Effect of amino acids on exoprotease synthesis by Bacillus thuringiensis

The influence of certain L-amino acids and their mixtures on the synthesis of exoprotease from Bacillus thuringiensis was studied. Physiological experiments showed that the mixture of 20 amino acids added to the artificial medium repressed the synthesis of exoprotease. Among the compounds studied there are both the compounds which stimulate the synthesis of exoprotease (glutamic and aspartic acids, glycine), and the compounds which repress the synthesis of the enzyme (proline, tryptophane, tyrosine, asparagine, serine, cystein). None of the amino acids caused a change in the exoprotease activity. It has been assumed that the repression of the protease synthesis in the presence of the amino acids is accomplished by ammonium ions, which are formed when using the amino acids of Bac. thuringiensis. The glutamine synthetase activity of cells was determined during the growth of Bac. thuringiensis both on a medium containing triptone and after the addition of certain amino acids to the cell suspension. The correlation between the influence of different amino acids on the synthesis of exoprotease and the glutamine synthetase activity was demonstrated.     

Proteinases in growth and spore formation of Bacillus thuringiensis]

Some serine proteases and leucine aminopeptidases were detected inside and outside the cells during the analysis of three crystalline and two acrystalline strains of Bac. thuringiensis var. galleriae. The data obtained on the protease formation during growth and sporulation and the level of their activity are indicative of intracellular proteases involvement in spore- and crystal formation. The enzymes isolated from the culture medium do not probably take part in these processes. The intracellular enzymes may account for the different crystal protein composition of various strains due to limited proteolysis of crystal proteins in the course of biosynthesis.     

Using a combined transcription-translation system for determining the role of cryptic plasmids of Bacillus thuringiensis

The possibility of entomocyde crystal protein synthesis was studied using a heterological cell-free system with Bacillus thuringiensis plasmid DNA as template. The high level of template activity is usual for Bac. thuringiensis plasmid DNA. Immunochemical studies of the in vitro synthesized polypeptides showed that Bac. thuringiensis plasmid DNA does not direct crystal protein synthesis.     

Specific phages used for differentiation of the entomopathogenic bacterium Bacillus thuringiensis]

The sensitivity to specific phages and morphological, physiological, and antigenic properties were compared among several strains of Bacillus thuringiensis isolated from insects inhabiting various geographical zones. All 43 cultures assigned to Bac. thuringiensis var. sotto and 198 among 170 cultures classed as Bac. thuringiensis var. dendrolimus were found to belong to Bac. thuringiensis var. dendrolimus. None of these cultures was resistant to its specific phage. The same was true of 22 studied cultures of Bac. thuringiensis var. galleriae. Only two among studied 45 cultures of Bac. thuringiensis var. thuringiensis were resistant to phages specific for this variety. Therefore, the abundance of variants resistant to specific phages in natural conditions differs among the varieties of Bac. thuringiensis. In most cases, cultures of the same variety of Bac. thuringiensis isolated from various insects inhabiting different geographical zones are identical by their sensitivity to specific phages and by other important characteristics.     

Changes in the level of bacitracin synthesis in autolysin-resistant strains of Bacillus licheniformis

Production of bacitracin by Bac. licheniformis 1001 and its spontaneous autolysin resistant variants was studied. It was found that the antibiotic activity of some variants was 1.5--2 times higher than that of the initial strain. No differences in the activity of serine exoprotease in the initial strain and resistant variants were observed. The latter variants lost their resistance to autolysis in 2--3 subcultures on solid and liquid nutrient media. their antibiotic activity in these cases decreased to the control level. The study indicates that there is a phenomenologic relation between the autolytic and antibiotic activities of Bac. licheniformis. The nature of the relation is not known yet. Possibly, it is due to changes in the specific metabolic steps connected with regulation of bacitracin synthesis.     

The multiple effect of plasmid RP4::Mu cts 62 in transcipient bacilli

Transfer of conjugative hybrid plasmid RP4::Mu cts 62 from Escherichia coli into Bac. cereus, Bac. thuringiensis, Bac. mesentericus and Bac. polymyxa cells led to the multiple effects on the structure and physiology of bacillus cells. It has resulted in a decrease of the bacillus vitality, in the accelerated autolytic decay of cells, in the delay of cell growth and reproduction rate in liquid and solid media, in the disruption of ultrastructure of the cell membrane and its surface layer.     

