The nucleotide sequence of the lipo-penicillinase gene of alkalophilic Bacillus sp. strain 170

The lipo-penicillinase (LIPEN) gene from an alkalophilic Bacillus sp. strain 170 was cloned in Escherichia coli using the vector pHSG399. A plasmid, pFAP121, was isolated from an ampicillin resistant transformant and the cloned LIPEN gene was found to be in a 2.2 kb DNA fragment. The nucleotide sequence of a 1.9 kb segment encoding the LIPEN was determined. This segment showed an open reading frame which would encode a polypeptide of 310 amino acids. The amino acid sequence of this LIPEN gene product has strong homology with those of the Bacillus cereus -lactamase III and Bacillus licheniformis penicillinase.     

In vitro synthesis of hydrophobic penicillinase in extracts of Bacillus licheniforms 749/C.

Extracts of $\text{Bacillus licheniformis}$ 749/C in an $\text{in vitro}$ protein synthesis system produced the hydrophobic penicillinase containing covalently-bound phospholipid. The hydrophilic penicillinase (exoenzyme) and the hydrophobic enzyme without the phospholipid were scarcely detectable.     

Heterologous protein secretion in Lactococcus lactis is enhanced by the Bacillus subtilis chaperone-like protein PrsA

The Bacillus subtilis lipoprotein PrsA enhances the yield of several homologous and heterologous exported proteins in B. subtilis by being involved in the posttranslocational stage of the secretion process. In this work, we have studied the effect of B. subtilis PrsA on the secretion of Bacillus amyloliquefaciens α-amylase (AmyQ), a target protein for PrsA, and Bacillus licheniformis penicillinase (PenP) a nontarget protein for PrsA, in Lactococcus lactis. Two compatible plasmids were constructed and introduced into L. lactis strain NZ9000: one high copy plasmid, expressing the AmyQ gene (amyQ) or the PenP gene (penP), and one low copy plasmid, expressing the PrsA encoding gene (prsA). When amyQ and prsA were simultaneously expressed under the nisin-inducible promoter P _nisA , Western blotting experiments revealed a 15- to 20-fold increase in the total yield of AmyQ and a sixfold increase in secreted AmyQ activity, compared to a control strain lacking prsA. When expressed under the same induction conditions, PrsA had no effect on the secretion or total yield of PenP. These results show that the secretion yield of some heterologous proteins can be significantly increased in L. lactis when coproduced with the B. subtilis PrsA protein.     

Environmental control of protein export of Bacillus licheniformis NCIB 6346 reveals two types of extracellular proteins

Teichuronic acid was the major anionic polymer of Bacillus licheniformis NCIB 6346 durign phosphate-limited (P-limited) growth in the chemostat. This polymer was also present in significant quantities when B. licheniformis was grown under carbon-limited (C-limited) or magnesium-limited (Mg-limited) conditions where teichoic acid predominated in the cell wall. However, the cell wall composition was not of significance in protein export and the parameters for the excretion process were found to be environmental. In particular, two types of extracellular proteins were identified: the first type of enzyme, penicillinase, was only weakly catabolite repressed; was maximally synthesized and secreted during P-limited growth; was unaffected by growth in high Na⁺ media but its production was inhibited by gramicidin. The second type of enzyme, -amylase, was strongly catabolite repressed and its export was markedly inhibited during P-limited growth or in the presence of Na⁺ or gramicidin. It is noteworthy that the penicillinase carries a glyceride-cysteine modification at its N-terminus whilst the -amylase does not.     

Central carbon metabolism influences cellulase production in Bacillus licheniformis

Bacillus licheniformis that can produce cellulase including endo glucanase and glucosidase is an important industrial microbe for cellulose degradation. The purpose of this research was to assess the effect of endo glucanase gene bglC and glucosidase gene bglH on the central metabolic flux in B. licheniformis. bglC and bglH were knocked out using homologous recombination method, respectively, and the corresponding knockout strains were obtained for ¹³C metabolic flux analysis. A significant change was observed in metabolic fluxes after ¹³C metabolic flux ratio analysis. In both of the knockout strains, the increased fluxes of the pentose phosphate pathway and malic enzyme reaction enabled an elevated supply of NADPH which provided enough reducing power for the in vivo synthesis reactions. The fluxes through tricarboxylic acid cycle and anaplerotic reactions increased fast in the two knockout strains, which meant more energy generated. The changed fluxes in central carbon metabolism provided a holistic view of the physiological status in B. licheniformis and possible targets for further strain engineering.

