Subtilis Growth Research Articles

Synthesis and Biological Evaluation of Chromium Bioorganometallics Based on the Antibiotic Platensimycin Lead Structure

The recent discovery of the natural product platensimycin as a new antibiotic lead structure has triggered the synthesis of numerous organic derivatives for structure-activity relationship studies. Herein, we describe the synthesis, characterization and biological evaluation of the first organometallic antibiotic inspired by platensimycin. Two bioorganometallic compounds containing (eta(6)-pentamethylbenzene)Cr(CO)(3) (2) and (eta(6)-benzene)Cr(CO)(3) (3), linked by an amide bond to the aromatic part of platensimycin, were synthesized. Their antibiotic activities were tested against B. subtilis 168 (Gram positive) and E. coli W3110 (Gram negative) bacterial strains. Both compounds were found to be inactive against E. coli but derivative 2 inhibits B. subtilis growth at a moderate MIC value of 0.15 mM. To test the intrinsic toxicity of chromium, several chromium salts along with {eta(6)-(3-pentamethylphenyl propionic acid)}Cr(CO)(3) (5) and {eta(6)-(3-phenyl propionic acid)}Cr(CO)(3) (6) were tested against both bacterial strains. No activity was observed against E. coli for any of the compounds; B. subtilis growth was not inhibited by Cr(NO(3))(3) and only very weakly by 5, K(2)Cr(2)O(7) and Na(2)CrO(4) at MIC values of 0.5, 0.68 and 1.24 mM, respectively. Compounds 2, 3, 5 and 4 (the pure organic analogue of 2) show similar cytotoxicity against HeLa, HepG2 and HT-29 mammalian cell lines. Furthermore, the cellular uptake and the intracellular distribution of compounds 2, 3 and Cr(NO(3))(3) in B. subtilis were studied using atomic absorption spectroscopy to gain insight in to the possible cellular targets. Compound 2 was found to be readily taken up and distributed almost equally among cytosol, cell debris and cell membrane in B. subtilis.

Toxicity Effect of Pb(II) on Two Different Kinds of Microbes Measured by Microcalorimetry

AbstractIn this work, microcalorimetric technique was used to analyze Pb(II) toxic action on the metabolic activities of Candida humicola and Bacillus subtilis. The experimental results revealed that Pb(II) had a stimulating effect on C. humicola and B. subtilis growth at a relatively low concentration (10.0 g·mL−1); while, C. humicola and B. subtilis were inhibited completely when the concentrations were up to 320.0 and 160.0 g·mL−1, respectively, and the relationships between growth rate constant (k) and doses of Pb(II) were approximately linear for the two microbes at certain concerntrations. At the same time, their cell dry weight and turbidity (OD600) during growth were also obtained. Their thermogenic curves of the growth coincided well with their turbidity curves, elucidating that the microcalorimetric method agreed with the routine microbiology methods. All of these corroborate the validity and sensitivity of the microcalorimetric technique to investigate the toxic effect of Pb(II) on soil microorganisms.

The toxic effect of cadmium on pure microbes using a microcalorimetric method and a biosensor technique

A microcalorimetric technique based on microbes heat-output was explored to evaluate the effect of Cd (II) on Bacillus subtilis and Candida humicola. The power-time curves of the growth metabolism of Bacillus subtilis and Candida humicola and the effect of Cd (II) on it were studied by using a TAM III microcalorimeter, ampoules method at 28°C. For the evaluation of toxic effect on pure micro-organisms, the maximum peak-heat output power (P max) in the growth phase, the growth rate constants (k), the log phase heat effects (Q log), and the total heat effect (Q T) for Bacillus subtilis and Candida humicola were determined. Dissolved oxygen and biochemical oxygen demand (BOD) were evaluated by a biosensor. Cadmium has been regarded as the essential biological trace element. Cd (II) solutions of different concentration have different effects on Bacillus subtilis and Candida humicola growth metabolism. The higher concentrations of Cd (II) inhibit the growth of Candida humicola (1600–3200 μ g·mL−1), Bacillus subtilis (240–480 μg·mL−1); the lower concentrations can promote the growth of both micro-organism. The values of cell dry weight is also showed in conformity in the cell dry weight changes to the micro-organisms' growth time. Comparison of growth curves of two micro-organisms showed that both the trends of biochemical oxygen demand were exhibiting regressive changes with the passage of time during their generation times (t G). Results from ultraviolet spectrophotometer and precision pH meter all showed that the control growth curves were visioning same trends with the thermodynamic curves of micro-organisms measured by microcalorimeter.

