Plasmid stability and alpha-amylase production in batch and continuous cultures of Bacillus subtilis TN106[pAT5

Cell growth and enzyme (alpha-amylase) production characteristics of Bacillus subtilis TN106 containing the recombinant plasmid pAT5 are investigated in batch and continuous cultures using a defined medium with glucose as the limiting nutrient. The batch culture studies demonstrate that the recombinant plasmid, reported earlier(1) to be stably maintained in the host, suffers from segregational and structural instabilities. The structural instability of this strain occurred during culture storage and can be eliminated in bioreactor experiments by using a modified inoculum preparation procedure. Such elimination allows an unbiased investigation of segregational instability via continuous culture studies. Such studies conducted with this fast growing microorganism, in the absence of antibiotic selection pressure, indicate a very efficient glucose utilization (very low residual glucose concentrations) over a wide range of dilution rates (0.16 h(-1) - 0.94 h(-1)). The nearly time-invariant and low residual glucose concentrations at each such dilution rate enable convenient estimation of growth parameters of the host and recombinant cells and frequency of segregational instability from transients in the resulting mixed cultures. The specific alpha-amylase activity exhibits an inverse relationship to the specific growth rate of recombinant cells. The growth of recombinant cells is not affected by the presence of antibiotic (kanamycin). The growth advantage of host cells over recombinant cells diminishes with increasing dilution rate.     

Signal peptide of Bacillus subtilis alpha-amylase

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by the removal of the NH2-terminal 41 amino acid sequence (41 amino acid leader sequence). DNA fragments coding for short sequences consisting of 28 (Pro as the COOH terminus) 29 (Ala), 31 (Ala), and 33 (Ala) amino acids from the translation initiator, Met, in the leader sequence were prepared and fused in frame to the DNA encoding the mature alpha-amylase. The secretion activity of the 33 amino acid sequence was nearly twice as high as that of the parental 41 amino acid sequence, whereas the activity of the 31 amino acid sequence was 75% of that of the parent. In contrast, almost no secretion activity was observed with the 28 and 29 amino acid sequences. The signal peptide cleavage site of the precursor expressed from the plasmid encoding the 33 amino acid sequence was located between Ala and Leu at positions 33 and 34 and that from the 31 amino acid sequence between Thr and Ala at positions 33 and 34. The NH2-terminal amino acid from the latter corresponded to the 3rd amino acid of the mature enzyme. These results indicated that the functional signal peptide of the B. subtilis beta-amylase consists of the first 33 amino acids from the initiator, Met.     

Cultivation of Endomyces magnusii yeasts under batch and continuous conditions at an acid pH

The energy parameters of Endomyces magnusii cells and mitochondria were studied under the conditions of batch and continuous cultivation at different pH of the medium containing ethanol. The yeast was found to be capable of growth in the chemostat regime at D=0.2 h-1. Changes in the pH of the medium from 3.0 to 5.6 almost did not change the parameters characterizing oxidative phoshorylation of the mitochondria (the respiration chain contained three phosphorylation points). This correlated with the nearly identical biomass yield and economical coefficient. The content of RNA, DNA and protein remained unchanged at different pH values whereas the content of lipids increased at acid pH.     

Analysis of metabolic fluxes in batch and continuous cultures of Bacillus subtilis

It is well recognized that metabolic fluxes are the key variables that must be determined in order to understand metabolic regulation and patterns. However, owing to difficulties in measuring the flux values, evaluation of metabolic fluxes has not been an integral part of the most metabolic studies. Flux values for metabolites of glycolysis, tricarboxylic acid (TCA) cycle, and hexose monophosphate (HMP) pathway were obtained for batch and glucose-limited continuous cultures of Bacillus subtilis by combining the information from the stoichiometry of key biosynthetic reactions with the experimental data on concentrations of glucose and metabolic by-products, CO(2) evolution, and oxygen uptake rates. The results indicate that (1) the metabolic fluxes and energetic yield as well as the extent of flux mismatch in metabolic activity of glycolysis and the TCA cycle reactions can be accurately quantified; (2) the flux through the TCA cycle in continuous culture is much in excess of cell energetic and biosynthetic demands for precursors; (3) for the range of growth rates examined the TCA cycle flux increases almost in proportion to growth rate and is significantly repressed only at very high growth rates of batch cultures; and (4) for continuous cultures the isocitrate dehydrogenase catalyzed reaction of the TCA cycle is the major source of the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) used in biosynthesis. (c) 1993 John Wiley & Sons, Inc.     

Autoregulation of alpha-amylase formation in Bacillus subtilis

The phenomenon of self-regulation of alpha-amylase formation in Bacillus subtilis is discovered. The dependence of regulatory effect upon the phase of culture development in shown.     

NH2-terminal processing of Bacillus subtilis alpha-amylase

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by removal of the NH2-terminal 41-amino acid sequence. To study the mechanism of this processing, the extracellular forms of alpha-amylase were analyzed for B. subtilis N7 alpha-amylase cloned and expressed in B. subtilis. The major form (form N34) isolated from log phase cultures in L-broth had an NH2 terminus corresponding to position 34 from the initiator Met but appeared to be microheterogeneous, as judged by native gel electrophoresis. The major forms from stationary phase cultures had NH2 termini at positions 40 (form N40) or 42 (form N42) and were homogeneous. The conversion of the larger to smaller forms could be achieved in culture supernatants or partially purified samples. The process N34----N40 was inhibited by EDTA; N40----N42 was facilitated by Ca2+. Phenylmethylsulfonyl fluoride inhibited the former but not the latter process. These results suggest that the signal peptidase cleavage site 30 decreases 35 is -Ala-Ala-Ala-Ser-Ala-Glu-Thr- (arrow or further upstream) and that proteolytic maturation occurs after secretion, which involves at least two different processing enzymes.     

