Properties of Corynebacterium diphtheriae cultivated in the medium prepared from inedible raw materials

A nutrient medium for the cultivation of C. diphtheriae toxigenic strain was developed on the basis of raw materials unsuitable for use as foodstuffs and its main physico-chemical and cultivation properties were studied. The morphological, cultural, biochemical and toxigenic properties of C. diphtheriae cultivated in the experimental medium with horse serum were evaluated. As revealed in this study, C. diphtheriae retained their properties after prolonged cultivation and storage in the newly developed medium, both liquid and with agar added. The medium has a number of advantages: it is economical, raw materials for its production are readily available, the medium is free of ballast substances.     

Effect of lysogenic conversion of Corynebacterium diphtheriae strain on its iron assimilation and spectrum of cellular proteins

Growth of the isogenic pair of C. diphtheriae strains, the nontoxigenic strain C7 (-) and the toxigenic strain C7 (beta), was studied under conditions of limited availability of the iron source. The growth of the toxigenic strain was shown to depend on the concentration of iron in the medium to a lesser degree than the nontoxigenic one. Lysogenic conversion results in the synthesis of additional iron-dependent proteins, absent in C. diphtheriae initial nontoxigenic strain C7 (-). Special attention was paid to proteins with a mol. wt. 66 kD, synthesized by the toxigenic strain irrespective of the concentration of iron in the medium, while in the toxigenic strain these proteins were detected only under conditions of iron deficiency.     

Growth characteristics of Escherichia coli HB101[pGEc47] on defined medium

This paper shows that differences in growth behavior of Escherichia coli strain HB101 and strain HB101[pGEc47] can be related to yeast extract-enriched medium rather than plasmid properties. An optimal medium for growth of E. coli HB101[pGEc47] was designed based on the individual yield coefficients for specific medium components (NH4+ 6 g g-1, PO43- 14 g g-1, SO42- 50 g g-1). The yield coefficient for L-leucine depends on the glucose content of the medium (20 g g-1 for 3% glucose, 40 g g-1 for 1% glucose) and the yield coefficient for L-proline depends on the cultivation mode (20 g g-1 for batch cultivation, 44 g g-1 for continuous cultivation). Growth on defined medium after medium optimization is as rapid as on complex medium (0. 42-0.45 h-1). The critical dilution rate (DR) in the defined medium above which undesired production of acetic acid occurs is in the range of 0.23-0.26 h-1.     

Control of the mutagenicity of a new immunomodulator

New immunostimulator STP, peptide isolated from the cultivation medium of Streptococcus species producer strain TOM-1606 by chromatographic purification, was controlled for mutagenicity. The preparation, introduced into mice in doses of 6.7 X 10(2) - 6.7 X 10(4) micrograms/kg, i.e. exceeding the stimulating dose 1000-fold, did not induce the appearance of micronuclei in polychromatophilic erythrocytes of the marrow, the fixation of the material being made 24, 48 and 72 hours after the injection. In Ames' test on Salmonella typhimurium strains TA 98 and TA 100 neither native STP, nor STP activated with the microsomal fraction of rat liver enzymes did not increase the frequency of reversions to histidine independence. The absence of mutagenic properties in STP was demonstrated by the parallel pronounced genotoxic action of a number of known mutagens used as positive controls.     

Optimization of casein-based semisynthetic medium for growing of toxigenic Corinebacterium diphtheriae in a fermenter

Diphtheria toxin is produced by growing Corinebacterium diphtheriae either in a semisynthetic casein-based medium or in the Pope-Lingood meat extract based medium. The World Health Organization advises the use of the semisynthetic one, as it has important advantages. Data on the composition of casein-based media and their ability to support high toxin production are not freely available. Important factors affecting toxin production during C. diphtheriae cultivation are the pH of the culture medium and the concentration of casein hydrolysate and of Fe2+. We established that the optimal pH for toxin production is 7.2. The highest yield of toxin was obtained using a casein hydrolysate concentration of 35.0 g/L and a Fe2+ concentration of 0.05-0.41 microg/mL. Under these conditions, diphtheria toxin with higher purity and yield compared with the batches obtained using the meat-based medium of Pope-Lingood was produced.     

