Degradation and turnover of bacterial cell wall mucopeptides in growing bacteria

The mucopeptide layer of the cell wall ofBacillus megaterium is broken down into separate components during growth of the cells. The released diaminopimelic acid is partly decarboxylated to lysine, which is incorporated in the proteins and partly used for cell wall resynthesis. The smaller portion of the degraded mucopeptide is released into the medium in the form of non-utilized fragments. The rate of the mucopeptide turnover is a function of the rate of growth of the culture. About 15–20% of the rigid layer of the cell wall is degraded during on cell division. The sensitivity ofBacillus megaterium to lysozyme and the rate of its conversion to protoplasts is also proportionate to the rate of growth of the culture. There is no measurable mucopeptide turnover in non-growing cells, either in the stationary phase of the culture or in starvation in nitrogen-free medium. The resistance of the cell wall to lysozyme also increases during the stationary phase. The rigid component of the cell wall is probably also broken down during growth ofBacillus cereus andEscherichia coli cultures.     

Turnover of mucopeptide during the life cycle ofBacillus megaterium

Sporogenic and asporogenicBacillus megaterium strains, as well asBacillus cereus degraded the murein component of the cell wall labelled with¹⁴C-diaminopimelic acid to TCA-soluble compounds during growth. The rate of murein turnover was about 15% during one generation in all three cases. The addition of chloramphenicol instantaneously markedly decreased the degradation rate, whereas in the presence of penicillin the degradation proceeded at the beginning at a rate comparable with that in the control and decreased only after a certain time interval. The cell wall degradation was considerably or completely stopped during the stationary phase of growth. In sporogenic strains ofBacillus megaterium andBacillus cereus the release of mature spores was associated with a new wave of the wall degradation, during which the wall of the sporangial cell was completely digested to TCA-soluble fragments. Free spores contained practically no mucopeptide component (cortex or spore wall) originating from the wall of the vegetative cell. A possible existence of a stable fraction of the cell wall not subject to turnover was investigated by measuring the³H/¹⁴C ratio in cells labelled simultaneously with³H (or¹⁴C)-diaminopimelic acid and¹⁴C (or³H)-leucine. The ratio changed during five generations, remaining constant later. This indicates that a certain portion of murein could be stable. The murein degradation during growth was not associated with secretion or release of a significant quantity of autolytic enzymes into the medium. The wall was apparently attacked from the inside. On the other hand, the release of the spore was accompanied by an increasing autolytic activity in the medium. This latter activity reached values corresponding to 3–8 μg lysozyme/ml. The results published here were presented at the 2nd Harden Conference “Cell walls and cell membranes”, Wye, Kent, England in September 1970.     

Reversible filamentous growth in the psychrophile Bacillus psychrophilus

At the near-maximum growth temperature of 32.5 °C, the psychrophile Bacillus psychrophilus loses the ability to septate and divide, resulting in the formation of filaments, which are four to six times longer than cells grown at 20 °C. DNA synthesis relative to growth occurs at the same rate both in the filaments at 32.5 °C, (which actually become multi-nucleated) and in normal-size cells at 20 °C, showing that the inhibition of DNA synthesis by the elevated temperature is not the cause of the filamentous growth, as has been found for other microorganisms. Similarly, temperature-sensitive cell-wall mucopeptide synthesis does not appear to be responsible. Reversal of filament production occurs when preformed filaments are incubated at 20 °C. Such reversal, i.e., septation of preformed filaments, requires the de novo synthesis of protein, probably throughout the reversal period.Filamentous cells are more nutritionally demanding than cells at 20 °C, with at least one substrate becoming limiting within 8 hr at 32.5 °C but not at 20 °C. However, such variation in nutritional requirement is not the cause of filament formation. KCl and NaCl stimulate cell division in cells growing at 32.5 °C but not in preformed filaments. Other membrane-active agents such as lysolecithin, dimethyl sulfoxide, ethanol, sodium oleate, and pantoyl lactone do not stimulate septum formation in filaments.     

Phosphorylation of skeletal muscle myosin light chain kinase by the catalytic subunit of cAMP-dependent protein kinase

Phosphatidylethanolamine (PE) was isolated from membranes of Bacillus megaterium. The organism was grown at 20°C and 55°C. The phase equilibria in PE/water systems were studied by ²H and ³¹P nuclear magnetic resonance, and by polarized light microscopy. PE isolated from B. megaterium grown at 20°C forms a lamellar liquid crystalline phase at the growth temperature, and at low water contents a cubic liquid crystalline phase at 58°C. The ratio iso/ante-iso acyl chains was 0.3 in this lipid. PE isolated from this organism grown at 55°C forms only a lamellar liquid crystalline phase up to at least 65°C. In this lipid the ratio iso/ante-iso acyl chains was 3.2.     

