Amino acid pool and protein turnover during differentiation (spherulation) ofPhysarum polycephalum

The composition of the amino acid pool during spherulation was determined. It changes in size and in composition, the concentration of each amino acid behaving individually. The first response to the onset of spherulation either by starvation or osmotic shock (0.5 M mannitol) always is a decrease of the pool's size, which during further starvation expands for a short period and then decreases again. During development induces by mannitol in the presence of external amino acids, the pool size increases continuously after the initial depletion.As shown by radioactive labeling, amino acids were actively released from the plasmodium into a medium containing amino acids, but retained by the microplasmodia in an amino acid-free medium. The kinetics of the uptake of radioactive amino acids from the medium is biphasic, indicating the existence of multiple pools. Even after a labeling period of 8 h the amino acid pool is not yet in equilibrium with the medium. The possibility of a compartimentation of the pool was confirmed by density labeling of two different enzymes.Whereas the turnover of total protein is only very low during growth, it is rather high in spherulating microplasmodia. At least 70% of the originally existing protein is degraded during this development, while, simultaneously, at least 50% of the protein present after 24 h starvation is newly synthesized during that period.     

Effect of L-cystine on macromolecular changes during spore and parasporal crystal formation inBacillus thuringiensis var.thuringiensis

High concentration of L-cystine (0.25%) when present in a glucose-mineral salt medium inhibited sporulation-specific events like protease production, calcium uptake and dipicolinic acid synthesis inBacillus thuringiensis var.thuringiensis. In addition, the enzymes of the Krebs cycle from aconitase onwards were completely inhibited by a high concentration of cystine. At a low concentration of cystine (0.05%), none of the above mentioned macromolecular changes were affected. Lipid synthesis monitored by [1,2¹⁴ C]-acetate incorporation into lipid as well as into whole cells was completely inhibited.     

Spore and crystal formation inBacillus thuringiensis var.thuringiensis during growth in cystine and cysteine

The effect of the addition of different concentratons of cystine and cysteine on sporulation and parasporal crystal formation inBacillus thuringiensis var.thuringiensis was studied. The effect was well pronounced when the cystine/cysteine additions were made after the stationary phase. Heat stable spores and crystals were formed when the culture was provided with a low concentration of cystine/cysteine (0.05 per cent w/v). At a moderate concentration of cystine or cysteine (0.15%), only heat labile spores were formed without the production of the crystal. When the cystine/cysteine concentration was high (0.25%), spore and crystal formation were completely inhibited. Partial reversal of inhibition of sporulation was brought about by sodium sulphate or Zinc sulphate and lead, copper, cadmium or cobalt acetate at 0.2 mM or at 0.2% of sodium or potassium pyruvate, citrate, cisaconitate, oxalosuccinate, ∞ -keto-glutarate, succinate, fumarate, malate, or oxalacetate. Glutamate (0.2%) overcame the inhibitory effect of cystine/cysteine completely. The structural changes observed using phase contrast microscopy were dependent upon the concentration of cystine/cysteine.     

L-lysine production at 65°C by auxotrophic-regulatory mutants ofBacillus stearothermophilus

Summary The amino acid L-lysine was produced from auxotrophic-regulatory mutants ofBacillus stearothermophilus at a temperature of 60–65°C. One of the mutants (AEC 12 A5, S-(2-aminoethyl)-cysteine^r, homoserine^–), produced L-lysine at the concentration of 7.5 g/l in shaken flasks in minimal medium containing 5% glucose. Culture conditions for optimizing L-lysine production were not investigated. The aspartokinase activity of the wild strainB. stearothermophilus Zu 183 was inhibited by lysine alone and by threonine plus lysine. AEC resistant mutants showed an aspartokinase activity genetically desensitized to the feedback inhibition. Optimal temperature and pH of aspartokinase were 45°C and 9.5, respectively. The data provide significant evidence that mutants of the speciesB. stearothermophilus have a potential value for amino acid production.     

Enhancement and regulation of extracellular protein production by Bacillus brevis 47 through manipulation of cell culture conditions

Bacillus brevis 47 was cultivated in 2 liter fermentors in semidefined media containing polypeptone with or without glucose or fructose. Neither sugar was essential for growth or extracellular (S-layer) protein production, and 2.5 to 3.0 g/L protein was accumulated in the medium. When present, glucose was used very slowly, however, fructose was used much more quickly. Dramatic changes in metabolic indicators (dissolved oxygen and pH) were seen when fructose became depleted, and protease was produced, decreasing the amount of protein ultimatelv accumulated in the medium. Using the change in dissolved oxygen as a marker for the time of addition, polypeptone, fructose, or both were used to stimulate protein production. With the addition of polypeptone, on stimulation was achieved, but protease production was suppressed. Addition of fructose did result in a small stimulation of protein production (to 5 g/L) if added once. Further additions resulted in more growth, but no increase in protein production. Various combinations of polypeptone and fructose were also used, with the most effective combination (fructose added early, fructose and polypeptone added later) resulting in an accumulation of 15 g/L protein in the medium. This is comparable to that seen when B. brevis 47 is grown in a complex glucose medium and stimulated with polypeptone addition at 21 hours. These results are discussed with respect to the structure and function of S-layer proteins, as well as the use of this organism for the production of heterologous proteins.     