Exoprotease Activity of Two Marine Bacteria during Starvation

Exoprotease activity during 120 h of total energy and nutrient starvation was examined in two marine bacteria, Vibrio sp. strain S14 and Pseudomonas sp. strain S9. The activity was determined by spectrophotometric measurement of the rate of release of soluble color from an insoluble azure dye derivative of hide powder (hide powder azure). Starved cells of both strains (5 h for S14, and 4 or 24 h for S9) showed greater extracellular proteolytic activity than at the onset of starvation. The exoprotease activity of cells starved for longer periods of time then decreased, but was found to be present at significant levels throughout the starvation period studied (120 h). The accumulation of exoprotease activity in the bulk phase during starvation indicated that both strains constitutively excreted extracellular proteases. As deduced from experiments with chloramphenicol, de novo protein synthesis during starvation was required for the production and/or release of the exoproteases into the surrounding environment. The degradation of hide powder azure allowed an immediate increase in respiration rate, also by long-term-starved cells. This suggests that metabolic systems are primed to respond to the availability of substrates, allowing the cells to recover rapidly. The regulation of exoprotease activity was also studied and found to be different in the two strains. Casamino Acids repressed exoprotease activity in Pseudomonas sp. strain S9, whereas a mechanism similar to catabolite repression was found for Vibrio sp. strain S14 in that glucose repressed activity and cyclic AMP reversed this effect. The exoproteases appeared to be metalloproteinases because the addition of EDTA to cell-free starvation supernatants from both strains significantly inhibited the activity of the proteases.     

Relationship between exoprotease activity, spore and crystal formation and virulence of bacteria of the group Bacillus thuringiensis]

Experiments were carried out to investigate the activity, spore and crystal formation and virulence of Bacillus thuringiensis. Bacteria of this group showed different exoprotease activity. Strains of one serotype differed in the level and time of occurrence of peak enzyme activity. Exoprotease activity of the bacteria cannot be indicative of their toxicity because it did not correlate with their virulence. Exoprotease activity can be used to characterize bacteria during their classification and to determine finer differences between strains within one serotype.     

Improvement of bioinsecticides production through adaptation of Bacillus thuringiensis cells to heat treatment and NaCl addition

AIMS: The present work aimed to increase yields of delta-endotoxin production through adaptation of Bacillus thuringiensis cells to heat shock and sodium chloride and to investigate their involvements in bioinsecticides production improvement. METHODS AND RESULTS: Growing B. thuringiensis cells were heat treated after different incubation times to study the response of the adaptative surviving cells in terms of delta-endotoxin synthesis. Similarly, adaptation of B. thuringiensis cells to sodium chloride was investigated. Adaptation to combined stressors was also evaluated. When applied separately in the glucose-based medium, 20-min heat treatment of 6-h-old cultures and addition of 7 g l(-1) NaCl at the beginning of the incubation gave respectively 38 and 27% delta-endotoxin production improvements. Heat shock improved toxin synthesis yields, while NaCl addition improved delta-endotoxin production by increasing the spore titres without significant effect on toxin synthesis yields. Cumulative improvements (66%) were obtained by combination of the two stressors at the conditions previously established for each one. Interestingly, when the similar approach was conducted by using the large scale production medium based on gruel and fish meal, 17, 8 and 29% delta-endotoxin production improvements were respectively, obtained with heat shock, NaCl and combined stressors. CONCLUSIONS: Heat treatment of vegetative B. thuringiensis cells and NaCl addition to the culture media improved bioinsecticides production. Heat treatment increased toxin synthesis yields, while addition of NaCl increased biomass production yields. Cumulative improvements of 66 and 29% were obtained in glucose and economic production media, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Overproduction of bioinsecticides by B. thuringiensis could be obtained by the combination of heat treatment of vegetative cells and addition of NaCl to the culture medium. This should contribute to a significant reduction of the cost of B. thuringiensis bioinsecticides production and utilization, and also manage for higher toxin content in the bioinsecticides, which is very interesting from a practical point of view because fewer spores would be disseminated into the ecosystem.     

Instability of the auxotrophic markers in Bacillus thuringiensis

Auxotrophic mutants of Bacillus thuringiensis have been isolated and compared with the mutants of Bacillus subtilis. It has been found that after spore formation and a prolonged storage, the considerable part of cells in a population of some strains display a genetically unstable additional requirement for arginine with high frequency. The correlation between tetracycline sensitivity and unstable requirement for arginine and other growth factors has been found. The possible reasons for instability of genetic markers in Bac. thuringiensis are discussed.     