Significance and Impact of the Study

Cellulase is very important in the field of agriculture and bioenergy because of its degrading effect on cellulosic biomass. This study presented the effect of central carbon metabolism on cellulase production in Bacillus licheniformis. The study also provided a holistic view of the physiological status in B. licheniformis. The shifted metabolism provided a quantitative evaluation of the biosynthesis of cellulase and a priority ranked target list for further strain engineering.     

Enthalpies and Entropies of Cd and Zn Adsorption onto Bacillus licheniformis and Enthalpies and Entropies of Zn Adsorption onto Bacillus subtilis from Isothermal Titration Calorimetry and Surface Complexation Modeling

Determining the thermodynamic driving force of metal-bacteria surface complexation is important for understanding why, from a thermodynamic perspective, these spontaneous reactions occur. We therefore determined the Gibbs energies, enthalpies, and entropies of Cd and Zn complexation onto Bacillus licheniformis and of Zn complexation onto Bacillus subtilis using surface complexation modeling and isothermal titration calorimetry. Our results indicated that Cd and Zn complexation onto Bacillus licheniformis is entropically driven at low pH and enthalpically driven at circumneutral pH. Zn complexation onto Bacillus subtilis is entropically driven, which suggests that Bacillus licheniformis has different donor ligands dominating reactivity around circumneutral pH.     

Role of the pre-pro-region of neutral protease in secretion in Bacillus subtilis

A thermostable neutral protease (NprT) from Bacillus stearothermophilus CU21 is translated as a precursor containing pre-pro-structure. Deletion and insertion mutations were introduced in the pro-region and the effects of the mutations on production of active NprT were studied. The pro-region was important for the production of active NprT and was effective only when it was combined with the signal sequence (pre-region) of NprT. To examine the role of the pre-region, a penicillinase gene from Bacillus licheniformis (penP) was fused with various nprT secretion vectors. It was also found that the pre-region of NprT can potentially function as a signal sequence but is more functional for the production of NprT when it was combined with a normal pro-region. We discuss how the processing between the pro-structure and the mature portion of the protease might be done autoproteolytically.     

Effect of environmental phosphate concentration on protein export by Bacillus licheniformis NCIB 6346

Bacillus licheniformis NCIB 6346 was grown under phosphate or magnesium limitation in continuous culture and forward and reverse transitions carried out to examine their effects upon extracellular enzyme production. Analysis of the observed kinetics allowed a distinction to be made between the effects of phenotypic variation and of genetic selection upon exoenzyme production. Furthermore, increases in the phosphate concentration (0.3–30 mM) were directly related to increased phenotypic expression of alpha-amylase. By contrast, penicillinase production was reduced under these conditions but analysis revealed that its phenotypic expression was only indirectly influenced by the concentrations of either magnesium or phosphate present during the transitions.     

A frameshift mutation that elongates the penicillinase protein of Bacillus licheniformis

A penicillinase mutant penP102, isolated after ICR (acridine mustard) mutagenesis of Bacillus licheniformis strain 749/C, retains about 50% of the wild-type penicillinase specific activity. The penicillinase produced by this mutant differs from the wild-type protein in its sensitivity to pH and its electrophoretic behaviour. The penP102 mutation appears to have several other phenotypic effects, including an increase in the efficiency of release of the extracellular form of the enzyme.The penP102 penicillinase has been purified and its amino acid sequence compared to that of the wild-type enzyme. The mutation has resulted in the replacement of the last three amino acids of the wild-type enzyme and the addition of 17 residues at the carboxy-terminus. Comparison of the wild-type and mutant amino acid sequences shows that the mutational event is a single nucleotide deletion from the codon for asparagine²⁶⁵. Consideration of the possible nucleotide sequence for the region beyond the carboxy-terminus of the wild-type protein shows that there are no possible termination codons until four and six triplets beyond the codon for the carboxy-terminal lysine, indicating that the carboxy-terminus of the wild-type extracellular penicillinase is generated by proteolytic cleavage of a larger precursor protein.     