A Procedure for High-Yield Spore Production by Bacillus s ubtilis

Bacillus subtilis spores have a number of potential applications, which include their use as probiotics and competitive exclusion agents to control zoonotic pathogens in animal production. The effect of cultivation conditions on Bacillus subtilis growth and sporulation was investigated in batch bioreactions performed at a 2-L scale. Studies of the cultivation conditions (pH, dissolved oxygen concentration, and media composition) led to an increase of the maximum concentration of vegetative cell from 2.6 x 10(9) to 2.2 x 10(10) cells mL(-)(1) and the spore concentration from 4.2 x 10(8) to 5.6 x 10(9) spores mL(-)(1). A fed-batch bioprocess was developed with the addition of a nutrient feeding solution using an exponential feeding profile obtained from the mass balance equations. Using the developed feeding profile, starting at the middle of the exponential growth phase and finishing in the late exponential phase, an increase of the maximum vegetative cell concentration and spore concentration up to 3.6 x 10(10) cells mL(-)(1) and 7.4 x 10(9) spores mL(-)(1), respectively, was obtained. Using the developed fed-batch bioreaction a 14-fold increase in the concentration of the vegetative cells was achieved. Moreover, the efficiency of sporulation under fed-batch bioreaction was 21%, which permitted a 19-fold increase in the final spore concentration, to a final value of 7.4 x 10(9) spores mL(-)(1). This represents a 3-fold increase relative to the highest reported value for Bacillus subtilis spore production.

Toxic Effect of Inorganic Arsenite [As(III)] on Metabolic Activity of Bacillus subtilis by Combined Methods

In this study, microcalorimetry and measurement of culture turbidity were applied to evaluate the As(III) toxic effect on the metabolic growth of Bacillus subtilis. Using a multichannel thermal activity monitor, the power-time curves of the metabolic activity of B. subtilis during growth in the absence and presence of various concentrations of As(III) were obtained and studied. The turbidity changes during B. subtilis growth with As(III) were investigated by ultraviolet-visible spectrophotometry and the data agree with the results obtained by microcalorimetry. As(III) of various concentrations has different effects on the metabolic growth of B. subtilis with biphasic dose-response relationships called hormesis [i.e., low-concentration stimulation (10 microg/mL) and high-concentration inhibition (20-160 microg/mL). Typical J-shapes of the relationship between the growth rate constant (k) and c, and toxicity at the half-inhibitory concentration (IC(50)) of 98.82 +/- 7.32 microg/mL were obtained. The similarity between the two methods corroborates the validity and sensitivity of the microcalorimetric technique to investigate the toxic effect of As(III) on microorganisms.

Involvement of PlsX and the acyl-phosphate dependent sn-glycerol-3-phosphate acyltransferase PlsY in the initial stage of glycerolipid synthesis in Bacillus subtilis

The gene responsible for the first acylation of sn-glycerol-3-phosphate (G3P) in Bacillus subtilis has not yet been determined with certainty. The product of this first acylation, lysophosphatidic acid (LPA), is subsequently acylated again to form phosphatidic acid (PA), the primary precursor to membrane glycerolipids. A novel G3P acyltransferase (GPAT), the gene product of plsY, which uses acyl-phosphate formed by the plsX gene product, has recently been found to synthesize LPA in Streptococcus pneumoniae. We found that in B. subtilis growth arrests after repression of either a plsY homologue or a plsX homologue were overcome by expression of E. coli plsB, which encodes an acyl-acylcarrier protein (acyl-ACP)-dependent GPAT, although in the case of plsX repression a high level of plsB expression was required. B. subtilis has, therefore, a capability to use the acyl-ACP dependent GPAT of PlsB. Simultaneous expression of plsY and plsX suppressed the glycerol requirement of a strict glycerol auxotrophic derivative of the E. coli plsB26 mutant, although either one alone did not. Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [14C]-labeled G3P to synthesize [14C]-labeled LPA, whereas those from DeltaplsY cells did not. The results indicate unequivocally that PlsY is an acyl-phosphate dependent GPAT. Expression of plsX corrected the glycerol auxotrophy of a DeltaygiH (the deleted allele of an E. coli homologue of plsY) derivative of BB26-36 (plsB26 plsX50), suggesting an essential role of plsX other than substrate supply for acyl-phosphate dependent LPA synthesis. Two-hybrid examinations suggested that PlsY is associated with PlsX and that each may exist in multimeric form.

