Licheniformis Cells Research Articles

Quantitative characterization of biotic iron oxides by analytical electron microscopy

Hydrous ferric oxides (HFO) play a tremendous role in the environment, and as a result, they have often been analyzed for their bulk chemical and mineralogical composition, but rarely at the particle level. This study used a combination of transmission electron microscopy (TEM), parallel electron energy loss spectroscopy (TEM-PEELS), and selected area electron diffraction (TEMSAED), to determine the morphology, stoichiometry, and mineralogy of synthetic biotic HFO and assess the effect of specimen preparation (i.e., dehydration) on their overall chemical composition. A mixture of synthetic lepidocrocite (γ-FeOOH) and goethite (α-FeOOH) was used as reference material for all TEM analyses. HFO were formed by the oxidation of Fe 2+ (pH = 8) in the presence of Bacillus subtilis (bacteria without extracellular polymers) and Bacillus licheniformis cells (with extracellular polymers). Biotic Fe-oxides occurred as small particles (5-10 nm diameter) on the bacterial cell walls and associated exopolymers and were identified as 2-line ferrihydrite by TEMSAED. Quantitative TEM-PEELS results showed that (1) freeze-dried abiotic samples had an Fe/O atomic ratio of 0.54 ± 0.03, whereas the Fe/O ratio of the biotic ferrihydrite formed in the presence of B. licheniformis and B. subtilis was 0.48 ± 0.06 and 0.54 ± 0.08, respectively; (2) air-dried biotic ferrihydrite possessed an Fe/O ratio close to 0.30, whereas the ratio of the abiotic air-dried HFO samples remained unchanged (i.e., 0.51 ± 0.04). These results indicate that quantitative TEM-PEELS can be used for the chemical characterization of Fe-oxides, since the measured Fe/O atomic ratios of the reference HFO are in agreement with their known stoichiometry (i.e., 0.50 for FeOOH). Our results also suggest that ferrihydrite dehydrates during freeze-drying and that specimen preparation is very important when assessing chemical composition

Biosynthesis of exopolysaccharide by a Bacillus licheniformis strain isolated from ropy cider

A strain of Bacillus licheniformis displaying a ropy phenotype was isolated from a French ropy cider. The influence of culture conditions on the production of exopolysaccharide (EPS) was investigated. When B. licheniformis was grown in Man, Rogosa and Sharpe (MRS) medium, the highest amount of EPS was observed at mid exponential growth phase whatever the carbon source, glucose, fructose or sucrose. Interestingly at mid exponential growth phase, EPS amounts did not increase with increasing sugar concentrations. Incubation of B. licheniformis cells in media supplemented with ethanol (1–7%, v/v) revealed that EPS production was enhanced by the presence of ethanol, in exponential as well as in stationary phase. High Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR) analysis of EPS composition indicated that it was a heteropolymer in which mannose was the predominant monosaccharide as it constituted more than 80% of total polysaccharide.

Accumulation of some metal ions on Bacillus licheniformis

Pure species of Bacilluslicheniformis was used to remove ions from aqueous and simulated waste solutions. Metal ion accumulation on B. licheniformis was fast. Maximum uptake occurred at pH 4±0.5 and at 25±3 °C. One gram of dry B. licheniformis was found to accumulate 115 mg cerium, 34 mg copper and 11 mg cobalt from aqueous solutions. The presence of certain foreign ions such as calcium, sodium and potassium decreased the uptake of ions by B. licheniformis, while citrate and EDTA prevent the uptake. Electron microscopic investigations showed that cerium (III), copper (II) and cobalt (II) accumulated extracellulary around the surface wall of B. licheniformis cells. A bio-adsorption mechanism between the metal ions and B. licheniformis cell wall was proposed.

Minimum structure of peptidoglycan required for induction of antibacterial protein synthesis in the silkworm, Bombyx mori

Various peptidoglycan fragments, different in mode of cross-linking and molecular size, were isolated, and the elicitor activity was tested for induction of antibacterial protein synthesis in larvae of Bombyx mori. Linear uncross-linked peptidoglycans from Bacillus licheniformis and Micrococcus luteus were effective elicitors, similar to the directly cross-linked peptidoglycan from B. licheniformis cell wall. The fragments of uncross-linked peptidoglycan with a sugar chain length of four or more were active elicitors, but the disaccharide unit had no elicitor activity. The minimum structure of peptidoglycan required for induction of antibacterial protein synthesis was determined to be two repeating N-acetylglucosamine- N-acetylmuramic acid units with peptide side chains.