Flavonoids in the Flowers of Gnaphalium affine D. Don

Thermonsenstivie division mutants were derived from Bacillus subtilis Marburg 168 thy trp₂ by means of membrane filtration after nitrosoguanidine mutagenesis. Among them, ts42 requiring uracil for normal growth at 48°C was investigated.

In the absence of uracil, the mutant cells grew normally at 37°C and stopped dividing after temperature shift to 48°C resulting in filaments of two to four times length of normal rods. The total cell number after temperature shift from 37 to 48°C, increased two to three fold in 90 min and remained constant thereafter. The viable count after the temperature shift to 48°C, increased 1.5 to 2 fold in initial 60 min and then decreased exponentially. A rapid restoration of colony forming ability was shown when the mutant cells were shifted back to the permissive temperature after 120 to 180 min of incubation at 48°C or when uracil was introduced to the culture at 48°C. This recovery of viability was partly observed even in the presence of chloramphenicol. The synthesis of RNA of this mutant was shown to decline 20 min after the temperature shift to 48°C whereas the syntheses of DNA and protein proceeded for more than 80 min at that temperature.

No newly isolated uracil requiring mutants formed filaments in the medium lacking uracil or showed growth pattern like ts42.     

Elevated content of hsp 70 does not induce tolerance of sporulation to higher temperature

The temperature permissive for sporulation (up to 42°C) inBacillus megaterium is by 4–5°C lower than that for its growth (up to 46–47°C). The ability ofB. megaterium cells to synthesize and degrade stress proteins under incubation in the sporulation medium was therefore investigated. The higher level of hsp 70, a typical stress protein induced by a temperature shock in postexponential growth phase, did not increase the permissive temperature of sporulation. The hsp 70 protein did not undergo a rapid turnover and its portion in the soluble protein fraction did not drop for at least 6 h at a temperature that was nonpermissive for sporulation (43.5°C). On the other hand, the elevated level of hsp 70 could not bring about the inhibition of sporulation as it was retained in the cells even after a shift of the temperature to 35°C, permitting sporulation of the culture.     

Preparation and evaluation of Bacillus megaterium-alginate microcapsules for control of rice sheath blight disease

Bacillus megaterium encapsulated in calcium alginate microcapsules was prepared and tested for its efficacy against sheath blight disease of rice. In laboratory conditions, the aqueous suspension (1:100, v/v in potato dextrose agar) of the bacterial microcapsules (10¹⁰ spores/ml) inhibited mycelial growth of Rhizoctonia solani (>99 %) after the microcapsules were produced and stored for 12 months at room temperature (28 ± 2 °C). The survival of the bacterium in the microcapsules in response to ultraviolet (u.v.) irradiation and high temperature was investigated. The survivability of the bacterium in the encapsulated form was greater than that of the fresh cells when it was subjected to u.v. (20-W General electric u.v. lamp from a 25 cm distance for 48 h) and a high temperature treatment (80 °C for 48 h). Cells of the bacterium were detected by scanning electron microscope on both the leaf sheath and the leaf blade (in pot tests in a greenhouse) after spraying encapsulated product. The number of bacteria on the surface of both rice tissues (5 Log. number/g of plant) after spraying with encapsulated product was not significantly different from that after spraying with fresh cells onto the rice seedlings. Spraying the encapsulated B. megaterium on rice plants in the greenhouse was as effective as spraying a chemical fungicide for suppressing rice sheath blight disease.     

Suppression of an exocellular proteinase synthesis inBacillus megaterium by increased temperature

During cultivation ofBacillus megaterium at 42 °C the amount of the exocellular protease produced by growing cells sharply decreases as compared with temperatures of 28 and 35 °C. Within the above range the growth rate and incorporation of amino acids increase with increasing temperature. The culture adapted to 42 °C does not produce more proteinase at this temperature than the non-adapted culture. The high temperature does not induce accumulation of the enzyme in the cells. Total protein excretion was slightly lower at 42 °C than at 28 and 35 °C.     

A quantitative study of phagocytosis by hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria

Fresh hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria were exposed to known concentrations of Bacillus megaterium, Escherichia coli, and Staphylococcus aureus in vitro and it was ascertained that all four types of cells of C. virginica and all three types of M. mercenaria became associated with the bacteria. Association is defined as either the first, i.e., contact and adherence, or second, i.e., engulfment, phase of phagocytosis. However, when the surfaces of each type of cell, as well as the percentages of each type in whole hemolymph, from both species of molluscs are taken into consideration, it is concluded that the granulocytes are the most important from the standpoint of phagocytosis.When hemocytes of M. mercenaria were exposed to Bacillus megaterium at 4°, 22°, and 37°C, it was found that the association indices were higher at the latter two temperatures. It is postulated, because of the results of Feng and Feng (1974), that nonself materials adhere with less frequency at 4°C and hence are not phagocytosed at this lower temperature.     