The role of bacteria on heavy-metal extraction and uptake by plants growing on multi-metal-contaminated soils

Four bacterial isolates were examined for their ability to increase the availability of water soluble Cu, Cr, Pb and Zn in soils and for their effect on metals uptake by Zea mays and Sorghum bicolor. Random Amplified Polymorphic DNA (RAPD) analysis was used to show that the bacterial cultures were genetically diverse. Bacterial isolates S3, S28, S22 and S29 had 16S rRNA gene sequences that were most similar to Bacillus subtilis, Bacillus pumilus, Pseudomonas pseudoalcaligenes and Brevibacterium halotolerans based on 100% similarity in their 16S rDNA gene sequence, respectively. Filtrate liquid media that had supported B. pumilus and B. subtilis growth significantly increased Cr and Cu extraction from soil polluted with tannery effluent and from Cu-rich soil, respectively, compared to axenic media. The highest concentrations of Pb (0.2 g kg−1), Zn (4 g kg−1) and Cu (2 g kg−1) were accumulated in shoots of Z. mays grown on Cu-rich soil inoculated with Br. halotolerans. The highest concentration of Cr (5 g kg−1) was accumulated in S. bicolor roots grown in tannery-effluent-polluted soil inoculated with a mixed inoculum of bacterial strains. These results show that bacteria play an important role in increasing metal availability in soil, thus enhancing Cr, Pb, Zn and Cu accumulation by Z. mays and S. bicolor.

Identification ofin VivoSubstrates of the Chaperonin GroEL fromBacillus subtilis

We investigated GroEL substrates from Bacillus subtilis 168 using the single-ring mutant of B. subtilis GroEL. We identified 28 candidates for GroEL substrates, of which Spo0B, Ald, Eno, SpoIIP, and FbaA were involved in spore formation, and Rnc, Tuf, Eno, Tsf, and FbaA were essential for B. subtilis growth. As observed at the protein level, the amount of SpoIIP interaction with GroEL increased at 3 h after initiation of sporulation.

Involvement of YkoN in production of a new phospholipid in Bacillus subtilis Marburg

To understand the physiological role of the ykoN gene of Bacillus subtilis, the gene was expressed from the IPTG-inducible spac promoter in B. subtilis cells. When lipid composition of the cells induced for ykoN expression was examined, a new spot of phospholipid was found on the thin layer chromatogram. Induction of ykoN in Escherichia coli cells produced a similar new spot. The new spot was produced in mutant E. coli cells lacking cardiolipin or phosphatidylethanolamine, but not in the cells lacking phosphatidylglycerol. The result suggests an involvement of phosphatidylglycerol in production of the new spot. Examination of the lipid composition during the stages of B. subtilis growth revealed that the new phospholipid was produced four hours after cessation of logarithmic growth, consistent with an increase in the promoter activity of ykoN in the late stage in sporulation. YkoN has the pentapeptide lipase/esterase motif (Gly-X-Ser-X-Gly), and shows a lipolytic activity (Matsuura et al., in press). The mutant YkoN (S205A) in which the central serine residue of the motif is replaced with alanine produced the new phospholipid in the amount as small as only 1/10 of the wild type, suggesting that the lipolytic activity of YkoN is involved in the new phospholipid production.

부패된 팥앙금으로부터 분리된 미생물의 동정과 그 균의 생리적 특성

In order to determine the causes of sweetened adzuki ann spoilage, the characteristics of microorganism isolated from spoiled adzuki ann were investigated. The isolated microorganism was gram-positive, rod-shaped and spore-forming bacteria; its surface was smooth and glazed. From the results of the assimilation test of 46 different biochemicals by the Vitec 2 Compact test and comparison of the cellular wall composition of fatty acid by the data bank of Midi sherlock system, the microorganism was identified as Bacillus subtilis. D-value of the B. subtilis spore was 4.85 min at 115℃, 0.69 min at 121℃ and 0.48 min at 125℃; Z-value was 9.71. The Bacillus subtilis growth was not observed below water activity of 0.92 at 45℃. However, bacteria growth increased gradually as water activity increased above 0.93.

Antibacterial lysine analogs that target lysine riboswitches

Lysine riboswitches are bacterial RNA structures that sense the concentration of lysine and regulate the expression of lysine biosynthesis and transport genes. Members of this riboswitch class are found in the 5' untranslated region of messenger RNAs, where they form highly selective receptors for lysine. Lysine binding to the receptor stabilizes an mRNA tertiary structure that, in most cases, causes transcription termination before the adjacent open reading frame can be expressed. A lysine riboswitch conceivably could be targeted for antibacterial therapy by designing new compounds that bind the riboswitch and suppress lysine biosynthesis and transport genes. As a test of this strategy, we have identified several lysine analogs that bind to riboswitches in vitro and inhibit Bacillus subtilis growth, probably through a mechanism of riboswitch-mediated repression of lysine biosynthesis. These results indicate that riboswitches could serve as new classes of antibacterial drug targets.

