Small RNA mediated repression of subtilisin production in Bacillus licheniformis.

The species Bacillus licheniformis includes important strains that are used in industrial production processes. Currently the physiological model used to adapt these processes is based on the closely related model organism B. subtilis. In this study we found that both organisms reveal significant differences in the regulation of subtilisin, their main natural protease and a product of industrial fermentation processes. We identified and characterized a novel antisense sRNA AprAs, which represents an RNA based repressor of apr, the gene encoding for the industrial relevant subtilisin protease. Reduction of the AprAs level leads to an enhanced proteolytic activity and an increase of Apr protein expression in the mutant strain. A vector based complementation of the AprAs deficient mutant confirmed this effect and demonstrated the necessity of cis transcription for full efficiency. A comparative analysis of the corresponding genome loci from B. licheniformis and B. subtilis revealed the absence of an aprAs promoter in B. subtilis and indicates that AprAs is a B. licheniformis species specific phenomenon. The discovery of AprAs is of great biotechnological interest since subtilisin Carlsberg is one of the main products of industrial fermentation by B. licheniformis.

Secreted protease mediates interspecies interaction and promotes cell aggregation of the photosynthetic bacterium Chloroflexus aggregans.

Interspecies interactions were studied in hot spring microbial mats where diverse species of bacterial cells are densely packed. The anoxygenic photosynthetic bacterium, Chloroflexus aggregans, has been widely found in the microbial mats as a major component in terrestrial hot springs in Japan at the temperature from 50 to 70°C. C. aggregans shows cellular motility to form a microbial mat-like dense cell aggregate. The aggregating ability of C. aggregans was affected by another bacterial species, strain BL55a (related to Bacillus licheniformis) isolated from the microbial mats containing C. aggregans. Cell aggregation rate of C. aggregans was promoted by the addition of culture supernatants of strain BL55a. Similar effects were also detected from other bacterial isolates, specifically Geobacillus sp. and Aeribacillus sp. Protease activity was detected from the culture supernatants from all of these isolates. The promoting effect of strain BL55a was suppressed by a serine protease inhibitor, phenylmethylsulfonyl fluoride. A purified serine protease, subtilisin obtained from B. licheniformis, showed a promoting effect on the cell aggregation. These results suggest that an extracellular protease, secreted from co-existing bacterial species promoted the aggregating motility of C. aggregans. This is the first report that exogenous protease affects bacterial cellular motility.

[Expression optimization of Bacillus pumilus subtilisin-like proteinase].

The characteristics of Bacillus pumilus subtilisin-like protease expression were investigated. Potential binding sites for the transcription factor DegU~P have been identified in the regulatory region of the enzyme gene, which have been optimized for interaction with the regulatory protein. The expression of the extracellular subtilisin-like protease modified gene has been examined. It was established that the modification of one of the sites has resulted in increased expression of the proteinase in 2 times. It is concluded that the optimization of the promoter led to increased expression of subtilisin-like protease.

Discovery of a new role of human resistin in hepatocyte low-density lipoprotein receptor suppression mediated in part by proprotein convertase subtilisin/kexin type 9.

OBJECTIVES: In this study, our goal was to determine if human resistin plays a role in regulating the uptake of atherogenic low-density lipoproteins in human hepatocytes. BACKGROUND: Serum levels of resistin, an adipose tissue-derived adipokine, are increased in human obesity and are positively correlated with atherosclerotic cardiovascular diseases. However, the function of resistin in humans is enigmatic. METHODS: Human hepatocytes (HepG2 and primary) were treated (24 h) with the following: 1) purified human resistin at various concentrations, with and without lovastatin; and 2) obese human serum with elevated resistin levels or serum from which resistin was removed via antibody-immunoprecipitation. The effect of the treatments on cellular low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) messenger ribonucleic acid and protein levels were determined by using real-time polymerase chain reaction and Western blotting, respectively. RESULTS: Resistin, at physiological levels observed in human obesity, down-regulated hepatocyte LDLR expression substantially (by 40%). A key mechanism by which human resistin inhibited LDLR levels was by increased cellular expression of the recently identified protease, PCSK9, which enhances intracellular LDLR lysosomal degradation. The quantitatively important role of human resistin in LDLR expression was demonstrated by antibody-immunoprecipitation removal of resistin in human serum, which decreased serum stimulation of hepatocyte LDLRs markedly (by 80%). Furthermore, resistin diminished statin-mediated up-regulation of the LDLR by 60%, implicating resistin in the relative ineffectiveness of statins in selective target populations. CONCLUSIONS: These results reveal for the first time that resistin is a highly attractive therapeutic target in ameliorating elevated serum low-density lipoprotein and, thereby, atherosclerotic cardiovascular diseases in obese humans.