Manipulation of B. megaterium growth for efficient 2-KLG production by K. vulgare

In the two-step Vitamin C fermentative production, its precursor 2-keto-l-gulonic acid (2-KLG) was synthesized by Ketogulonicigenium vulgare through co-culture with Bacillus megaterium. The rates of K. vulgare cell growth and 2-KLG production were closely related with B. megaterium concentration in the co-culture system. To enhance the 2-KLG production efficiency, a strategy of manipulating B. megaterium growth in the co-culture system and properly releasing its intracellular components was introduced. Lysozyme was used specifically to damage B. megaterium cell wall structure and subsequently inhibit its cell growth. When 10,000 U mL⁻¹ lysozyme was fed to the co-culture system at 12 h, the growth rate of K. vulgare, sorbose consumption rate, and 2-KLG productivity could increase 27.4%, 37.1%, and 28.2%, respectively.     

Regulation of Bacterial Cell Division: Genetic and Phenotypic Analysis of Temperature-Sensitive, Multinucleate, Filament-Forming Mutants of Escherichia coli

Three mutants of Escherichia coli K-12 which form filaments during 42 C incubation have been characterized. The mutant strains AX621, AX629, and AX655 continued to grow and to synthesize deoxyribonucleic acid at 42 C for 150 to 180 min, after which time growth ceased. When cultures of the mutants were transferred from 42 to 28 C, septation of the filaments began after a 25- to 30-min period and continued at a greater than normal rate until no filaments remained. Addition of chloramphenicol at the time of transfer from 42 to 28 C prevented cell division in strain AX655 and caused lysis of strains AX621 and AX629. The temperature sensitivity mutation in each strain mapped near leu. For strain AX621, the mutation was specifically located between leu and nadC by P1 transduction. Properties of these strains are compared with those of other cell division mutants.     

Effects of temperature on the cell wall and osmotic properties in dark-grown rice and azuki bean seedlings

The mechanism inducing the difference in growth rate under various temperature (10–50 °C) conditions was analyzed using rice and azuki bean seedlings. The growth rate of rice coleoptiles and azuki bean epicotyls increased as temperature increased up to 40 and 30 °C, respectively, and the elongation was retarded at a higher temperature. The cell wall extensibility of rice coleoptiles and azuki bean epicotyls also showed the highest value at 40 and 30 °C, respectively, and became smaller as the temperature rose or dropped from the optimum. The opposite tendency was observed in the minimum stress-relaxation time of the cell wall. On the other hand, the cellular osmotic concentration of rice coleoptiles and azuki bean epicotyls was lower at the temperature optimum for growth at 40 and 30 °C, respectively. When rice and azuki bean seedlings grown at 10, 20, 40, or 50 °C were transferred to the initial temperature (30 °C), the growth rate of coleoptiles and epicotyls was mostly elevated, concomitant with an increase in the cell wall extensibility. The growth rate was correlated with the cell wall mechanical parameters in both materials. These results suggest that the environmental temperature modulates the growth rate of plant shoots by affecting mainly the mechanical properties of the cell wall. Electronic Publication     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Activation of Bacillus spores at moderately elevated temperatures (30–33 °C)

The time/temperature profiles experienced by spores on the track from their natural sporulation environment to consumable food products may be highly diverse. Temperature has been documented as an important factor that may activate spores, i.e. potentiates spores to germinate. There is, however, limited knowledge about the relationship between the expected temperature history and the subsequent germination characteristics of bacterial spores. We show here that the germination rate of five different Bacillus spore populations, represented by strains of Bacillus cereus, Bacillus weihenstephanensis, Bacillus pumilus, Bacillus licheniformis and Bacillus subtilis could be increased following 1 week storage at moderately elevated temperatures, 30–33 °C, compared to spores stored at 3–8 °C. The results imply that spores contamination routes to foods, specifically the temperature history, could be highly relevant data in predictive modeling of food spoilage and safety. Activation at these moderately elevated temperatures may be a native form of spore activation in their natural habitats, knowledge that also could be useful in development of decontamination strategies for mildly heated foods.     

Influence of glass microbeads on growth,activity and morphological changes of Bacillus megaterium

Cells of Bacillus megaterium growing in the presence of glass microbeads with average diameters of 29 and 53 μm were frequently filamentous and sometimes reached lengths of 600 μm. Some of the filaments were nonseptate. The formation of filaments was prevented by magnesium but not by several other cations. In media with supplemental magnesium, the time required before active proliferation commenced was inversely related to the diameter of the particles. B. megaterium growing in media with the smaller size beads consumed oxygen and utilized glucose at greater rates than bacteria in media with the larger spheres or in bead-free solutions, and the uptake of oxygen was maintained for a longer period.     