Purification and structural characterization of siderophore (corynebactin) from Corynebacterium diphtheriae

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin.     

Optimization of cultivation medium for the production of Bacillus intermedius 3-19 glutamyl endopeptidase

The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of glutamyl endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of glutamyl endopeptidase.     

Optimization of Cultivation Medium for the Production of Bacillus intermedius 3-19 Glutamyl Endopeptidase

The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of glutamyl endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of glutamyl endopeptidase.     

A new dried nutrient medium for the isolation of Corynebacterium diphtheriae (its development and experimental study)

A new dried diagnostic medium for the isolation of C. diphtheriae has been developed on the basis of aminopeptide. This new aminopeptide-based medium compares favorably with Buchin's medium in its growth and inhibitory properties.     

Regulation of toxinogenesis in Corynebacterium diphtheriae: mutations in the bacterial genome that alter the effects of iron on toxin production

Mutants of Corynebacterium diphtheriae C7(beta) that are resistant to the inhibitory effects of iron on toxinogenesis were identified by their ability to form colonies surrounded by toxin-antitoxin halos on agar medium containing both antitoxin and a high concentration of iron. Chromosomal mutations were essential for the altered phenotypes of four independently isolated mutant strains. During growth in deferrated liquid medium containing various amounts of added iron, these mutants differed from wild-type C. diphtheriae C7(beta) in several ways. Their growth rates were slower under low-iron conditions and were stimulated to various degrees under high-iron conditions. The concentrations of iron at which optimal toxin production occurred were higher for the mutants than for wild-type C. diphtheriae C7(beta). Toxin production by the mutants during growth in low-iron medium occurred throughout the period of exponential growth at nearly constant rates that were proportional to the bacterial growth rates. In contrast, toxin production by wild-type C. diphtheriae C7(beta) in similar low-iron cultures occurred predominantly during the late exponential phase, when iron was a growth-limiting nutrient. Additional studies demonstrated that these mutants had severe defects in their transport systems for ferric iron. We propose that the altered regulation of toxinogenesis by iron in our mutants was caused by the severe defects in their iron transport systems. As a consequence, the mutants exhibited a low-iron phenotype during growth under conditions that permitted wild-type C. diphtheriae C7(beta) to exhibit a high-iron phenotype.     

Physiologo-biochemical properties of a strain of Beijerinckia mobilis 1phi Phn+--a degrader of polycyclic aromatic hydrocarbons

Beijerinckia mobilis 1f capable of degrading polycyclic aromatic hydrocarbons (PAHs) was isolated from a soil contaminated with creosote. Strain 1f could utilize phenanthrene and naphthalene as the sole sources of carbon. The mean rate of phenanthrene degradation during culture growth was 7-8 micrograms/(ml h). After cultivation under nonselective conditions, strain 1f retained its ability to degrade phenanthrene. Cometabolism considerably widened the range of PAHs that could be transformed by strain 1f. The strain was able to grow in a mineral medium with creosote as the sole source of carbon. After 30 days of cultivation in this medium, the total concentration of PAHs decreased from 665.5 mg/l to 170 mg/l.     

Mutants of Corynebacterium diphtheriae PW8 that produce toxin in medium with excess iron.

Two mutants that produce toxin in medium with excess iron were isolated from the PW8 strain of Corynebacterium diphtheriae. These mutants produced as much toxin in medium containing excess iron (3 mug of Fe2+ per ml) as did the parent PW8 strain in iron-depleted medium, and they will be very useful for easy production of toxin.     