Inhibition of Bacillus anthracis and Potential Surrogate Bacilli Growth from Spore Inocula by Nisin and Other Antimicrobial Peptides

The ability of nisin, synthetic temporin analogs, magainins, defensins, and cecropins to inhibit Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus subtilis growth from spore inocula was determined using well diffusion assays. Nisin, magainin II amide, and defensins were inhibitory in screening against B. anthracis Sterne or B. cereus ATCC 7004, but only nisin inhibited virulent B. anthracis strains. The MICs of nisin against the 10 Bacillus strains examined were 0.70 to 13.51 μg/ml. Synthetic temporin analogs also inhibited B. anthracis but were not as potent as nisin. None of the strains examined were appropriate B. anthracis surrogates for testing sensitivity to antimicrobial peptides.

The synthesis and role of the mechanosensitive channel of large conductance in growth and differentiation of Bacillus subtilis

A translational lacZ fusion of the Bacillus subtilis mscL gene that encodes the mechanosensitive channel of large conductance (MscL) was expressed at significant levels during log phase growth of B. subtilis, and the level of mscL-lacZ expression was increased 1.5-fold by growth in medium with high salt (1 M NaCl). However, in growth media with either low or high salt, mscL-lacZ expression fell drastically beginning in the late log phase of growth, and fell to even lower levels during sporulation, although a significant amount of beta-galactosidase from mscL to lacZ was accumulated in the developing spore. Deletion of mscL had no effect on B. subtilis growth, sporulation or subsequent spore germination. The DeltamscL strain also grew as well as the wild-type parental strain in medium with 1.2 M NaCl. While log phase wild-type cells grown with 1.2 M NaCl survived a rapid 0.9 M osmotic downshift, log phase DeltamscL cells rapidly lost viability and lysed when subjected to this same osmotic downshift. However, by the early stationary phase of growth, DeltamscL cells had become resistant to a 0.9 M osmotic downshift.

The GTP-binding Protein YlqF Participates in the Late Step of 50 S Ribosomal Subunit Assembly in Bacillus subtilis

Bacillus subtilis YlqF belongs to the Era/Obg subfamily of small GTP-binding proteins and is essential for bacterial growth. Here we report that YlqF participates in the late step of 50 S ribosomal subunit assembly. YlqF was co-fractionated with the 50 S subunit, depending on the presence of noncleavable GTP analog. Moreover, the GTPase activity of YlqF was stimulated specifically by the 50 S subunit in vitro. Dimethyl sulfate footprinting analysis disclosed that YlqF binds to a unique position in 23 S rRNA. Yeast two-hybrid data revealed interactions between YlqF and the B. subtilis L25 protein (Ctc). The interaction was confirmed by the pull-down assay of the purified proteins. Specifically, YlqF is positioned around the A-site and P-site on the 50 S subunit. Proteome analysis of the abnormal 50 S subunits that accumulated in YlqF-depleted cells showed that L16 and L27 proteins, located near the YlqF-binding domain, are missing. Our results collectively indicate that YlqF will organize the late step of 50 S ribosomal subunit assembly.

Secretion of functional human interleukin-3 from Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis is well-known for its huge capacity to produce secreted bacterial enzymes. Nevertheless, the secretion of pharmaceutically interesting recombinant proteins by this organism is frequently inefficient. This paper documents for the first time on the optimisation of B. subtilis for the production of human interleukin-3 (hIL-3), a four-helix bundle cytokine, which stimulates the proliferation and differentiation of a broad range of blood cells. By developing a host-vector system on the basis of the multiple protease-deficient B. subtilis strain WB700 and a multicopy plasmid containing two tandemly positioned strong promoters plus an efficient signal sequence, the hIL-3 protein was efficiently produced and secreted into the growth medium. As verified by SDS-PAGE, mass spectrometry and cross-linking experiments with a thiol-specific reagent, intact and properly folded hIL-3 was purified from the B. subtilis growth medium. Bioactivity tests showed that the isolated hIL-3 was able to specifically induce proliferation of the hIL-3-dependent leukaemia cell line MO7e. Using the eight-fold protease-deficient strain WB800 the hIL-3 accumulation in the growth medium was increased to levels up to 100 mg l(-1).