Thermostable 1-amylase production by Bacillus licheniformis cells immobilized on polyacrylates with cyclic carbonate groups in the side chain

A considerable increase (up to 35%) of thermostable a-amylase yield was achieved by Bacillus licheniformis cells, immobilized on polymers containing cyclic carbonate groups in comparison to the synthesis by free cells. The increase of enzyme production was dependent on the manner of cell immobilization and on the kind of polymers used. The a-amylase yield by immobilized “growing” cells was higher than that reached by immobilized "washed" cells. The homopolymer poly [(2-oxo-1,3-dioxolan-4-yl) methyl 2-propenoate] and especially its methyl derivative were found to be more suitable for bacterial cell immobilization than the copolymer N-vinylpyrrolydone poly [(2-oxo-1,3-dioxolan-4-yl) methyl2-propenoate]. After 576 h (4 cycles) of repeated batch fermentations, all biocatalysts used showed approximately equal a-amylase activity (2000 U/ml), corresponding to 60-80% residual activity depending on the kind of the biocatalyst.

Anaerobic metabolism in Bacillus licheniformis NCIB 6346.

SUMMARYThe products of anaerobic metabolism of glucose and its derivatives sorbitol, gluconate and glucuronate by Bacillus licheniformis have been determined by proton NMR. Glucose was fermented through mixed-acid fermentation pathways to acetate, 2,3-butanediol, ethanol, formate, lactate, succinate and pyruvate. However, the bacterium was incapable of fermenting the three glucose derivatives. When B. licheniformis cells were incubated anaerobically with glucose in the presence of nitrate, the reduced products and formate did not appear and acetate was formed as the major metabolite. Growth and formation of acetate was also observed when B. licheniformis cells were incubated anaerobically with each of the three glucose derivatives, in the presence of nitrate. A formate-nitrate oxido-reductase system was induced under anaerobic conditions, with increased activities when nitrate was added to the anaerobic growth medium. However no activity was detected when cell; were grown in the presence of molecular oxygen. Formate-nitrate oxido-reductase activity was absent in chlorate-resistant mutants isolated spontaneously or following Tn917 insertional mutagenesis. The spontaneous mutants fermented glucose in the presence of nitrate suggesting that they were incapable of nitrate respiration, due to a deficiency in one or more components of the formate-nitrate oxido-reductase system. Two insertional mutants exhibited elevated β-galactosidase activity when grown in the presence of nitrate.

Screening of a growing cell immobilization procedure for the biosynthesis of thermostable α-amylases

Studies were carried out on α-amylase production with immobilized cells of twoBacillus strains. High yields of thermostable αamylases were obtained byBacillus licheniformis 44MB82-G, resistant to glucose catabolite repression and a thermophileBacillus brevis 174, after repeated batch cultivation (270–600 h) of the immobilized biocatalysts. Various cell immobilization techniques were compared, including entrapment in gel matrices (Ca-alginate,x-carrageenan, agar, and their combinations with polyethylene oxide), adsorption on cut disks of polymerized polyethylene oxide, and fixation on formaldehyde activated acrylonitrile-acrylamide membranes. The optimal immobilization parameters (gel and biocatalyst concentration, initial cell quantity) were determined. Among the gels and supports tested, agar,x-carrageenan, agar/polyethylene oxide gels, and the membranes were found to be suitable for immobilization and biocatalysts with high operational stabilities were obtained. An enzyme yield of 2750 U/mL culture medium was reached in the fifth repeated batch run with membrane-immobilizedBacillus licheniformis cells. This activity represented 176% of the corresponding yield obtained in batch fermentation with free cells. Higher amylase yields than the activity of the control were reached in all experiments and repeated batch runs with immobilizedBacillus brevis cells.

