twice that observed in exponential growth. The total messenger RNA in each organism was resolved into two components, that involved in the formation of cell proteins and that concerned in extracellular protein production, by determining the relative rates of incorporation of l-[¹⁴C]valine into the two protein fractions. In both cases the cell protein component was the same and remained a relatively constant proportion of the total cellular material throughout the growth cycles. The exoprotein mRNA paralleled exoprotein secretion in each species, remaining at a constant low level in B. subtilis and undergoing a tenfold increase after the end of exponential growth in B. amyloliquefaciens. Applying a serial hybridization procedure to B. amyloliquefaciens, no evidence was obtained for the accumulation of a specific component of the messenger RNA in the exponential or post-exponential phase of growth, which was not detected by hybridization.     

Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains

Bacillus thuringiensis and Bacillus cereus belong to the B. cereus species group. The two species share substantial chromosomal similarity and differ mostly in their plasmid content. The phylogenetic relationship between these species remains a matter of debate. There is genetic exchange both within and between these species, and current evidence indicates that insects are a particularly suitable environment for the growth of and genetic exchange between these species. We investigated the conjugation efficiency of B. thuringiensis var. kurstaki KT0 (pHT73-Em^R) as a donor and a B. thuringiensis and several B. cereus strains as recipients; we used one-recipient and two-recipient conjugal transfer systems in vitro (broth and filter) and in Bombyx mori larvae, and assessed multiplication following conjugation between Bacillus strains. The B. thuringiensis KT0 strain did not show preference for genetic exchange with the B. thuringiensis recipient strain over that with the B. cereus recipient strains. However, B. thuringiensis strains germinated and multiplied more efficiently than B. cereus strains in insect larvae and only B. thuringiensis maintained complete spore germination for at least 24 h in B. mori larvae. These findings show that there is no positive association between bacterial multiplication efficiency and conjugation ability in infected insects for the used strains.     

Catabolite repression in Bacillus subtilis

The d-gluconate transport system of Bacillus subtilis is optimally induced by exposure of cells for 2 h to 5 mM d-gluconate in the growth medium. d-gluconate transport is subject to catabolite repression, as distinct from inducer exclusion or catabolite inhibition, in a manner parallel to the repression of inducible histidase synthesis, suggesting that the repression is not specific to this transport system. Maximum repression with the repressing carbon source (10 mM) added to cells grown in either casein hydrolysate or amino acid medium is achieved within two doubling times. Urea, the only non-carbon source tested for a repressing effect, was found to act solely by inducer exclusion. The ability of a sugar carbon source to evoke catabolite repression appears to be unrelated to its suitability as a substrate for the sugar: phosphoenolpyruvate phosphotransferase system but nonetheless the conversion to a phosphorylated derivative of the sugar seems essential. Repressed cells fail to synthesize, or do so to a more limited extent, an as yet unidentified phosphorylated compound (probably a highly phosphorylated nucleotide) which is accumulated in the medium of non-repressed cells. Mutant studies imply that inosinic acid synthesis is necessary for catabolite repression whereas the adenosine highly phosphorylated nucleotides required for spurulation are not.     

Isoprenoids of Bacillus acidocaldarius

-The isoprenoids of Bacillus acidocaldarius, a notably acidophilic thermophilic organism, were investigated. Besides normal bacterial isoprenoids such as menaquinone and polyprenols, which latter were resolved in a-cis and a-trans fractions, and some minor components (a-tertiary-prenols and the corresponding anhydroderivatives), not fully characterized, and probably ‘natural’ artefacts, we have isolated and characterized squalene and pentacyclic triterpene hydrocarbons belonging to the hopane class, which must be accounted as unusual. Radioactivity from mevalonate-[2-¹⁴C] is incorporated into hopene-b (the major triterpene component), thus establishing the origin of cyclized squalene derivatives in B. acidocaldarius as a result of de novo synthesis.     

