Trehalose Metabolism by Bacillus popilliae

Trehalose was found to be utilized more readily than glucose for the growth of Bacillus popilliae NRRL B-2309MC. The pathway of degradation of trehalose was elucidated and found to differ from that reported for other organisms. Trehalase and trehalose phosphorylase activities could not be detected. Rather, trehalose was found to undergo phosphoenolpyruvate (PEP)-dependent phosphorylation, and the resulting trehalose 6-phosphate was cleaved by a phosphotrehalase to equimolar amounts of glucose and glucose 6-phosphate. The phosphotrehalase was purified 34-fold and shown to have a pH optimum of 6.5 to 7.0 and a K(m) for trehalose 6-phosphate of 1.8 mM. A mutant missing the phosphotrehalase failed to grow on trehalose but grew normally on other sugars. The mutant accumulated [(14)C]trehalose as [(14)C]trehalose 6-phosphate. Phosphorylation of trehalose by dialyzed extracts was at least 25 times faster with PEP than with adenosine 5'-triphosphate, and the phosphorylation activity was associated primarily with the particulate fraction. These data and the results of studies of [(14)C]trehalose uptake suggest that trehalose is transported into the cell as trehalose 6-phosphate by a PEP:sugar phosphotransferase system. Cell extracts of other strains of B. popilliae were also found to produce [(14)C]sugar phosphate from [(14)C]trehalose and to have phosphotrehalase activity.     

Selective Medium for Quantitation of Bacillus popilliae in Soil and in Commercial Spore Powders

A medium consisting of MYPGP agar supplemented with vancomycin was found to be highly selective for Bacillus popilliae, especially for strains originally isolated from Japanese beetle larvae. The medium has proven to be useful for the quantitation of B. popilliae spores in commercial spore powder and in soil.     

Superoxide Dismutase in Bacillus popilliae

Vegetative cells of Bacillus popilliae were devoid of catalase but had high levels of superoxide dismutase. This provides further support of a theory that oxygen tolerance by an organism is more dependent on superoxide dismutase than on catalase.     

Spore formation by Bacillus polymyxa during the inhibition of polymyxin biosynthesis

When amino acids are added to the culture of Bacillus polymyxa growing under the conditions of active spore formation, the rate of polymyxin biosynthesis and the quantity of spores being formed decrease. All cells in the population start to produce spores under these conditions, but only some of them pass through all of the differentiation stages. Most of the cells remain in the state of prospores. Some cells which have started to differentiate are characterized by ultrastructural anomalies, do not form prospores and undergo early lysis.     

Sporogenicity of yeast autolyzates and casein hydrolyzates for Bacillus popilliae in liquid cultures

Bacillus popilliae NRRL B-2309S forms several million refractile spores/ml in liquid shaken cultures. A suitable medium includes glucose, K₂HPO₄, water, and three selected ingredients-activated carbon, a yeast autolyzate, and a casein hydrolyzate. Only 4 out of 26 lots of commercial yeast autolyzates tried were sporogenic. However, spore formation in the presence of the four was poor and erratic unless a compatible casein hydrolyzate also was present. Five of eight lots of casein hydrolyzates improved the sporogenicity of selected yeast products to various degrees. A comparison of amino acid compositions of yeast and casein lysates sheds no light on differences between suitable and unsuitable ones. Less refined yeast and casein products seem preferable.     

Spore Production by Bacillus stearothermophilus

[This corrects the article on p. 690 in vol. 14.].     

Propagation of Bacillus popilliae in laboratory fermentors

The insect pathogen Bacillus popilliae Dutky causes a fatal milky disease of Japanese beetle larvae. Spores of the bacterium offer a biological means of controlling this insect. While satisfactory sporulation in vitro has not yet been accomplished, conditions have been developed for the proliferation of vegetative cells in shaken flasks and aerated fermentors. Vegetative cultures are maintained by frequent transfer or by lyophilization. Media based on yeast extract are used routinely, but corn steep liquor and casein hydrolyzates afford comparable yields of 5 × 10⁸ cells/ml. in 16–24 hr. Nutritional requirements have been established for growth in a synthetic medium. Oxygen availability affects the pathway of carbohydrate catabolism and is necessary for optimal growth. In rapidly growing cultures, a short period of maximum viability is characteristically followed by rapid death of the cells. When inoculum size and transfer time are suitably manipulated, viable cell yields reach 1–2 × 10⁹/ml. Alternative methods of propagation, including the addition of particulate carbon, and procedures designed either to neutralize acids or to remove metabolic products by dialysis, do not markedly enhance the yield of cells per volume of medium, although viability may be prolonged.     