Burkholderia pseudomallei Requires Zn(sup2+) for Optimal Exoprotease Production in Chemically Defined Media

The effects of various medium components on exoprotease production by Burkholderia pseudomallei were studied in order to understand the production of virulence factors by this pathogenic organism. FeSO(inf4), NaCl, and MgSO(inf4) do not significantly affect exoprotease activity, and CaCl(inf2) has only a slight influence. Conversely, ZnCl(inf2) plays a fundamental role since it drastically increases exoprotease production.     

Inhibition of the Plaquing Efficiency of T4r+ Bacteriophage by Subtilisin

The mechanism by which the replicative cycle of T4r(+) phage is inhibited by certain nonhost bacterial systems was investigated. Some Bacillaceae, especially Bacillus subtilis, decreased the plaquing efficiency of this virus more than 95% within 24 hr of exposure. Sarcina lutea and Micrococcus sp. both failed to cause any significant change in the infectivity of T4r(+) phage. Preliminary investigations into the nature of the inhibitory substance(s) suggested that an extracellularly elicited protein was at least partially responsible for this effect. Further analysis has implicated subtilisin, an exoprotease from B. subtilis, as the cause of some, if not all, of the observed decrease in plaquing efficiency. Gel-filtration chromatography of control and treated (14)C-labeled T4r(+) phage showed a wide dispersal of phage-specific material of these particles after 24 hr of exposure to pure subtilisin or to expended medium exoprotease from B. subtilis. It was concluded that B. subtilis exoprotease is capable of chemically altering the structure of the phage capsid, thus causing a decrease in its plaquing efficiency.     

对双歧杆菌DM9227株的实验研究 Ⅲ.双歧杆菌DM9227株与蜡样芽胞杆菌DM423株共生关系的初步研究

本文对厌氧的双歧杆菌DM9227株与需氧的蜡样芽胞杆菌DM423株的共生关系进行了初步研究。将DM9227株与DM423株分别单独及按一定比例等量混合接种于BS肉汤内进行培养,间隔一定时间检测两菌在培养基内菌数的消长情况。结果表明DM423株与DM9227株之间呈偏生关系,即DM423株对DM9227株有促进作用,且在两菌比例适宜(1:100)情况下促进作用更明显,而DM9227株对DM423株有抑制作用。进一步将接种DM423株的Nb琼脂平板与接种DM9227株的BS琼脂平板在普通环境下一起密闭培养,可见厌氧的DM9227株生长良好,提示DM423株可能是通过消耗氧气,降低培养基的Eh,从而促进DM9227株生长的。     

Genetic analysis of the pAD1 hemolysin/bacteriocin determinant in Enterococcus faecalis: Tn917 insertional mutagenesis and cloning

Thirty-seven nonhemolytic/nonbacteriocinogenic mutations in Enterococcus (Streptococcus) faecalis plasmid pAD1 were generated by Tn917 insertion. All were found to belong to one of two complementation classes. Each class of mutants secreted either hemolysin/bacteriocin (Hly/Bac) component A or L into the culture medium. DNA encoding Hly/Bac was cloned in Escherichia coli in which both components of the hemolysin were expressed individually and collectively. The region encoding components A and L was further defined by deletion analysis and physically mapped. A total of approximately 8.4 kilobases of pAD1 DNA were observed to be required for hemolysin expression. Hly/Bac activity of the wild-type and the inactive L substance was observed to be heat stable. Active Hly/Bac resulting from incubating separately secreted components A and L was also found to be heat stable. The results indicate that component A activates component L and that activated component L possesses the Hly/Bac activity. Component A was also observed to be associated with host immunity to the Hly/Bac.     

Cloning and comparative characteristics of genes coding two structurally distant delta-endotoxins of Bacillus thuringiensis var. galleriae and kurstaki

Representative total DNA libraries of Bac. thuringiensis var. kurstaki (strain Dipel) and galleriae (strain 11-67) have been constructed on the basis of phasmid vector lambda pSL5. Recombinant phasmid clones, carrying delta-endotoxin-coding genes of both subspecies have been isolated by means of immunoenzyme screening. Restriction mapping and partial nucleotide sequence determination have demonstrated that phasmid lambda pOC2, isolated from var. kurstaki DNA library, contains the complete delta-endotoxin-coding gene, identical to the one, described by Schnepf H.E. et al. J. Biol. Chem. 1985. V. 260. P. 6264. Recombinant phasmids lambda pOC10 and 11 have been shown to contain the full-sized gene, coding delta-endotoxin of Bac. thuringiensis var. galleriae. The protein products of the cloned genes have been characterized by Western-blot analysis and bioassays. The absence of substantial homology of two genes, evidenced by Southern-blot hybridisation, correlates with sufficiently big differences in biological specificity of the corresponding proteins. This is in accordance with our previous data on N-terminal amino acid sequence determination of delta-endotoxins of those subspecies of Bac. thuringiensis.     