Purification and characterization of a cellulase-free xylanase of a moderate thermophile Bacillus licheniformis A99

Two cellulase-free xylanases were secreted by a thermophile, Bacillus licheniformis A99. Of the two, the predominant one was purified to homogeneity. The enzyme was optimally active at 60 °C, pH 6–7.5, and had a molecular weight of about 45 KDa and isoelectric point of 7.0 ± 0.2. The K _m (for birchwood xylan) and V _max were 3.33 mg/ml and 1.111 mmols mg^–1 protein min^–1 respectively. The half-life of the enzyme was 5 h at 60 °C. All cations except Hg²⁺ and Ag⁺ as well as EDTA were well tolerated and did not adversely affect xylanase activity. However, SDS inhibited the enzyme activity. The release of reducing sugars from unbleached commercial pulp sample on treatment with the enzyme indicated its potential in prebleaching of paper pulp. The enzyme caused saccharification of lignocellulosics such as wheat bran, wheat straw and sawdust. This is the first report on purification and characterization of cellulase-free xylanase from a moderate thermophile Bacillus licheniformis.     

Distribution and variation of bacitracin synthetase gene sequences in laboratory stock strains of Bacillus licheniformis

Distribution and variation of bacitracin synthetase gene (bac) sequences in 22 laboratory stock strains of Bacillus licheniformis were studied by Southern hybridization of bac gene probes from B. licheniformis ATCC 10716 to genomic PstI or HindIII restriction fragments. Eleven strains gave hybridization signals. These hybridization patterns were classed into two types. Eight strains showed similar patterns to that of ATCC 10716 and all, except one, produced bacitracin. The three strains showed fairly different hybridization patterns from that of ATCC 10716, and one (ATCC 33632) of them produced bacitracin. None of the remaining 11 strains, including ATCC 14580 (type strain), gave any hybridization signals. All strains carrying bac gene sequences were erythromycin resistant. With one exception, all strains without bac gene sequences were erythromycin sensitive. These results show that B. licheniformis strains are divided into two groups with respect to presence of bac gene sequences and erythromycin resistance. Received: 5 September 2001 / Accepted: 19 October 2001     

Two acidothermotolerant lipases from new variants of Bacillus spp.

Two acidothermotolerant lipases from new isolates of Bacillus stearothermophilus SB-1 and Bacillus licheniformis SB-3 are reported. In addition, a thermotolerant, neutral lipase from Bacillus atrophaeus SB-2 that hydrolyses castor oil is also reported. The lipase from B. stearothermophilus SB-1 retained 70% activity and that from B. licheniformis SB-3 retained 50% activity at pH 3.0 at 50 °C. In addition, at 100 °C B. stearothermophilus SB-1 lipase had a half life of 25 min at pH 3.0 and 15 min at pH 6.0. Lipase activity was markedly stimulated by glycerol in case of B. stearothermophilus SB-1 and by diethylether in cases of B. atrophaeus SB-2 and B. licheniformis SB-3. The lipases varied in their substrate specificity towards triacylglycerols. The rate of hydrolysis of neem oil with B. stearothermophilus SB-1 and B. atrophaeus SB-2 lipases was, respectively, nearly 4-fold and 2-fold more than with olive oil.     

Membrane-bound and secreted forms of penicillinase from Bacillus licheniformis

Three forms of penicillinase from Bacillus licheniformis have been isolated. Two are secreted into the extracellular medium and one is membrane-bound. The secreted proteins are water-soluble; one has been previously described and sequenced, the other contains an amino-terminal extension of eight amino acid residues. The membrane-bound form behaves in all respects as a typical amphiphilic membrane protein. It binds one micelle of Triton XI00 and reassociates with egg lecithin to lipid vesicles into which the protein is incorporated. No lipids are covalently associated with the purified protein. Membrane penicillinase contains an amino-terminal peptide extension as compared to the exo forms. This tail is the most likely explanation to its amphiphilic properties.     

Studies on Bacillus licheniformis endo-\-1,3-1,4-d-glucanase: characterization and kinetic analysis

Summary A new purification procedure for endo-\-1,3-1,4-d-glucanase from Bacillus licheniformis is described. The secreted enzyme was purified both from B. licheniformis and from recombinant Escherichia coli harbouring the cloned gene by ion exchange chromatography on a CM-Sepharose matrix at pH 5.6. The mature enzyme was resistant to proteolysis by trypsin and chymotrypsin but it was slowly digested by protease V8. It showed a continuous trimming where no large-limit polypeptides were noticeable thus supporting a monodomain structure. Former appearing peptides have been assigned theoretically according to the protein sequence and predictive methods of accessible areas. Kinetic parameters for the hydrolysis of barley \-glucan and lichenan by measuring the net release of reducing sugars at the optimum pH (7.02) and temperature (55° C) are k _cat=3500 ±800 s^–1 (turnover number) and K _m=1.45±0.21 mg/ml for barley \-glucan and k _cat=3000±750 s^–1 and K _m=1.98±0.40 mg/ml for lichenan. Correspondence to: E. Querol     