Survival of Lyophilized Bacillus popilliae in Soil

Survival of lyophilized vegetative Bacillus popilliae in dry soil and in soil kept in atmospheres with different relative humidities (RH) was determined. Cells survived for at least 1 year when the RH was 22% or lower, but an RH of 33% or higher reduced viability. At 33% RH, no bacteria were recovered after 5 months of storage; at 42% or above, no bacteria were recovered after 1 month of storage.     

Fatty Acids in Bacillus larvae, Bacillus lentimorbus, and Bacillus popilliae

The types of fatty acids produced by two strains each of Bacillus larvae, B. lentimorbus, and B. popilliae, and their distribution patterns, were studied by gas-liquid chromatography. All six organisms produced eight major fatty acids: six branched (iso-C(14), -C(15), -C(16), and -C(17), and anteiso-C(15) and -C(17)), two normal (n-C(14) and -C(16)), and two minor (n-C(15) and monounsaturated n-C(16)). In addition, some other trace acids were produced. Branched-chain fatty acids accounted for 54 to 85% of the total fatty acids. These compositions are similar to those previously found with 26 strains of 12 species of the genus Bacillus. Thus, an abundance of branched-chain fatty acids seems to be a characteristic of the biochemical nature of the genus Bacillus. It is noteworthy that marked differences between the nutritional requirements of the three insect pathogens used in the present study and those of the other 12 species of the genus Bacillus studied previously are not significantly reflected in their fatty acid composition.     

Oligosaccharide formation byTrichoderma harzianum in lactose containing medium

Summary A soil isolate ofTrichoderma harzianum produced oligosaccharides in lactose containing medium. The yield of trisaccharides was 32.1 per cent based on total saccharides. The main product formed was 6-galactosyllactose.     

Superoxide Dismutase in Bacillus popilliae, a Catalaseless Aerobe

Bacillus popilliae, a cytochrome-containing aerobic organism that lacks catalase and peroxidase, was examined for superoxide dismutase activity. The activity was quite high relative to a wide variety of organisms previously surveyed and was induced by oxygen. No correlation could be made between superoxide dismutase activity and the unexplained death of this organism after completion of exponential growth.     

Macromolecular Sieving by the Dormant Spore of Bacillus cereus

The threshold surface porosity in the dormant spore of Bacillus cereus strain T was assessed by measuring passive permeabilities to a series of polydisperse polyethylene glycol samples which increased in average molecular size. The apparent exclusion threshold at diffusional equilibrium corresponded to a polymer of number-average molecular weight ( M(n)) = 150,000 and equivalent hydrodynamic radius ( r(ES)) = 16 nm, which confirmed a previous report. However, analytical gel chromatography before and after uptake by the spores revealed that only the low molecular weight fractions in a polymer sample distribution were taken up. From graphical analyses of the changes in molecular weight distributions, a quasi-monodisperse exclusion threshold was determined corresponding to M(n) = 8,000 and r(ES) = 3.2 nm. Thus, the equivalent porosity in the limiting outer integument appeared much more restrictive than heretofore shown for spores, although still more open than the monodisperse equivalent for the cell wall of vegetative bacilli.     

Changes in the chemical composition of Bacillus popilliae vegetative cells in the course of their growth on a chemically defined medium

The growth, development and changes in the chemical composition of Bacillus popilliae cells were studied in the course of their growth on a liquid chemically defined medium containing casein hydrolysate, complex vitamin mixtures and glucose. Growth of the culture on this medium was accompanied with diauxia which could be registered by changes in the rate of growth and synthesis of the main cellular polymers (DNA, RNA, protein, phospholipids). The rate of protein synthesis correlated with the specific growth rate which was maximal when the reserve substances of the cells or the compounds of partly digested biomass were used. The rate of protein synthesis in the experiment depended mainly on the protein synthesizing activity of RNA rather than on its content in the cells. The low rates of growth and protein synthesis, which suggest that the growth of the culture is being interested with in the course of utilization of the components of the nutrient medium, may account for the absence of spore formation by this strain on the medium used.     

The in vivo production of Bacillus popilliae var. rhopaea spores

The effect of various factors on the yield of Bacillus popilliae var. rhopaea spores formed in Rhopaea verreauxi larvae have been studied. Lack of adequate food, temperatures above and below 23°C, and infecting doses above 10⁶ spore larva, all significantly lowered spore yield per larva. Larval age had a pronounced effect; second-instar and young third-instar larvae produ ed about 1 × 10¹⁰ spores while old third-instar larvae produced about 4 × 10¹⁰ spores. Incubation of larvae for longer than 4 weeks did not increase spore yield per larva. Yields were similar whether larvae were infected by injection or per os. Three other host species could be used to mass-produce B. popilliae var. rhopaea spores but all were less efficient than R. verreauxi. Milky third-instar R. verreauxi larvae, which were field collected, yielded 1.57 × 10¹⁰ spores per larva.