Isolation and characteristics of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase secreted by a recombinant strain of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> at various growth phases

The recombinant strain of Bacillus subtilis bearing B. intermedius glutamyl endopeptidase gene in multicopy plasmid Δ58.21 secretes the enzyme to the medium at the phase of slowing of growth and the stationary growth phase with accumulation maxima at 24 and 48 h. Enzyme samples were isolated from the culture liquid after 24 and 48 h of culturing of and were purified up to homogeneity by ion exchange chromatography on carboxymethyl cellulose and HPLC on a MonoS column. The molecular weight of the corresponding proteins was 29 kDa. Both preparations had identical structure, but differed in affinity to the specific substrate Z-Glu-pNA. The effects of Ca²⁺ ions and specific low-molecular and protein inhibitors on the activity of the enzyme corresponding to various growth phases has been studied.     

Synthesis and Secretion of Proteinases by Bacillus intermedius in the Late Stages of Sporulation

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 -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.     

Isolation and Characterization of Glutamyl Endopeptidase 2 from Bacillus intermedius 3-19

The culture filtrate of Bacillus intermedius 3-19 was used for isolation by chromatography on CM-cellulose and Mono S columns of a proteinase that is secreted during the late stages of growth. The enzyme is irreversibly inhibited by the inhibitor of serine proteinases diisopropyl fluorophosphate, has two pH optima (7.2 and 9.5) for casein hydrolysis and one at pH 8.5 for Z-Glu-pNA hydrolysis. The molecular weight of the enzyme is 26.5 kD. The K(m) for Z-Glu-pNA hydrolysis is 0.5 mM. The temperature and pH dependences of the stability of the proteinase were studied. The enzyme was identified as glutamyl endopeptidase 2. The N-terminal sequence (10 residues) and amino acid composition of the enzyme were determined. The enzyme hydrolyzes Glu4-Gln5, Glu17-Asp18, and Cys11-Ser12 bonds in the oxidized A-chain of insulin and Glu13-Ala14, Glu21-Arg22, Cys7-Gly8, and Cys19-Gly20 bonds in the oxidized B-chain of insulin.     

The cellular location of proteolytic enzymes ofBacillus intermedius

The activities of proteinases in the culture fluid and cellular fractions ofBacillus intermedius 3–19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. The production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2-to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of C0Cl₂ (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted.     

Heterologous expression of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> gene of glutamyl endopeptidase in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains defective in regulatory proteins

Expression of the gene of glutamyl endopeptidase from Bacillus intermedius (gseBi) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5–5-fold decrease in glutamyl endopeptidase activity. It has been concluded that expression of the B. intermedius glutamyl endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.     

Membrane-Bound Forms of Serine Proteases in Bacillus intermedius

Proteolytic proteins solubilized from the membrane of Bacillus intermedius were studied by electrophoresis. The content of membrane-bound proteinases was lower in cells grown in the presence of glucose. Proteinase enzymograms revealed four molecular forms of subtilisin and four molecular forms of glutamyl endopeptidase. The electrophoretic mobility of one of the molecular forms was similar to those of the mature extracellular proteinases. Chromatography of membrane proteins on a MonoS column yielded four protein fractions that caused hydrolysis of Z-Glu-pNA and four fractions that caused hydrolysis of Z-Ala-Ala-Leu-pNA, which is in agreement with the results of electrophoresis. The molecular forms of proteinases identified in the membrane may reflect various stages of biogenesis of the corresponding extracellular enzymes.     

Hydrolytic Enzymes and Sporulation in Bacillus intermedius

The investigation of the activity of extracellular hydrolytic enzymes and sporulation in the bacterium Bacillus intermedius 3-19 showed that the activity of ribonuclease is maximal in the glucose-containing growth medium, in which sporulation is suppressed. At the sporulation stages II–IV, the synthesis of phosphatase was not regulated by the factors that influence this synthesis in the phase of growth retardation. Caseinolytic activity exhibited two peaks. The first peak was observed when thiol-dependent proteinase began accumulating in the medium. The second peak corresponded to the late stages of sporulation, i.e., the stages of spore maturation and the autolysis of sporangium. The regulatory relationship between proteinase synthesis and sporulation and the possible role of extracellular phosphatases and proteinases in the sporulation are discussed.     