Extrachromosomal DNA in Bacillus thuringiensis var. kurstaki, var. finitimus, var. sotto, and in Bacillus popilliae

Four entomopathogenic bacteria contained extrachromosomal deoxyribonucleic acid (DNA) molecules of various sizes. Bacillus thuringiensis var. kurstaki contained twelve elements banding on agarose gels that ranged from 0.74 to > 50 × 10⁶ daltons, three of which were giant extrachromosomal DNA elements. B. thuringiensis var. sotto contained one giant extrachromosomal DNA element with a molecular size of about 23.5 × 10⁶ daltons and two lesser elements of 0.80 and 0.62 × 10⁶ daltons. B. thuringiensis var. finitimus harbored two giant DNA elements corresponding to >50 × 10⁶ daltons and two lesser bands with relative small size (0.98 and 0.97 × 10⁶ daltons). B. popilliae contained no giant extrachromosomal DNA elements but did contain two smaller elements corresponding to 4.45 and 0.58 × 10⁶ daltons. The possible use of extrachromosomal DNA elements that prove to be autonomous replicons for recombinant DNA studies is discussed.     

Bidirectional chromosome replication in Bacillus subtilis

We have examined autoradiographically the pattern of DNA replication following the germination of Bacillus subtilis spores in [³H]thymidine. Thymine-requiring spores were germinated in low specific activity medium and diluted into higher specific activity medium soon after the first round of replication was expected to start. After a further short period, replication was stopped and the chromosomal structures examined by autoradiography. From the pattern of labelling within individual replicating loops it is clear that the majority (≥75%) expand by growth at two positions that are opposite, i.e. expand bidirectionally. The loops continue to expand at approximately equal rates in both directions until at least 20% of the chromosome has been replicated.From a consideration of the other structural forms that become visible, it seems likely that most chromosomes replicate bidirectionally.     

Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Sporeless mutants of Bacillus thuringiensis

Five sporeless mutant strains of Bacillus thuringiensis were selected after treatment with the mutagen N-methyl-N′-nitro-N-nitrosoguanidine. Two mutant strains were derived from alesti and three from aizawai. All mutants were completely lacking in ability to form spores. Bipyramidal crystalline bodies of the mutants were very regular in shape, as seen with the parent strains, and lay free in the culture broth with autolysis. The insecticidal activity of mutants was, in principle, the same as that of original strains.Cells of the mutants tended to autolyze easily at the end of cultivation. However, 1–10% of cells still remain living. They are completely killed by heat treatment, e.g., 60°C for 30 min, which, however, causes a slight but nonsignificant reduction of toxicity.Thus, use of these sporeless mutants of Bacillus thuringiensis as microbial insecticide having no viable cells is suggested. They may serve as a biochemical starting material for β-endotoxin.     

Polysaccharide deacetylases serve as new targets for the design of inhibitors against Bacillus anthracis and Bacillus cereus

Peptidoglycan N-acetylglucosamine (GlcNAc) deacetylases (PGNGdacs) from bacterial pathogens are validated targets for the development of novel antimicrobial agents. In this study we examined the in vitro inhibition of hydroxamate ligand N-hydroxy-4-(naphthalene-1-yl)benzamide (NHNB), a selective inhibitor of histone deacetylases-8 (HDAC8), against two PGNGdacs namely BC1974 and BC1960 from B. cereus, highly homologous to BA1977 and BA1961 of B. anthracis, respectively. Kinetic analysis showed that this compound functions as a competitive inhibitor of both enzymes with apparent K_i’s of 8.7?μM (for BC1974) and 66?μM (for BC1960), providing thus the most potent CE4 inhibitor reported to date. NHNB was tested in antibacterial assays and showed bactericidal activity against both examined pathogens acting as a multi-target drug. This compound can serve as lead for the development of inhibitors targeting the conserved active sites of the multiple polysaccharide deacetylases (PDAs) of both pathogens.     

Evolution of pathogenicity in the Bacillus cereus group

The Bacillus cereus group of bacteria comprises soil-dwelling saprophytes but on occasion these bacteria can cause a wide range of diseases in humans, including food poisoning, systemic infections and highly lethal forms of anthrax. While anthrax is almost invariably caused by strains from a single evolutionary lineage, Bacillus anthracis, variation in the virulence properties of strains from other lineages has not been fully addressed. Using multi-locus sequence data from 667 strains, we reconstructed the evolutionary history of the B. cereus group in terms of both clonal inheritance and recombination. The strains included 155 clinical isolates representing B. anthracis, and isolates from emetic and diarrhoeal food poisoning, septicaemia and related infections, wound, and lung infections. We confirmed the existence of three major clades and found that clinical isolates of B. cereus (with the exception of emetic toxin-producing strains) are evenly distributed between and within clades 1 and 2. B. anthracis in particular and emetic toxin-producing B. cereus show more clonal structure and are restricted to clade 1. Our characterization of the patterns of genetic exchange showed that there exist partial barriers to gene flow between the three clades. The pathogenic strains do not exhibit atypically high or low rates of recombination, consistent with the opportunistic nature of most pathogenic infections. However, there have been a large number of recent imports in clade 1 of strains from external origins, which is indicative of an on-going shift in gene-flow boundaries for this clade.     