Mating duration and egg production of the predaceous mite Neoseiulus californicus (Acari: Phytoseiidae) vary with temperature

Effects of constant temperature on mating duration and total fecundity of Neoseiulus californicus females mated once were investigated at 18 °C, 25 °C, 30 °C, and 35 °C with a photoperiod of 16L:8D. Adult mites grown and maintained at 25 °C mated for 315.3 min on average and produced 46.1 eggs per female. These values varied significantly by temperature: 553.6 and 13.9 (18 °C), 261.2 and 26.6 (30 °C), and 253.6 and 23.9 (35 °C), respectively. Duration of copulation was negatively correlated with temperature. However, total egg production peaked at 25 °C and decreased at lower and higher temperatures. Reduced sperm transfer and/or survival rate of sperm in the female body may account for decreased egg production when temperatures are not optimal.     

Conducting High acetic acid and temperature acetification processes by Acetobacter pasteurianus UMCC 2951

In this study Acetobacter pasteurianus strain UMCC 2951 was tested as a microbial starter to conduct acetification processes by repeatedly cultivation cycles under high temperature acetification at 40 ± 1 °C. Acid production and acetification rate increased with repeated cultures under high temperature acetification as adaptation period increased, but were still lower than acetification at 30 ± 1 °C. However, the addition of 0.15 % calcium chloride reduced the negative effects of 40 ± 1 °C on both acid production and acetification rate compared to 30 ± 1 °C. A strong decrease in fatty acids and phosphatidylethanolamine and increases in phosphatidylcholine and phosphatidylglycerol in cell membranes were found under high acid and high temperature acetification. In addition, transmission electron microscope images reveal a more compact cell wall when calcium chloride was added to the cultivation medium. The strategy used in this study confirmed that the use of acetic acid bacteria as microbial starters could be effective also at temperature above the optimal values, when acetification processes are managed through repeated semi-continuous cycles.     

Extracellular proteases modify cell wall turnover in Bacillus subtilis.

The rate of turnover of peptidoglycan in exponentially growing cultures of Bacillus subtilis was observed to be sensitive to extracellular protease. In protease-deficient mutants the rates of cell wall turnover were greater than that of wild-type strain 168, whereas hyperprotease-producing strains exhibited decreased rates of peptidoglycan turnover. The rate of peptidogylcan turnover in a protease-deficient strain was decreased when the mutant was grown in the presence of a hyperprotease-producing strain. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to cultures of hyperprotease-producing strains increased their rates of cell wall turnover. Isolated cell walls of all protease mutants contained autolysin levels equal to or greater than that of wild-type strain 168. The presence of filaments, or cells with incomplete septa, was observed in hyperprotease-producing strains or when a protease-deficient strain was grown in the presence of subtilisin. The results suggest that the turnover of cell walls in B. subtilis may be regulated by extracellular proteases.     

The response of bacterial spores to vacuum treatments. II. Germination and viability studies.

The viability of Bacillus megaterium spores has been determined after exposure to vacuum dehydration at temperatures between 0 and 65 °C, for periods up to 24 hr. A curvilinear relationship has been demonstrated between viability and drying temperature, with minimum viability occuring around 15 °C and increases in viability being shown above 35 °C. In contrast to vegetative bacteria, reequilibration of the dried spores to 2 × 10^?3 or 10 Torr aqueous vapor pressure, and/or subsequent exposure to oxygen had no effect on viability. Dehydration, rehydration and oxygen treatments had no effect on the time for outgrowth of the spores or on the growth rate of the resultant vegetative cells. Physical loss of spores from samples was not demonstrated during any of these treatments. Evidence has been presented for a novel type of spore activation, which occurs during vacuum dehydration at high temperatures, to an extent that is dependent upon drying time. The mechanism of this activation is unlike that of conventional heat or chemical activation but is oxygen independent and unaffected by reequilibration to 2 × 10^?3 or 10 Torr.     

Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019

This study demonstrated the improved polyhydroxybutyrate (PHB) production via high cell density cultivation of Bacillus megaterium BA-019 with balanced initial total sugar concentration and carbon to nitrogen (C/N) weight ratio. In the 10 L stirred fermentor operated at 30 °C, pH 7.0, 600 rpm, and 1.0 vvm air, with the initial total sugar concentration of 60 g/L and urea at the C/N weight ratio of 10:1, 32.48 g/L cell biomass with the corresponding PHB weight content of 26.94 % and volumetric productivity of 0.73 g/L h were obtained from batch cultivation. Continuing cultivation by intermittent feeding of the sugarcane molasses along with urea at the C/N weight ratio of 12.5:1 gave much improved biomass and PHB production (90.71 g/L biomass with 45.84 % PHB content and 1.73 g/L h PHB productivity). Similar biomass and PHB yields were obtained in the 90 L stirred fermentor when using the impeller tip speed as the scale-up criterion.