Induction of cystine transport activity in human fibroblasts by oxygen

The transport activity for cystine in cultured human fibroblasts decreased after incubation of the cells under a low oxygen concentration. After the incubation for 48 h under 3% oxygen, the Vmax of the transport was decreased to less than one-third of that of the control cells, with little change in Km. The similar transport activity was observed in the cells cultured under 3% oxygen for 10-40 days with several times of passages. When these low oxygen-cultured cells were incubated under room air, the activity was enhanced with a lag of about 4 h and was almost completely restored within 24 h. This restoration required protein synthesis. The cystine transport activity increased by 50% after exposure of the cells to hyperoxia (40% oxygen). From these results it is concluded that the transport activity for cystine is induced by oxygen. In contrast, little change in the transport activities for alanine and leucine occurred in the cells exposed to the corresponding hypoxia or hyperoxia. Since the cystine transported into the cells is reduced to cysteine and the cysteine readily exits to the culture medium where it autoxidizes to cystine, a cystine-cysteine cycle across the plasma membrane has been postulated. Since the autoxidation of cysteine in the culture medium was markedly slowed down under the low oxygen concentration, the change in the cystine transport activity in response to the oxygen concentration was regarded as pertinent. Induction of the cystine transport activity may constitute a protective mechanism against the oxidative stress, to which the culture cells are exposed, by providing the cells with cysteine which is mainly incorporated into glutathione.     

Protein composition of crystals (delta-endotoxin) of different serotypes of Bac. thuringiensis]

The crystals of entomopathogenic protein from Bac. thuringiensis contain admixtures of proteinases adhering to their surfaces. A newly developed technique of protease inactivation allowed to estimate the true protein composition of the crystals of various strains of Bac. thuringiensis. It was shown that the crystals of all strains (with the exception of V and VIII) are composed of only one protein with molecular weights of 145,000, 135,000 and 130,000, depending on the strain. The crystals of serotype VIII and the majority of the V serotype strains have two proteins with molecular weights of 135,000 and 130,000. A method for estimation of the protein composition of crystal without their preliminary isolation from a crystal--spore mixture is proposed.     

Effect of pancreatic DNAse on DNA synthesis in Bacillus subtilis]

An addition of pancreatic DNAse to the cultural medium is found to stimulate DNA synthesis and proliferation of Bacillus subtilis cells. Pancreatic DNAse induces a single-stranded disruption of Bac. subtilis DNA, which may act as a mechanism of DNA synthesis increase and of the culture growth acceleration.     

The regulation of hydroxymethylglutaryl-CoA reductase in cultured cells

Growth-stimulated synchronized cells exhibit a rapid increase in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.88) activity prior to the onset of DNA synthesis. Under normal culture conditions, HMG-CoA reductase activity exhibits wide variations among experiments. To determine whether this phenomenon is dependent on cell replication, we used J774 macrophage-like cells to compare changes in reductase activity in cells synchronized by serum deprivation and then growth-stimulated by fresh media containing serum to unsynchronized cells treated with fresh media and serum. Under these conditions, no increase in [3H]thymidine incorporation into cell DNA was seen in unsynchronized cells, but a large increase was observed in synchronized cells 10-12 h after media change. Although the growth characteristics differed between the cells, reductase activity was low at the time of media change and increased 10 to 20-fold 5-10 h after media change, returning to basal levels by 24 h in both synchronized and unsynchronized cells. This pattern of reductase activity was observed in unsynchronized cells from a variety of cell lineages, although the magnitude of the changes varied. Fluctuations of [14C]acetate incorporation into cholesterol were observed in parallel to alterations in reductase activity. LDL receptor expression also paralleled the changes in reductase activity, but scavenger receptor expression was not affected. Addition of lipoproteins at the time of media change inhibited the rise in reductase activity by 80-90%. The increase in reductase activity was not due to a stimulation of cholesterol efflux into the medium, but evidence for the secretion into the media of an inhibitory factor was obtained. These results suggest that cell requirements for cholesterol are not always directly related to replication, and that standard culture conditions induce transient fluctuations in reductase activity and lipoprotein receptor expression.