Effect of oxygen on steady-state product distribution in Bacillus polymyxa fermentations

Bacillus polymyxa ferments glucose to 1-2,3 butanediol, acetoin, ethanol, acetic acid, lactic acid, and formic acid. This research investigates product formation as a function of oxygen availability. A predictive model that simulates product distribution at known oxygen transfer rates is developed on the hypothesis that, in an energy-limited environment, B. polymyxa utilizes glucose and oxygen in the most efficient manner. The efficiency of utilization of glucose and oxygen is measured in terms of the ATP yields of each oxidative pathway. The identity of the products constituting the profile at the given oxygen transfer rate is determined by comparing the ATP production and consumption rates. While the ATP generated is calculated from a knowledge of the oxygen transfer rate and ATP yields of the oxidative pathways, the ATP consumption is estimated by the Pirt expression in terms of growth- and nongrowth-associated components. The product formation rates are obtained by solving ATP and NAD balance equations. They equate the production and consumption rates of these intermediates and are derived from the pseudo-steady-state hypothesis. The model is applied to continuous culture systems that are both open and closed with respect to biomass. At a given oxygen transfer rate, dilution rate, and inlet glucose concentration, the model predicts steady-state concentrations of two dominant fermentation endproducts with the help of four parameters that can be determined from independent experiments. In contrast with earlier approaches, the experimental studies are carried out in continuous culture. Product profiles are obtained at various oxygen transfer rates, fer rates, inlet glucose concentrations, and dilution rates. The effect of pH on the relative distribution of products is also demonstrated. Results indicate that the model is fairly successful in predicting product profiles as a function of oxygen availability. (c) 1992 John Wiley & Sons, Inc.     

Preparation of a flowable liquid bacterial insecticide based on Bacillus sphaericus

Fermenter-produced Bacillus sphaericus 2362 was formulated into a thick, dark flowable liquid concentrate containing 4.8×10⁹ c.f.u./ml and charcoal as protector against ultraviolet light. The potencies of the formulation against L₄ Culex pipiens quinquefasciatus before and after storage for 2 years were 5714 and 5862 International Toxic Units (ITU), respectively, when compared with a standardized B. sphaericus from the WHO at 1000 ITU. In field trials, treatment at 1.01/ha gave 96 to 100% control of mosquito larvae. B. sphaericus could be re-isolated in 5% of the samples 9 months after application.The authors are at the Department of Applied Microbiology & Brewing, Anambra State University of Technology, P.M.B. 5025, Awka, Nigeria.     

Phenotypic variations in re-lysogenization ofBacillus licheniformis with temperate phage

A bacitracin-producing strainBacillus licheniformis ATCC 10716 harbors two types of inducible phages (LP52 and DLP 10716). 156 strains re-lysogenized with phage LP52 were independently isolated from a cured strain UM12 ofB. licheniformis. Those strains were divided into 12 groups based on colony morphology and pigment production. Some of the re-lysogenized strains grew faster than UM12 and others produced more bacitracin than the cured strain. For example, the production of bacitracin by one of the relysogenized strains, L89, was enhanced by about 70% in comparison with UM12. The phenotypic variations observed with re-lysogenized strains might be due to the re-insertion of the phage genome at different sites of the chromosome in addition to the pleiotropic effect assumed.Abbreviations ATCC American Type Culture Collection - DNA Deoxyribonucleic acid - MC Mitomycin C - OD Optical density - PFU Plaque forming unit - rpm Revolutions per minute - UOD Unit of optical density - UV Ultraviolet Definition Specific growth rate (h^-1) - t time (h) - X cell concentration (g/l)     

Cell wall turnover in phosphate and potassium limited chemostat cultures of Bacillus subtilis W23

Turnover in phosphate and potassium limited chemostat cultures of Bacillus subtilis W23 results in the release of over 80% of the wall material present at the time of chasing equilibrium-labelled cultures. The rate at which turnover proceeds is faster in potassium limited cultures than in phosphate limited cultures but in both cases a fraction of the wall material appears to be conserved, or to undergo turnover at a lower rate. Previously we have shown that the polar wall is less active metabolically than the cylindrical wall and it is possible that the apparently conserved wall is that present in the pole.     

Biocontrol: Bacillus penetrans and Related Parasites of Nematodes

Bacillus penetrans Mankau, 1975, previously described as Duboscqia penetrans Thorne 1940, is a candidate agent for biocontrol of nematodes. This review considers the life stages of this bacterium: vegetative growth phase, colony fragmentation, sporogenesis, soil phase, spore attachment, and penetration into larvae of root-knot nematodes. The morphology of the microthallus colonies and the unusual external features of the spore are discussed. Taxonomic affinities with the actinomycetes, particularly with the genus Pasteuria, are considered. Also discussed are other soil bacterial species that are potential biocontrol agents. Products of their bacterial fermentation in soil are toxic to nematodes, making them effective biocontrol agents.