Stability of rapidly labelled messenger ribonucleic acid in Bacillus amyloliquefaciens during the phases of minimum and maximum extracellular enzyme formation.

The stability of rapidly labelled hybridizable messenger RNA in both exponential and post-exponential phase cells of Bacillus amyloliquefaciens was measured in terms of the rate of loss of its radioactivity. In the exponential phase, where 96% of the mRNA was specific for cell proteins and only 4% was exoprotein mRNA, the label was lost exponentially from the rapidly labelled hybridizable mRNA fraction with a half-life of six minutes at 30 °C. The antibiotic rifampicin, at a concentration of 10 μg/ml, had no effect on the characteristics of decay of this exponential-phase mRNA. In the post-exponential phase, where there were equal amounts of cell protein and exoprotein-specific mRNA, rapidly labelled hybridizable mRNA decayed exponentially in the presence of rifampicin (10 μg/ml), with a half-life of six minutes at 30 °C. In the absence of rifampicin the characteristics of decay were more complex. The evidence available suggested that this was due to the superimposition of a component attributable to reincorporation of degradation products of radioactive RNA on the characteristic exponential decay pattern of the post-exponential mRNA.Measurement of the stability of active mRNA, by studying the loss of ability to incorporate l-[¹⁴C]leucine into protein in the presence of rifampicin (10 μg/ml), gave half-lives of 4.5 minutes and six minutes, respectively, for exponential and post-exponential material.     

Evidence for extrusion of unfolded extracellular enzyme polypeptide chains through membranes of Bacillus amyloliquefaciens.

The production of extracellular alpha-amylase and protease by protoplasts of Bacillus amyloliquefaciens has been achieved. The production of enzymically active protease was totally dependent on a high concentration of either Mg2+, Ca2+, or spermidine, but production of active alpha-amylase was not. This cation dependence of protease production was seen immediately upon addition of lysozyme to intact cells. The cations could prevent the inactivation of protease and alter the cytoplasmic membrane configuration of protoplasts. Production of active alpha-amylase and protease by protoplasts was totally inhibited by proteolytic enzymes such as trypsin, alpha-chymotrypsin, or the organism's purified extracellular protease. The evidence suggests that these degradative enzymes act specifically on the emerging polypeptide of the extracellular enzyme and that the polypeptide emerges in a conformation different from that of the native molecule.     

Accumulation of messenger RNA for extracellular enzymes as a general phenomenon in Bacillus amyloiquefaciens

Recently it has been established that in harvested Bacillus amyloliquefaciens there exists a pool of messenger RNA which can support de novo extracellular protease production for 80 minutes Both, McInnes, Hanlon, May &; Elliott 1972. The present study shows that this is not peculiar to protease; the other two extracellular enzymes under study, α-amylase and ribonuclease, show similar characteristics. Synthesis of all three extracellular enzymes is insensitive to rifampicin and actinomycin D for extended time periods after cessation of RNA and cellular protein synthesis.     

Processing of the prepropeptide portions of the Bacillus amyloliquefaciens neutral protease fused to Bacillus subtilis alpha-amylase and human growth hormone during secretion in Bacillus subtilis.

A set of nested 3'-terminal deletions of the prepropeptide of the Bacillus amyloliquefaciens neutral protease gene was constructed. Alpha-amylase and human growth hormone were secreted using these truncated genes in Bacillus subtilis. The level of the secreted alpha-amylase varied with the region for the truncated prepropeptide contained in the fusion gene but was independent of its length. Even though length of the prepropeptide varied, the mobilities of secreted alpha-amylases were the same as that of the control alpha-amylase derived from the alpha-amylase clone, pTUB4 (Yamazaki et al., 1983). Analyses of the secreted N-terminal amino acid sequences confirmed that they were all identical to that of the authentic one. Precursor proteins of the alpha-amylase were found in the cell-associated fraction, suggesting that the prepropeptide portion was processed during secretion. On the other hand, the N-terminus of hGH secreted using one of these prepropeptide portions varied by 1 to 4 additional N-terminal amino acid residues derived from the junction sequence between the sequence for propeptide portion and mature hGH or from C-terminal region of the propeptide portion. These results suggest that the prepropeptide portion can be generally processed even in the heterogeneous fusion. A probable mechanism of processing and maturation of the fusion gene products is also discussed.     