The effect of the nature of the iron sources on the growth of different Corynebacterium diphtheriae strains

In the study of the influence of organic and inorganic sources of iron on the growth of 5 C. diphtheriae clinical isolates bacterial growth was found to depended on the nature of the source of iron and its concentration. Differences between the strains in the level of growth, observed when ferric sulfate was used as the only source of iron in the medium, were established. Quantitative differences in the concentrations of inorganic and organic sources of iron, necessary for growth, were determined. The influence of three chemical chelators on the growth of C. diphtheriae under the conditions of iron deficiency in the culture medium was studied. The results of the study are indicative of the possibility of the differentiation of C. diphtheriae isolated according to the level of iron consumption.     

Development of the biological preparation enatin with broad-range antimicrobial action

Physiological and biochemical traits of epiphytic spore forming bacteria Bacillus pumilis BIM V-263 were examined. The nutrient medium and conditions for submerged cultivation of the strain were selected. The growth dynamics and antagonistic activity during cultivation in a laboratory fermenter ANKUM-2M were studied. The results provide grounds for development of the biological preparation Enatin with broad-range antimicrobial effect. The plant-protective and growth-stimulating effect of Enatin was examined in laboratory and field experiments. The preparation holds promise as means for biological control of crop pathogens.     

The regulation of Bacillus intermedius glutamyl endopeptidase biosynthesis in the recombinant Bacillus subtilis strain during sporulation

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th h of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30-150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50-100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different.     

Preparation and characteristics of protoplasts of Streptomyces kanamyceticus

To prepare actively regenerating protoplasts of S. kanamyceticus, the influence of the conditions of the mycelium cultivation, the culture age, lytic conditions, composition of the regeneration medium, the procedure of the culture inoculation to the regeneration medium and other parameters were studied. The study resulted in development of optimal conditions for preparation of S. kanamyceticus protoplasts in a number of 1.10(9) protoplasts per ml. The cultivation on the ST medium with 10 to 15% sucrose and addition of glycine up to 1% for 30 hours (the stationary growth phase) followed by treatment of the culture with lysozyme in an amount of 2 mg/ml for 1 hour at 32 degrees C provided preparation of up to 100% of actively regenerating protoplasts free of mycelium fragments. The size of the protoplasts increased up to 1.5 micron against the usually observed size of 0.7 to 1.0 micron with using modified lyzing buffer with 20% of sucrose according to the method recommended for S. erythreus. However, 50 to 70% of the protoplasts had point of linear regions in the cell walls, which suggested that spheroplasts were mainly forming and the phenomenon was associated with the characteristic properties of the strain cell wall structure.     

一株产纤维素酶细菌紫外线诱变研究

以一株产纤维素酶细菌为主要研究对象进行紫外线诱变研究,通过考察紫外线诱变时间、诱变距离、菌体浓度和菌龄对其产纤维素酶能力的影响,并用纤维素刚果红培养基进行复筛,然后进行液体静置发酵产酶试验,确定了最适紫外线诱变组合条件。结果表明最适紫外线诱变组合条件为:诱变时间180 s、诱变距离25 cm、菌体浓度为10-5、菌龄为24h,在此条件下进行2d液体静置发酵,其酶活最高值为38.30U/mL,与原始出发菌株相比酶活力提高了40.08%,该研究对提高纤维素酶活性具有一定参考价值和借鉴意义。     

Development of the Biological Preparation Enatin with Broad-Range Antimicrobial Action

Physiological and biochemical traits of the epiphytic spore-forming bacteria Bacillus pumilusBIM B-263 were examined. The nutrient medium and conditions for submerged cultivation of the strain were selected. The growth dynamics and antagonistic activity during cultivation in an ANKUM-2M laboratory fermenter were studied. The results provide grounds for development of the biological preparation Enatin, with broad-range antimicrobial effect. The plant-protective and growth-stimulating effect of Enatin was examined in laboratory and field experiments. The preparation holds promise as means for biological control of crop pathogens.