Genetic diversity and involvement in bread spoilage of Bacillus strains isolated from flour and ropy bread

AIMS: To study Bacillus contamination of wheat flour and ropy bread, to analyse genetic diversity of isolated strains and to evaluate the ability of these strains to produce ropy bread. METHODS AND RESULTS: Classical and molecular methods [16S rDNA sequencing and random amplified polymorphic DNA (RAPD)-PCR] were used to identify and type-isolated strains. The predominant species isolated were Bacillus subtilis and B. licheniformis. RAPD analysis demonstrated that the same sample may harbor different strains. Ten of 15 strains of B. subtilis and four of six strains of B. licheniformis were able to cause rope spoilage of the laboratory-baked bread. CONCLUSION: RAPD typing can be useful in the tracking of Bacillus strains during bakery processing and in the understanding of the role of different Bacillus strains in the rope spoilage of bread. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate the variability of Bacillus strains isolated from flour and responsible for rope spoilage of bread.     

Co-linear scaffold of the Bacillus licheniformis and Bacillus subtilis genomes and its use to compare their competence genes

We have established the co-linear regions of Bacillus licheniformis, an industrially important bacterium, and Bacillus subtilis, a model bacterium. In the co-linear regions, revealed by PCR, gene content and order are presumed to be conserved. These regions constitute approximately 60% of the compared chromosomes. Sequencing of the competence genes of B. licheniformis allowed us to validate the approach, and to demonstrate how it can be used for the comparative analysis of complex genetic systems. A new insertion sequence, designated IS3Bli1, was discovered in the competence region of the analyzed B. licheniformis strain.     

Production of antimicrobial substances by Bacillus subtilis LFE-1, B. firmus HO-1 and B. licheniformis T6-5 isolated from an oil reservoir in Brazil

AIMS: Forty Bacillus strains isolated from a Brazilian oil reservoir were tested against each other to select strains producing antimicrobial substances (AMS). Three strains, Bacillus subtilis (LFE-1), Bacillus firmus (H2O-1) and Bacillus licheniformis (T6-5), were selected due to their ability to inhibit more than 65% of the Bacillus strains tested. These three strains were also investigated for their capability to inhibit sulphate-reducing bacteria (SRB). Furthermore, physiological and biochemical characteristics of the antimicrobial compounds produced by the selected strains were determined. METHODS AND RESULTS: Among the forty strains tested, 36 (90%) strains were able to inhibit at least one Bacillus strain used as indicator in plate assays and three of them (LFE-1, T6-5 and H2O-1) were able to inhibit 65, 70 and 97.5% of the 40 strains studied here respectively. Clear zones of inhibition were observed when H2O-1 was tested against SRB-containing consortium T6-lab and Desulfovibrio alaskensis strain NCIMB 13491, while strain T6-5 was able to inhibit only the D. alaskensis strain. The three substances showed to be insensitive to different enzymes and chemicals, were heat stable and the substances produced by strains T6-5 and H2O-1 were active over a wide pH range. CONCLUSIONS: Three different AMS produced by Bacillus strains from an oil reservoir, two of them with activity against SRB, are presented here. SIGNIFICANCE AND IMPACT OF THE STUDY: The preliminary characterization of these AMS points to their potential use as biocides in the petroleum industry for controlling problems associated with SRB.     

Characterization of a Theta Plasmid Replicon with Homology to All Four Large Plasmids ofBacillus megateriumQM B1551

A replicon from one of an array of seven indigenous compatible plasmids ofBacillus megateriumQM B1551 has been cloned and sequenced. The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551. The cloned 2374-bpHindIII fragment was sequenced and contained two upstream palindromes and a large (>419-amino-acid) open reading frame (ORF) truncated at the 3′ end. Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF. The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication. The ORF product was shown to act intrans.A small region with similarity to theB. subtilischromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins. Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream. The predicted ORF protein of >48 kDa was basic and rich in glutamate + glutamine (16%). There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF. The data suggest that this is a theta replicon with an expressedrepgene required for replication. The replicon contains its iterons within the gene and has no homology to reported replicons. It is the first characterization of aB. megateriumreplicon.     