Thermostable extracellular protease of Bacillus stearothermophilus: factors affecting its production

A strain of protease-producing Bacillus stearothermophilus has been isolated. Glycerol was the best carbon source for production whereas yeast extract was the best nitrogen source. The bacterium could grow up to 70°C but optimum protease production was at 60°C. Best initial pH for protease production was 5. Alkaline pH inhibited production. The enzyme was stable at 60°C for 18 h and was inhibited by EDTA, PMSF and HgCl₂.The authors are with the Enzyme and Microbial Technology Group, Faculty of Science and Environmental Studies, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor, Malaysia     

The role of the promoter and leader sequences of extracellular ribonuclease from Bacillus amyloliquefaciens in regulation of enzyme biosynthesis

It is shown that in the medium rich with inorganic phosphate there is a stimulation of biosynthesis of ribonuclease from B. amyloliquefaciens (barnase) by actinomycin D, while biosynthesis of ribonucleases from B. intermedius (binase) and B. pumilus (KNase Bpu) in these conditions was suppressed. Features of biosynthesis of binase and RNase Bpu, directed by the barnase promoter, and also expression of chimeric gene of RNase Bpu with leader peptide of barnase were investigated. It was established that stimulation of synthesis of extracellular ribonuclease from B. amyloliquefaciens in the presence of actinomycin D was defined by structure of leader sequences.     

Dynamics of the formation of extracellular protease and the ATP pool in a thermophilic strain of Bacillus brevis]

The dynamics of varying ATP concentration was studied in the cells of the thermophilic strain of Bacillus brevis 224 synthesizing a neutral exoprotease. Some irregularities during the primary and secondary growth of the culture corresponded to the changes in the amount of ATP. A sharp decrease (by a factor of ca. 100) in the level of ATP pool was detected in the bacterial cells after 2.5 hours of their exponential growth; their growth ceased and the synthesis of protease was depressed. A decrease in the rate of the enzyme synthesis coincides with an increase in the concentration of ATP in the cells.     

The effect of rifampicin on the stability of the messenger ribonucleic acid of Bacillus amyloliquefaciens as determined by DNA:RNA hybridization.

Rifampicin at a concentration of 10 mug/ml completely inhibited protein synthesis in exponential-phase cultures of Bacillus amyloliquefaciens. At this same concentration the drug was shown to have no effect on the stability of mRNA, determined as the functional and hybridizable material, when compared with hybridizable mRNA in an uninhibited system. In each case, the half-life of the mRNA had a value in the range 5 +/- 1 min, at 30 degrees C.     

Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli

alpha-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J. L., C. N. McGrath, K. R. Smith, and D. A. Agard. 1988. Gene (Amst.). 69: 237-244). The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D. R. 1970. Methods Enzymol. 19:599-613). Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325) or in trans (Silen, J. L., and D. A. Agard. 1989. Nature (Lond.). 341:462-464) for the proper folding and extracellular accumulation of the enzyme. The maturation process is temperature sensitive in E. coli; unprocessed precursor accumulates in the cells at temperatures above 30 degrees C (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325). Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E. coli outer membrane. The active site mutant Ser 195----Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures. When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane. However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium. Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful. Taken together, these results suggest that only properly folded and processed forms of alpha-lytic protease are efficiently transported to the medium.     

Purification of the extracellular protease of Bacillus licheniformis and its inhibition by bacitracin.