Origin of cyclohexanecarboxylic acid in Bacillus acidocaldarius

In Bacillus acidocaldarius, shikimic acid is converted into the cyclohexancearboxylic acid precursor of fatty acids by way of cyclohexene-l-carboxylic acid, but not by way of cyclohexene-3- or -4-carboxylic acid or benzoic acid.     

Ecology of Bacillus thuringiensis in storage moths

A disease-free stock of Plodia interpunctella was produced by a continuous rearing technique. In dense populations of this stock, 10⁴ or more spores of H serotype V Bacillus thuringiensis applied at one point on the surface of 200 g of food were required to cause epizootics, compared with 10⁷ or more when spread evenly over the surface. In infected populations, spores contaminated the surfaces of all stages of the insect. In diseased larval cadavers there were 5.6–42.2 × 10⁸ spores/g of dry insect (P. interpunctella, Ephestia cautella, Anagasta kuehniella, Ephestia elutella, and Galleria mellonella). Larvae did not cannibalize live larvae while food was present though they sometimes ate cadavers. This is the most potent means of natural spread of the disease. Occurring mainly in protected situations such as food stores, natural infections are usually light, but occasionally spectacular surface accumulations of dead larvae occur, possibly associated with stress, physiological condition of the larvae, serotype of the bacterium, or behavior pattern such as migration. Natural disease may curb infestations in debris, but it attacks too late to prevent excessive damage to stored food. A prophylactic, even admixture of 2 × 10⁹ spores/200 g of food is required for effective insect control.     

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.     

Hemolysis induced by Bacillus cereus sphingomyelinase

Bacillus cereus sphingomyelinase (Bc-SMase) induces hemolysis of sheep erythrocytes which contain large amounts of sphingomyelin. We investigated the mechanism of this hemolysis in comparison to that induced by Clostridium perfringens alpha-toxin. Pertussis toxin, a Gi-specific inhibitor, N-oleoylethernolamine, a ceramidase inhibitor, and dihydrosphingosine, a sphingosine kinase inhibitor, did not inhibit the hemolysis by Bc-SMase, but did inhibit that by alpha-toxin. Bc-SMase broadly bound to whole membranes, and alpha-toxin specifically bound to the detergent-resistant membrane fractions, lipid rafts. The level of ceramide production induced by Bc-SMase in sheep erythrocytes was 6- to 15-fold that induced by alpha-toxin, when the extent of the hemolysis by Bc-SMase was the same as that by the toxin. However, the level of ceramide production induced by Bc-SMase in SM-liposomes was equal to that triggered by the toxin, when the carboxyl fluorescein-release from liposomes induced by Bc-SMase was the same as that induced by alpha-toxin. Confocal laser microscopy showed that treatment of the cells with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that treatment of the cells with Bc-SMase leads to a reduction in membrane fluidity. These results show that Bc-SMase-induced hemolysis of sheep erythrocytes is related to the formation of interface between ceramide-rich domains and ceramide-poor domains through production of ceramide from SM.     

Ultrastructure of the nematode pathogen, Bacillus penetrans

The ultrastructure and development of Bacillus penetrans in root-knot nematodes, Meloidogyne spp., was studied with a transmission electron microscope. Host infection was by a germ tube from the cup-shaped sporangium containing the endospore. The prokaryotic vegetative cells contained septa formed by an ingrowth of the inner layer of the trilaminate cell wall and were associated with mesosomes. Structure of the endospore was similar to other bacteria with a spore protoplast enclosed within two cortical layers and three spore coats. An exosporium which may function in attachment and host specificity surrounded the endospore. Ultrastructural changes accompanying sporulation were similar to those reported for other endospore-forming bacteria but with some parasite specialization. The filamentous vegetative growth was characteristic of some Actinomycetales. Endospore development at the apices of dichotomously branched filaments of the thallus resembled the genus Actinobifida.