Cloning of a fibrinolytic enzyme (subtilisin) gene from Bacillus subtilis in Escherichia coli.

Several investigations are being pursued to enhance the efficacy and specificity of fibrinolytic therapy. In this regard, microbial fibrinolytic enzymes attracted much more medical interests during these decades. Subtilisin, a member of subtilases (the superfamily of subtilisin-like serine proteases) and also a fibrinolytic enzyme is quite common in Gram-positive bacteria, and Bacillus species stand out in particular, as many extracellular and even intracellular variants have been identified. In the present work, the subtilisin gene from Bacillus subtilis PTCC 1023 was cloned into the vector pET-15b and expressed in Escherichia coli strain BL21 (DE3). Total genomic DNA were isolated and used for PCR amplification of the subtilisin gene by means of the specific primers. SDS-PAGE and enzyme assay were done for characterizing the expressed protein. A ~1,100 bp of the structural subtilisin gene was amplified. The DNA and amino acid sequence alignments resulting from the BLAST search of subtilisin showed high sequence identity with the other strains of B. subtilis, whereas significantly lower identity was observed with other bacterial subtilisins. The recombinant enzyme had the same molecular weight as other reported subtilisins and the E. coli transformants showed high subtilisin activity. This study provides evidence that subtilisin can be actively expressed in E. coli. The commercial availability of subtilisin is of great importance for industrial applications and also pharmaceutical purposes as thrombolytic agent. Thus, the characterization of new recombinant subtilisin and the development of rapid, simple, and effective production methods are not only of academic interest, but also of practical importance.

[Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62].

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The activity of different regulatory mechanisms for the synthesis of this proteinase is assumed at the early and late stationary stages of growth.

In vitro fermentation studies for selection and evaluation of Bacillus strains as starter cultures for the production of okpehe, a traditional African fermented condiment.

Selected Bacillus and Enterococcus strains, isolated from traditional okpehe fermentations, were studied for their suitability as starter cultures in laboratory-scale fermentations of Prosopis africana seeds for the production of okpehe, a traditional fermented vegetable product of Nigeria. The strains were selected on the basis of highest proteolytic activity, as determined with the APIZYM (BioMerieux) test. The choice of starter strains was narrowed to Bacillus subtilis strains BFE 5301 and BFE 5372. These were determined as the best starter combination because of rapid growth, high amylolytic and proteolytic activities, high levels of polyglutamic acid production by strain BFE 5372, as well as bacteriocin production by strain BFE 5301. Other mixed culture fermentations did not yield sensorically acceptable products. Although a monoculture fermentation, using only B. subtilis strain BFE 5372, produced okpehe with very good sensory characteristics, the growth of B. cereus could be detected after 48 h fermentation, indicating that this starter did not sufficiently contribute to product safety. Mixed culture fermentation with the combination of bacteriocin-producing starter B. subtilis BFE 5301 and the non-bacteriocin-producing B. subtilis BFE 5372, produced a product with good sensory characteristics, in which growth of B. cereus was delayed. The bacteriocin produced by B. subtilis strain BFE 5301 was identified as subtilisin, using subtilisin-specific primers and PCR amplification of the subtilisin gene. The bacteriocin was heat-stable at 100 degrees C for 10 min and exhibited highest activity at pH values lower or equal to pH 6.0. The bacteriocin was sensitive to the proteolytic enzymes trypsin and alpha-chymotrypsin at concentrations of 10 mg/ml.

Characterization of an extracellular subtilisin protease of Rhizopus microsporus and evidence for its expression during invasive rhinoorbital mycosis.