Thermostable ?-amylase production by immobilized Bacillus licheniformis cells in agar gel and on acrylonitrile/acrylamide membranes

Thermostable ?-amylase production by immobilized Bacillus licheniformis cells in agar gel and on acrylonitrile/acrylamide membranes

Thermostable α-amylase production by immobilized Bacillus licheniformis cells in agar gel and on acrylonitrile/acrylamide membranes

Immobilized cells of Bacillus licheniformis 44MB82-G were used for the production of thermostable alpha-amylase. The immobilization was carried out by entrapment in agar gel or by binding to formaldehyde-activated acrylonitrile/acrylamide membranes. The alpha-amylase production after 144 h of cultivation of membrane immobilized cells was 40% higher in comparison with the free cells. The respective value for the agar-entrapped cells was 22%. Similar trends were observed in the repeated batch fermentations performed with the immobilized cells. The scanning electron micrographs (SEM) of the immobilized cells gave additional information about their binding to the respective carriers.

Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwlL, of Bacillus licheniformis.

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38,994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-L-alanine amidase, which has a M(r) value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.

Molecular cloning of a sporulation-specific cell wall hydrolase gene of Bacillus subtilis

Southern hybridization analysis of Bacillus subtilis 168S chromosomal DNA with a Bacillus licheniformis cell wall hydrolase gene, cwlM, as a probe indicated the presence of a cwlM homolog in B. subtilis. DNA sequencing of the cwlM homologous region showed that a gene encoding a polypeptide of 255 amino acids with a molecular mass of 27,146 Da is located 625 bp upstream and in the opposite direction of spoVJ. The deduced amino acid sequence of this gene (tentatively designated as cwlC) showed an overall identity of 73% with that of cwlM and of 40% with the C-terminal half of the B. subtilis vegetative autolysin, CwlB. The construction of an in-frame cwlC-lacZ fusion gene in the B. subtilis chromosome indicated that cwlC is induced at 6 to 7 h after sporulation (t6 to t7). The spoIIIC (sigma K) mutation and earlier sporulation mutations greatly reduced the expression of the cwlC-lacZ fusion gene. Northern hybridization analysis using oligonucleotide probes of the cwlC region indicated that a unique cwlC transcript appeared at t7.5 and t9. Transcriptional start points determined by primer extension analysis suggested that the -10 region is very similar to the consensus sequence for the sigma K-dependent promoter. Insertional inactivation of the cwlC gene in the B. subtilis chromosome caused the disappearance of a 31-kDa protein lytic for Micrococcus cell walls, which is mainly located within the cytoplasmic and membrane fractions of cells at t9. The CwlC protein hydrolyzed both B. subtilis vegetative cell walls and spore peptidoglycan.

Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase.

A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27,513. The gene was designated as cwlM, for cell wall lysis. The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region. Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity. The gene product purified from E. coli cells harboring a cwlM-bearing plasmid exhibited a M(r) value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28). The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B. licheniformis and B. subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference. CWLM prepared from B. subtilis cells harboring a plasmid containing cwlM had a similar M(r) value to that from E. coli. Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.

Enzymes of Glutamate and Glutamine Biosynthesis in Bacillus licheniformis

This chapter discusses the purification and characterization of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase and an examination of the pools of ammonia assimilatory metabolites in Bacillus licheniformis . B licheniformis contains an NADP + -specific glutamate dehydrogenase that apparently serves an anabolic function. The enzyme was purified and it was found to be distinct from the glutamate dehydrogenase-like activity of glutamate synthase. The glutamate synthase from B. licheniformis was purified to apparent homogeneity and it showed to be quite similar, in physical properties, to the enzyme from E. coli and K. aerogenes . The glutamine synthetase of B. licheniformis was purified to near homogeneity and shown to be a dodecamer of 616,000 MW. In B. licheniformis , the enzymes of ammonia assimilation appear to serve biosynthetic functions only. The kinetic characteristics of the glutamate synthase and glutamine synthetase appear to be in harmony with their ability to provide relatively high pools of glutamate and glutamine under all growth conditions. Although B. licheniformis cells respond to the nitrogen source in the growth medium by altering growth rates and levels of nitrogen catabolic enzymes, glutamine synthetase does not appear to be involved in the control of these responses.

Stereological analysis of plasmolysis in logarithmic-phase Bacillus licheniformis.