Membrane-bound and soluble extracellular alpha-amylase from Bacillus subtilis.

Extracellular alpha-amylase was purified to homogeneity from a Marburg strain of Bacillus subtilis. The enzyme is a single polypeptide chain of molecular weight approximately 67,000. Its NH2-terminal amino acid sequence is Leu-Thr-Ala-Pro-Ser-Ile-Lys. A membrane-derived alpha-amylase was solubilizing from membrane vesicles by treatment with Triton X-100 and was highly purified by chromatography on an anti-alpha-amylase-protein A-Sepharose column. Membrane-derived alpha-amylase was indistinguishable from the soluble extracellular enzyme by sodium dodecyl sulfate-gel electrophoresis and radioimmunoassay. The membrane-derived enzyme contains phospholipid. Approximately 30 to 80% of the phospholipid was extracted from the purified enzyme by chloroform:methanol. The extracted phospholipid was predominately phosphatidylethanolamine. Treatment with phospholipase D released phosphatidic acid. Membrane-bound alpha-amylase was latent in membrane vesicles. Release of membrane-bound alpha-amylase from vesicles by an endogenous enzyme was maximal at pH 8.5, was inhibited by metal chelators and diisopropyl fluorophosphate and was stimulated by Ca2+ and Mg2+. The amount of membrane-bound alpha-amylase was related to the level of secretion.     

Development of membrane lipids in the surfactin producer Bacillus subtilis

Processes occurring in the cytoplasmic membrane of the surfactin producer Bacillus subtilis were examined during a 3-d cultivation. The fatty acid composition was found to be almost stable within this interval, except for the early stationary phase when the nonbranched, mostly C(16:0) and C(18:0) (high melting fatty acids), prevailed transiently in the membrane. As for phospholipids, phosphatidylglycerol and phosphatidylethanolamine, representing 73 % of the total in the membranes of exponential cells were partly replaced by cardiolipin, which gradually rose from 5 to 28 % at the end of cultivation. In parallel, steady-state fluorescence anisotropy (r (s)) measurements with 1,6-diphenyl-1,3,5-hexatriene (DPH) indicated a remarkable increase of r (s) DPH during the long-term cultivation and implied a continuous rigidization of the membrane interior. By contrast, the almost constant values of r (s) 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene 4-toluenesulfonate (TMA-DPH) reflected stable microviscosity of the membrane surface region. Thus, the significant increase of high melting fatty acids and cardiolipin in the cytoplasmic membrane together with the progressive rigidization of the membrane interior reflected the cell adaptation to adverse conditions.     

Biodegradation of p-aminoazobenzene by Bacillus subtilis under aerobic conditions

Biological oxidation of organic dyes is important for textile industry wastewater treatment. The aim of this work was to assess the biodegradation kinetics of a specific azo-dye, p-aminoazobenzene. The degradation of p-aminoazobenzene by Bacillus subtilis was examined through batch experiments in order to investigate the effect of p-aminoazobenzene on the bacterial growth rate and elucidate the mechanism of dye degradation. The results proved that B. subtilis cometabolizes p-aminoazobenzene in the presence of glucose as carbon source, producing aniline and p-phenylenediamine as the nitrogen–nitrogen double bond is broken. The azo-dye was found to act as an inhibitor to microbial growth. A mathematical model was developed that describes cellular growth, glucose utilization, p-aminoazobenzene degradation and product formation. Received 26 July 1996/ Accepted in revised form 14 May 1997     

Action of Bacillus subtilis alpha-amylase on native wheat starch

Native starch granules from wheat have been subjected to enzymatic depolymerization with an alpha-amylase from Bacillus subtilis. Crystallites made from short-chain amylose and residues from mild acid hydrolysis have been also tested. Electron microscopy, particle size analysis, DSC, and x-ray diffractometry reveal that enzymatic degradation occurs granule by granule. Gel permeation chromatography shows off the macromolecular nature of the remaining material. In contrast, acid erodes simultaneously all the granules, leading to a splitting into small particles. Crystalline fractions are completely degraded by alpha-amylase. These results support evidence for an active disentanglement of chains, carried out by the different subsites of alpha-amylase molecules. A simple mathematical treatment is proposed to explain the results of the kinetics.     

Biosynthesis of nisin in the subtilin producer Bacillus subtilis ATCC6633

Summary Plasmids having the structural gene of nisin (nis A) combined with the subtilin or two hybrid subtilin-nisin leaders were integrated into the subtilin operon in the chromosome of a nisin-resistant and subtilin-producing strain of B. subtilis by single crossing over. Nisin was produced only when the leader consisted mainly of the nisin part. This indicates that nisin and its leader sequence might work as a couple that makes a recognizable conformation for the subtilin modification enzymes. Therefore recognition does not depend on the primary structure of the leader sequence itself.