An endoprotease Arp (alkaline Rhizopus protease) was identified and purified to virtual homogeneity from the culture supernatant of an isolate of Rhizopus microsporus var. rhizopodiformis recovered from a non-fatal case of rhinoorbital mucormycosis. N-terminal sequencing of the mature native enzyme was obtained for the first 20 amino acids and revealed high homology to serine proteases of the subtilisin subfamily. Arp migrated in SDS-PAGE with an estimated molecular mass of 33 kDa and had a pI determined to be at pH 8.8. Arp is proteolytically active against various substrates, including elastin, over a broad pH range between 6 and 12 with an optimum at pH 10.5. After invasive mucormycosis, specific antibodies against Arp were detected in stored serum samples taken from the patient from whom the R. microsporus strain of this study had been isolated. Furthermore, in search of factors involved in thrombosis as a typical complication of mucormycosis, a procoagulatory effect of the enzyme has recently been shown. Altogether, these data substantiate the expression of Arp during human rhinoorbital mucormycosis and suggest a role of the enzyme in pathogenesis.

[Growth conditions and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain].

The effect of the components of the nutrient medium on growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. The production of proteinase was found to be dependent on the composition of the nutrient medium and showed two peaks, at the 28th and 48th h of growth. The concentrations of the main components of the nutrient medium (peptone and inorganic phosphate) optimal for the biosynthesis of subtilisin-like serine proteinase at the 28th and 48th h of growth were determined in factorial experiments. Complex organic substances, casein at concentrations of 0.5-1%, gelatin at concentrations of 0.5-1%, and yeast extract at a concentration of 0.5%, stimulated the production of subtilisin-like serine proteinase by the recombinant strain. The study of the sporulation dynamics in this strain showed that the proteinase peaks at the 28th and 48th h of growth correspond, respectively, to the initial stage of sporulation and to the terminal stages of endospore formation (V-VII stages of sporulation).

[Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain].

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.

Selection of cultivation medium for production of late stationary phase serine proteinases from Bacillus intermedius.

B. intermedius have been shown previously to secrete two serine proteinases: glutamyl endopeptidase 2 and subtilisin 2 during the late stationary phase, with maximal levels of the enzymes activities recorded at the 40th and 44th hours of growth, respectively. In the current study, we analyzed the impact of various culture medium components on biosynthesis of these proteinases. Yeast extract and gelatin did not stimulate the enzymes biosynthesis. However, on the medium containing 0.1% casein subtilisin 2 production increased to reach 140%. Biosynthesis of both serine proteinases, produced by B. intermedius at the late stationary phase, were found to be inhibited by individual amino acids, and to be insensitive to catabolite repression. In order to maximise enzyme production, the presence of Ca(2+) and Mg(2+) at concentration of 5 mM was shown to be necessary. Based on the results of this work, the composition of a complex culture media for the effective production of late stationary phase proteinases by B. intermedius was developed.

Construction and analyses of mutant ftsH alleles of Bacillus subtilis involving the ATPase- and Zn-binding domains.

The ftsH gene, present in all eubacterial species, is anchored in the cytoplasmic membrane and contains an ATP- and a Zn-binding domain that are both part of a metalloprotease activity. The Bacillus subtilis ftsH is not essential, but null mutants exhibit a pleiotropic phenotype including filamentous growth; hypersensitivity towards heat and salt stress and a failure to sporulate. To find out whether one or the other functional domain is involved in these different phenotypes, point mutations were introduced into the coding region for both domains leading to a replacement of conserved amino acid residues. The mutant alleles were fused to a xylose-inducible promoter and integrated ectopically into two different strains, one expressing the wild-type ftsH allele and the other carrying a ftsH knockout. While none of the strains exhibited a growth defect in rich medium at 37 degrees C, those strains expressing only the mutant alleles did not resume growth after heat or salt stress challenge. Furthermore, none of the mutant alleles promoted sporulation. While only those purified mutant FtsH proteins with an intact Walker A box exhibited ATPase activity, all of them failed to degrade beta-casein.

[Synthesis and secretion of Bacillus intermedius proteinases in the late stages of sporulation]. / Sintez i sekretsiia proteinaz Bacillus intermedius na pozdnikh stadiiakh sporoobrazovaniia.

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating beta-galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites.