Resting cells of Bacillus popilliae and B. lentimorbus catabolize glucose with the production of C02 , lactic acid, acetic acid, glycerol, ethanol, and trace amounts of acetoin and acetaldehyde. The first three products are the major ones, and their ratios may be varied by controlling the availability of oxygen. Practically no lactic acid is produced when oxygen is not limiting, whereas it may comprise up to 80% of the total acid when oxygen is greatly limited. However, no glucose is catabolized by resting cells in the absence of molecular oxygen. Isotope and inhibitor studies and assays for key enzymes of the established metabolic routes all indicate that these organisms utilize both the Embden-Meyerhof and hexosemonophosphate pathways for glucose dissimilation. With a concentrated resting-cell suspension, the extent of participation of the latter route was estimated to be as high as 40% in an atmosphere of pure oxygen, and as low as 2% in air. Acetate was oxidized by only one of the cultures of B. popilliae tested, which is apparently a mutant. Cells of this strain from stationary phase cultures oxidized acetate at pH 7.0 or higher, but not at pH 6.0; however, they oxidized succinate, fumarate, and malate more rapidly at pH 6.0 than at 7.0. The oxidation of tricarboxylic acid cycle intermediates, the presence of condensing enzyme in extracts of cells capable of oxidizing acetate, and the complete inhibition of acetate oxidation by arsenite and partial inhibition by malonate all indicate that terminal oxidation of acetate by this strain of B. popilliae is via the tricarboxylic acid cycle. Bacillus popilliae and B. lentimwrbus both cause the "milky disease" of the Japanese beetle (Popillia japonica) larvae (Dutky, 1940). The parasitic cycle, as it appears in the larvae, 1 Journal article no. 3224, Michigan Agricultural Experiment Station.
Bacillus popilliae was found to be unique among aerobic microorganisms in that it was deficient in a hydrogen peroxide-scavenging system. Neither catalase nor peroxidase was found. At the same time, a system for producing hydrogen peroxide during oxidation of reduced nicotinamide adenine dinucleotide (NADH2) was consistently present in the soluble fraction of extracts of cells from older cultures. Cells harvested from 9-hr cultures did not produce a significant amount of peroxide. The soluble NADH2 oxidase was apparently a flavoprotein, since it was stimulated by flavin nucleotides, insensitive to cyanide and azide, and inhibited by Atabrine. Also, difference spectra demonstrated the presence of a reducible flavin in the soluble fraction of cell extracts. The particulate fraction of cell extracts was shown by difference spectra to contain cytochrome bi ; the strong inhibition of NADH2 oxidation by cyanide, azide, and carbon monoxide indicated that a terminal cytochrome oxidase was also present. This system was also flavin-dependent, since it was strongly inhibited by Atabrine. The specific activity of the NADH2 oxidase in the particulate fraction was lower in extracts of cells from older cultures than in those from exponentially growing cultures. Cytochrome c was not found in extracts of these cells. It is believed that the increased participation of the hydrogen peroxide-generating NADH2 oxidase in cells of older cultures may be responsible for the rapid loss in cell viability noted in stationary-phase cultures. Bacillus popilliae was reported by Dutky (1940) as preferentially anaerobic. This was based on growth tests with an agar medium, and the method used to exclude oxygen for anaerobic incubation was not described. However, more recent studies (Steinkraus, 1957; Pepper and Costilow, 1964; Costilow et al., unpublished data) have demonstrated that this organism responds greatly to forced aeration of broth cultures. It requires the presence of a carbohydrate for significant growth in artificial media, and it is unable to ferment glucose in the complete absence of oxygen. Thus, B. popilliae appears to be a strict aerobe. One of the primary differences found between this species and other aerobic microorganisms is its failure to produce catalase (Steinkraus, 1957). This was thought to be a possible reason for the rapid loss in cell viability which occurs very soon after cultures of B. popilliae attain the stationary phase of growth in artificial media. Furthermore, it was thought that perhaps the failure of this species to sporulate in vitro could be related to I Journal article no. 3425, Michigan Agricultural Experiment Station.
aspects of ionizing radiations as a means of sterilization. Report prepared for Industrial Liaison Conference on Food Sterilization.
Bacillus popilliae and B. lentimorbus grew most rapidly and to the greatest extent in aerated cultures at 30 to 32 C with oxygen absorption rates of 1 mmole of 02 per min per liter, or above. The control of pH also increased the maximal populations attained. Media were developed which consistently produced cell populations of about 109 within 24 to 48 hr in aerated cultures of these two species. The acetic acid produced in highly aerated cultures was shown not to be responsible for the rapid loss of cell viability in stationary phase cultures. However, H202 was very lethal to cells of B. popilliae, and this species is known to have the capacity to produce it. Stationaryphase cells were partially stabilized by reducing the availability of oxygen after 24 hr of incubation on a shaker, and the addition of low levels of glucose further stabilized the cells. The most stable cells were those produced in a medium in which 4% Trypticase (BBL) and 0.1 % barbituric acid were incorporated. A high percentage of these cells contained refractile bodies visible under a phase microscope. Although these bodies were not heat-resistant and lacked other characteristics of endospores, cells in cultures containing them had reasonably high viability for extended periods, as compared with those in control cultures.
Spores of Bacillus popilliae from infected larvae and refractile bodies produced in a Trypticase-barbiturate medium were similar but distinct from vegetative cells of this organism in protein, nucleic acid, and enzyme composition. The spores and refractile bodies were found to have catalase activity, some of which was heat-resistant. This enzyme was not found in the vegetative cells. The spores contained dipicolinic acid, but the refractile bodies did not. The latter were similar to cells in having considerably higher levels of phosphate extractable with cold trichloroacetic acid and of poly-f-hydroxybutyrate than had the spores. Electron microscopy demonstrated conclusively that the refractile bodies are distinctly different from either cells or spores of B. popilliae. The possibility that these bodies are formed as a result of an aborted sporulation process is discussed. Practically every cell of Bacillus popilliae produces a spore during production of "milky disease" in Japanese beetle larvae. In contrast, the best efforts published to date have resulted in a maximum of about 0.3%7o sporulation in vitro (30) on a solid medium. More recently, a strain has been isolated (NRRL B-2309M) in which about 15 % sporulation has been observed on a solid medium (E. S. Sharpe and G. St. Julian, Bacteriol. Proc., 1967, p. 10). For several years we have sought to ascertain any unique characters which might provide a key to sporulation of B. popilliae. This organism is a strict aerobe (24); glucose is catabolized by both the glycolytic and hexosemonophosphate pathways, producing acetate, lactate, and C02 primarily (4); acetate is not oxidized to a significant extent except by a single mutant strain (4); the electron transport system is deficient in cytochrome c (5); stationary-phase cultures generate considerable hydrogen peroxide and lack the ability to degrade it (5); cells are quite sensitive to 0.01 M H202 (8); the organism is auxotrophic for 11 amino acids and for thiamine (36); and barbituric acid is required for consistent growth in a synthetic medium (36). During these studies, a medium was developed 1 Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article 4058.
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