Sporulating cells of Bacillus licheniformis excrete three seryl proteases that are of similar size, 28,000 daltons, but of different charge at pH 6. The peptide antibiotic bactracin is released from the cells at the same time and exists, in part, as a bacitracin-protease complex that is stable throughout chromatographic procedures employed in enzyme purification. However, preextraction of crude protease with CHCl3 and subsequent gel filtration effect separation of the antibiotic and the enzyme. Three purified, bacitracin-free proteases, designated CMC I, CMC II, and CMC III and whose ratios of total activity are 1:3.7:10.3, respectively, are obtained by chromatography on carboxymethyl cellulose. The major component, CMC III, is inhibited by commercial bacitracin at near-physiological concentrations of the antibiotic.     

Bacillus subtilis var. amyloliquefaciens biosynthesis of extracellular protease possessing coagulase activity and formed under conditions of limiting the nitrogen sources in the medium

An exocellular protease having the activity of coagulase was synthesized by Bacillus subtilis var. amyloliquefaciens when the growth medium contained no nitrogen sources. The removal of a nitrogen source from the medium was found to induce the synthesis of exoproteases by washed bacterial cells. Protein as a sole source of nitrogen in the medium inhibited rather than induced the biosynthesis of proteases possessing the activity of coagulase by the cells. The production of exoproteases with the coagulase activity was inhibited when a mixture of amino acids was used as a sole nitrogen source.     

Isolation of the specific endonuclease BamH1 in the multicyclic cultivation of Bacillus amyloliquefaciens

The present article deals with the study of the multicyclic process of the cultivation of B. amyloliquefaciens in a liquid medium prepared from natural raw materials and with obtaining restrictase Bam H1 from the biomass with different microbiological characteristics. The selected medium has been found to be suitable for the realization of the multicyclic cultivation process on an industrial scale. The tendency towards a drop in the level of nonspecific nucleases simultaneously with a decrease in the specific growth rate has been revealed. A simplified scheme for the purification of the restricting endonuclease is proposed.     

DNA-[N4-cytosine]-methyltransferase from Bacillus amyloliquefaciens: mechanism of action derived from steady state kinetics

Kinetic analysis of methyl group transfer from S-adenosyl-L-methionine (SAM) to the 5'-GGATCC recognition site catalyzed by the DNA-[N4-cytosine]-methyltransferase from Bacillus amyloliquefaciens [EC 2.1.1.113] has shown that the dependence of the rate of methylation of the 20-meric substrate duplex on SAM and DNA concentration are normally hyperbolic, and the maximal rate is attained upon enzyme saturation with both substrates. No substrate inhibition is observed even at concentrations many times higher than the Km values (0.107 microM for DNA and 1.45 microM for SAM), which means that no nonreactive enzyme-substrate complexes are formed during the reaction. The overall pattern of product inhibition corresponds to an ordered steady-state mechanism following the sequence SAM decreases DNA decreases metDNA increases SAH increases (S-adenosyl-L-homocysteine). However, more detailed numerical analysis of the aggregate experimental data admits an alternative order of substrate binding, DNA decreases SAM decreases, though this route is an order of magnitude slower.     

Annexin A2 recognises a specific region in the 3'-UTR of its cognate messenger RNA

Annexin A2 is a multifunctional Ca(2+)- and lipid-binding protein. We previously showed that a distinct pool of cellular Annexin A2 associates with mRNP complexes or polysomes associated with the cytoskeleton. Here we report in vitro and in vivo experiments showing that Annexin A2 present in this subset of mRNP complexes interacts with its cognate mRNA and c-myc mRNA, but not with beta(2)-microglobulin mRNA translated on membrane-bound polysomes. The protein recognises sequence elements within the untranslated regions, but not within the coding region, of its cognate mRNA. Alignment of the Annexin A2-binding 3'-untranslated regions of annexin A2 mRNA from several species reveals a five nucleotide consensus sequence 5'-AA(C/G)(A/U)G. The Annexin A2-interacting region of the 3'-untranslated region can be mapped to a sequence of about 100 nucleotides containing two repeats of the consensus sequence. The binding elements appear to involve both single and double stranded regions, indicating that a specific higher order mRNA structure is required for binding to Annexin A2. We suggest that this type of interaction is representative for a group of mRNAs translated on cytoskeleton-bound polysomes.