Effect of Plantagomajor on cell proliferation in vitro

Plantago major (Plantaginaceae) is popularly used to treat tumors, infections and as a blood purifier. Aqueous, methanol, chloroform and hexane extracts of the aerial parts (leaves and seeds) were added to CD 1 mice bone marrow and spleen cultures incubated at 37 °C for 72 h, and also added to Escherichia coli, Bacillus subtilis and Candida albicans cultures, while methanol extract dilutions were added to HTC-15, OVCAR, UISO and KB cell line cultures. Doses of 0.4 and 0.2 mg/mL of aqueous and methanol extracts increased the bone marrow cell concentration by 2.70- and 3.15-fold, respectively, and increased the spleen cell concentration by 3.38- and 6.39-fold, respectively ( p < 0.001). Aqueous extract inhibited Bacillus subtilis growth from 78 to 21%; hexane extract inhibited the growth of Escherichia coli, and methanol and chloroform extracts weakly inhibited the growth of Bacillus subtilis and Escherichia coli, respectively. Methanol extract (1 μg/mL) decreased the UISO and OVCAR cell concentrations to 59 and 82%, respectively. Data demonstrate for the first time that Plantago major has hematopoietic activity in vitro.

Growth and antibacterial activity of Lentinula edodes in liquid media supplemented with agricultural wastes

Antibacterial activity of Lentinula edodes against Bacillus subtilis was evaluated in cell-free filtrates obtained after growth in 14 different culture media. The highest B. subtilis growth inhibition was promoted by filtrates of growth media supplemented with rice bran, vermiculite or molasses. L. edodes dry mycelial biomass in liquid culture with 0.5% added rice bran was 3.2 mg/ml, after growth for 30 days at 25oC without shaking, and 4.3 mg/ml under orbital shaking (150 rpm). However, antibacterial activity, detected between 20 and 24 days of incubation of stationary cultures, was absent in filtrates of aerated cultures. Temperatures of 20-25oC enhanced both growth and antibacterial activity. Optimum pH for L. edodes mycelial growth was 3.0-3.5, while for production of antibacterial substance(s) it was 4.5. Our results indicated that incubation conditions that enhance mycelial growth are quite different from those necessary for production of antibacterial substance(s) by L. edodes.

The antimicrobial activity of extracts of the lichen Cladonia foliacea and its (-)-usnic acid, atranorin, and fumarprotocetraric acid constituents.

The antimicrobial activity of the chloroform, diethyl ether, acetone, petroleum ether, and ethanol extracts of the lichen Cladonia foliacea and its (-)-usnic acid, atranorin, and fumarprotocetraric acid constituents against 9 bacteria and fungi has been investigated. The extracts and pure compounds alone were found active against the same bacteria and the same yeasts. Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, Proteus vulgaris, Listeria monocytogenes, Aeromonas hydrophila, Candida albicans, and Candida glabrata growth were inhibited. In addition, the MICs of the extracts, (-)-usnic acid, atranorin and fumarprotocetraric acid were determined.

Genome biology of a model bacterium, Bacillus subtilis

Bacillus subtilis has served as a model bacterium in biochemical, genetic and molecular biological studies. The complete genome sequence of B. subtilis was determined by an international consortium, and a collection of knockout mutants of genes whose specific functions were not known was also generated by an international effort. As a result, 2810 knockout and 135 IPTG-dependent mutants have been constructed. It has been estimated that 275 genes, including 25 genes of unknown function, are essential for B. subtilis growth in rich medium at a moderate temperature in aerobic conditions. The B. subtilis genome has 17 sigma factors and about 250 DNA binding transcriptional regulators. In order to develop a comprehensive picture of the regulatory network of gene expression in B. subtilis, a systematic effort to collect expression profiles of wild type cells and knockout mutants of regulator genes is in progress. Mutant resources and genome-wide functional data sets will further assist in elucidation of a complete picture of the functional network involving all B. subtilis gene products.

Kinetics of the Action of Na2SeO3 on Bacillus subtilis Growth as Studied by Microcalorimetry

AbstractMicrocalorimetric bioassay for acute cellular toxicity is based on metabolic heat production from cultured cells. The biological response to toxicants is the inhibition of the heat production rate in cells, and toxicity is expressed as the concentration of toxicant that is 50% effective in this inhibition (IC50). In mis paper, the effect of Na2SeO3 on Bacillus subtilis growth was investigated at 37 °C by microcalorimetry. The relationship between growth rate constants (k) and concentration of Na2SeO3 (c) shows a logarithmic normal distribution, and lC50 is 20.3 μg/mL. All these thermokinetic information is readily obtained by an LKB 2277–204 heat conduction microcalorimeter. Microcalorimetry is a quantitative, inexpensive, and versatile method for toxicology research.