The expression of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase gene in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> recombinant strains

The gene encoding for B. intermedius glutamyl endopeptidase (gseBi) has previously been cloned and its nucleotide sequence analyzed. In this study, the expression of this gene was explored in protease-deficient strain B. subtilis AJ73 during stationary phase of bacterial growth. We found that catabolite repression usually involved in control of endopeptidase expression during vegetative growth was not efficient at the late stationary phase. Testing of B. intermedius glutamyl endopeptidase gene expression with B. subtilis spo0-mutants revealed slight effect of these mutations on endopeptidase expression. Activity of glutamyl endopeptidase was partly left in B. subtilis ger-mutants. Probably, gseBi expression was not connected with sporulation. This enzyme might be involved in outgrowth of the spore, when germinating endospore converts into the vegetative cell. These data suggest complex regulation of B. intermedius glutamyl endopeptidase gene expression with contribution of several regulatory systems and demonstrate changes in control of enzyme biosynthesis at different stages of growth.     

Inhibitory effects of Bacillus probionts on growth and toxin production of Vibrio harveyi pathogens of shrimp

Aim:  To investigate the effects of Bacillus subtilis , Bacillus licheniformis and Bacillus megaterium in terms of toxin and growth of pathogenic Vibrio harveyi .
Methods and Results:  Three Bacillus probionts were isolated from probiotic BZT aquaculture and identified using a 16S rDNA sequence. Growth inhibition assay showed that supernatants from the 24-h culture of three Bacillus species were able to inhibit the growth of V. harveyi (LMG 4044); B. subtilis was the most effective based on the well diffusion method. Results of a liquid culture model showed that B. subtilis was also widely effective in inhibiting three strains of V. harveyi (isolated from Thailand, the Philippines and LMG 4044), and that both B. licheniformis and B. megaterium inhibit the growth of V. harveyi isolated from the Philippines. Moreover, a haemolytic activity assay demonstrated that V. harveyi (IFO 15634) was significantly decreased by the addition of B. licheniformis or B. megaterium supernatant.
Conclusions:  Bacillus subtilis inhibited Vibrio growth, and both B. licheniformis and B. megaterium suppressed haemolytic activity in Vibrio .
Significance and Impact of the Study:  The cell-free supernatants produced by Bacillus probionts inhibit Vibrio disease, and Bacillus probionts might have an influence on Vibrio cell-to-cell communications.     

The identification of polypeptides synthesised during the acquisition of teichoic acid synthetic activity in Bacillus licheniformis

An attempt has been made to identify proteins synthesised during induction of teichoic acid synthesis in Bacillus licheniformis ATCC 9945. The proteins are recognised as those produced on the change from teichuronic acid to teichoic acid synthesis that occurs after the transfer of the bacteria from phosphate-limited to phosphate-rich conditions. B. licheniformis was grown in phosphate-limiting conditions in the presence of threonine to stimulate threonine uptake. The bacteria were then transferred to phosphate-rich conditions and were pulsed-labelled with [¹⁴C]threonine during the change to teichoic acid synthesis. All of the proteins were extracted from the cells with sodium dodecyl sulphate and were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Radioactive polypeptides were identified by fluorography of the polyacrylamide gels. The radioactive polypeptides that were formed on change from teichuronic acid to teichoic acid synthesis were compared with the polypeptides present in a membrane sub-fraction that had high teichoic acid-synthesising activity. The labelling of nine polypeptides with [¹⁴C]threonine was dependent on new RNA synthesis. Of these nine polypeptides, five were also present in the membrane sub-fraction with the highest teichoic acid-synthesising activity.