The plasmolytic response of Bacillus licheniformis 749/C cells to the increasing osmolarity of the surrounding medium was quantitated with stereological techniques. Plasmolysis was defined as the area (in square micrometers) of the inside surface of the bacterial wall not in association with bacterial membrane per unit volume (in cubic micrometers) of bacteria. This plasmolyzed surface area was zero when the cells were suspended in a concentration of sucrose solution lower than 0.5 M, but increased linearly when the sucrose molarity rose above 0.5 M, reaching a plateau value of 3.61 micrometers2/micrometers3 in 2 M sucrose. In contrast, when the bacterial cells were treated with lysozyme plasmolysis increased abruptly from 0.06 micrometers2/micrometers3 in 0.75 M sucrose to 4.09 micrometers2/micrometers3 in 1 M sucrose. When the time of exposure was prolonged, the degree of plasmolysis increased gradually for the duration of the experiment (30 min) after exposure to 1 M sucrose without lysozyme, whereas with lysozyme plasmolysis reached a maximum (4.09 micrograms2/micrometers3) in 2 to 5 min. The examination of ultrastructure showed that the protoplast bodies of lysozyme-treated cells in 1 M sucrose and untreated cells in 2 M sucrose are maximally retracted from the intact wall of the bacteria; hardly any retraction of protoplasts could be seen for untreated cells in 1 M sucrose. The data suggest that the B. licheniformis cells are isoosmotic to 800 to 1,100 mosM solutions, but are able to withstand much greater osmotic pressure with no signs of plasmolysis because the cell wall and the plasma membrane are held in close association, perhaps by a covalent bond. It is likely that lysozyme weakens this bond by degradation of the peptidoglycan layer. Cellular autolysis also weakens this wall-membrane association.

In vitro production of bacitracin by proteolysis of vegetative Bacillus licheniformis cell protein.

The action of a sporulation-specific seryl protease on antibiotic-free extracts of Bacillus licheniformis cells yields a peptide that is identified as bacitracin by its biological activity, its spectral properties, and its comigration with genuine bacitracin in both paper and thin-layer chromatography. During proteolysis, a chemical structure is generated with the spectral properties of a delta-2 thiazoline ring. The yield in vitro, 4 microgram of bacitracin per mg of protein, is less than the maximal yield from sporulating cells, 75 microgram of bacitracin per mg of cell protein, but is a linear function of the amount of protein in the reaction system. Approximately 30% of the protein yielding the antibiotic is ribosomal associated, and only 25% of that amount can be removed by washing with 1 M NH4Cl. The substrate protein is a constant fraction of the cell protein throughout exponential growth and very early sporulation stages of culture development.

Enzymatic adaptation by bacteria under pressure.

A study of enzymic adaptation under hydrostatic pressure by moderately barotolerant bacteria that can grow at pressure up to about 500 atm revealed that some adaptive processes are relatively insensitive to pressure, whereas others are sufficiently barosensitive to compromise survival capacity in situations requiring adaptation to new substrates under pressure. Examples of the former include adaptation of Escherichia coli to arabinose catabolism for growth and adaptation of Streptococcus faecalis to catabolism of lactose, ribose, or maltose. Examples of the latter include derepression of the lac operon in Escherichia coli and induction of penicillinase synthesis by Bacillus licheniformis. For both these barosensitive systems, pressure had little effect on enzyme levels in constitutive strains or in bacteria that had previously been induced at 1 atm. Moreover, it had no detectable effect on penicillinase secretion. However, pressures of 300 to 400 atm were found to reduce markedly rates and extents of enzyme synthesis by bacteria undergoing derepression or adaptation. This inhibitory effect of pressure was reflected in greater barosensitivity with extended lag and slower growth of initially unadapted Escherichia coli cells inoculated into minimal medium with lactose as sole source of carbon and fuel, and by major reductions in the minimal inhibitory concentrations of penicillin G for unadapted B. licheniformis cells inoculated into complex, antibiotic-containing media. Cyclic adenosine 5'-monophosphate did not reverse pressure inhibition of derepression of the lac operon, and catabolite repression was complete under pressure. However, derepression of the lac operon was more sensitive to pressure at low concentrations of inducer than at high concentrations. Apparent volume changes for derepression were 94 and 60 ml/mol at inducer concentrations of about 0.5 and 5 mM, respectively. Pressure was found not to be inhibitory for uptake of beta-galactosides; in fact, it was somewhat stimulatory. Therefore, results were interpreted in terms of inducer binding and subsequent conversion of an operator-inducer-repressor complex to inactive repressor and operator. Both reactions appeared to result in an increase in volume, the former more so than the latter. We found also that 200 atm was actually stimulatory for growth of Escherichia coli in minimal media, and the bacterium was in a sense barophilic.

53b] β-Lactamase (Bacillus licheniformis)

Publisher Summary This chapter discusses the purification procedure of β-lactamase (Bacillus licheniformis). The most convenient and effective method of releasing cell bound β-lactamase appears to be the treatment of lysed cells with trypsin. However, exposure of B. licheniformis cells to the action of chymotrypsin, pronase, or an aqueous solution of butan-1-ol (4% v/v) also releases the greater part of the enzyme attached to the cells. It is found that membrane-bound β-lactamase may also be released by the action of sodium deoxycholate. Trypsin-released β-lactamase can be purified by a combination of ammonium sulfate fractionation and DEAE-cellulose chromatography. The use of affinity chromatography has been introduced for the small-scale purification of B. licheniformis β-lactamase.

Selective extraction of polymers from cell walls of gram-positive bacteria.

Brief heating of Bacillus Licheniformis cell walls at 100 degrees C in aqueous buffers of pH3.0-4.0 removes some polymers but not others from the mucopeptides. For example, relatively undegraded teichuronic acid can be extracted at 100 degrees C in 20min at pH3.0 whereas the teichoic acids are not removed. Similar specificity can be shown with walls from three other species of micro-organism.

18Oxygen Probes of Protein Turnover, Amino Acid Transport, and Protein Synthesis in Bacillus licheniformis

Abstract Experiments were performed in which protein turnover was measured by incorporation of 18O from H218O into protein. Since proteolysis causes the incorporation of 18O into the carboxyl group of amino acids and the incorporation of this amino acid leads to protein with the peptide carboxyl oxygen labeled, turnover can be determined quantitatively. The rate of protein turnover during the first 5 hours after growth was found to be about 22% per hour. Spores contain an amount of 18O that allows the suggestion that the average protein turns over two or more times during sporulation. To examine the possibility of oxygen exchange during protein synthesis, Bacillus licheniformis cells were grown through eight generations, a 200-fold increase in mass, on a medium of glucose plus a mixture of amino acids that were labeled with 18O in the carboxyl groups. The protein of the cells contained oxygen, after appropriate corrections, at an atom per cent excess similar to that of the added amino acids. It is concluded that neither amino acid transport nor protein synthesis involve mechanisms that lead to significant loss or exchange of amino acid carboxyl oxygens.

The regulation of aspartokinase in Bacillus licheniformis

The specific activity of aspartokinase (ATP: l-aspartate 4-phosphotransferase, EC 2.7.2.4) decreases rapidly to a 10 to 20-fold lower level at the end of the growth phase in Bacillus licheniformis grown on a minimal glucose-salts medium. Concurrent with this decrease in specific activity is a loss of sensitivity to feedback inhibition by l-lysine and a slower decrease in the concerted inhibition caused by l-lysine and l-threonine. Growth of the cells in the minimal medium plus l-lysine and l-threonine caused the production of aspartokinase with characteristics similar to those of enzyme from cells harvested 4 h after the end of growth. Growth in the presence of either l-lysine or l-threonine did not affect the specific activity of the enzyme but did alter its inhibition sensitivity. The addition of l-lysine to the medium during growth on the minimal medium caused a rapid loss of sensitivity to lysine inhibition, 50% desensitization occurring in 15 min. These latter conditions did not produce an alteration in the specific activity of the enzyme or in the concerted inhibition by lysine and threonine. Despite the marked alteration in feedback-inhibition properties, changes in physical or kinetic properties of purified aspartokinase were not observed. Throughout the purification steps there was no evidence that more than one aspartokinase was present in B. licheniformis cells. It is concluded that this enzyme is unusually plastic and assumes alternate forms depending on the physiology of the cell.