Action of antimicrobial substances produced by different oil reservoir Bacillus strains against biofilm formation

Microbial colonization of petroleum industry systems takes place through the formation of biofilms, and can result in biodeterioration of the metal surfaces. In a previous study, two oil reservoir Bacillus strains (Bacillus licheniformis T6-5 and Bacillus firmus H₂O-1) were shown to produce antimicrobial substances (AMS) active against different Bacillus strains and a consortium of sulfate-reducing bacteria (SRB) on solid medium. However, neither their ability to form biofilms nor the effect of the AMS on biofilm formation was adequately addressed. Therefore, here, we report that three Bacillus strains (Bacillus pumilus LF4—used as an indicator strain, B. licheniformis T6-5, and B. firmus H₂O-1), and an oil reservoir SRB consortium (T6lab) were grown as biofilms on glass surfaces. The AMS produced by strains T6-5 and H₂O-1 prevented the formation of B. pumilus LF4 biofilm and also eliminated pre-established LF4 biofilm. In addition, the presence of AMS produced by H₂O-1 reduced the viability and attachment of the SRB consortium biofilm by an order of magnitude. Our results suggest that the AMS produced by Bacillus strains T6-5 and H₂O-1 may have a potential for pipeline-cleaning technologies to inhibit biofilm formation and consequently reduce biocorrosion.     

Chitinase production by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>: Their potential in antifungal biocontrol

Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na⁺, Mg²⁺, Cu²⁺, and Ca²⁺ caused enhancement of enzyme activities whereas they were markedly inhibited by Zn²⁺, Hg²⁺, and Ag⁺. In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.     

Antibacterial Potential of 2,4-Di-tert-Butylphenol and Calixarene-Based Prodrugs from Thermophilic Bacillus licheniformis Isolated in Algerian Hot Spring

The present investigation reports the isolation, molecular identification and structure elucidation of antibacterial produced by two thermophilic spore-forming bacteria from hot spring (98?°C) of Guelma (Algeria). Morphological, biochemical and physiological characteristics were carried out. The molecular identification by 16S rRNA and 16-23S rRNA ITS-PCR sequencing identified the thermophilic strains as Bacillus licheniformis with 99% of similarity with GenBank accession numbers KX100031 and KX100032. Phenotypic characterization has mentioned several differences between thermophilic isolates and Bacillus licheniformis ATCC 14580. The ability of the thermophilic spore- forming bacteria to produce antibacterial compounds against two multidrug resistance bacteria Pseudomonas aeruginosa (NR_0754828.1) and Staphylococcus aureus (NR_075000.1) in pure and mixed culture was investigated by Radial Diffusion Assay at 55?°C. Structural elucidation of actives compounds was carried out using gas chromatography–mass spectrometry analyses. Antibacterial potency of the thermophilic isolates might be due to the association between two phenolic compounds: 2,4-Di-tert-butyl-phenol as principal active compound and p-tert-butylcalix[4]arene as prodrugs comparing between gas chromatography–mass spectrometry analysis of pure and mixed extract. To the best of our knowledge, this is the first report showing production of p-tert-butylcalix[4]arene and 2,4-Di-tert-butyl-phenol as extremolytes compounds from thermophilic Bacillus licheniformis at 55?°C.     

Bioemulsifier production by a halothermophilic Bacillus strain with potential applications in microbially enhanced oil recovery

A halothermotolerant Gram-positive spore-forming bacterium was isolated from petroleum reservoirs in Iran and identified as Bacillus licheniformis sp. strain ACO1 by phenotypic characterization and 16S rRNA analysis. It showed a high capacity for bioemulsifier production and grew up to 60°C with NaCl at 180 g l⁻¹. The optimum NaCl concentration, pH and temperature for bioemulsifier production were 4% (w/v), 8.0, and 45°C, respectively. Although ACO1 did not utilize hydrocarbons, it had a high emulsifying activity (E ₂₄ = 65 ± 5%) on different hydrophobic substrates. Emulsification was optimal while growing on yeast extract as the sole carbon source and NaNO₃ as the nitrogen source. The efficiency of the residual oil recovery increased by 22% after in situ growth of B. licheniformis ACO1 in a sand-pack model saturated with liquid paraffin.     

On the industrial use of Bacillus licheniformis: a review

We document here that in those rare cases where disease has been related to Bacillus licheniformis, infection was associated with bypassing the normal biological protective barriers or severely debililated patients. No case suggests any invasive properties of this bacterium. B. licheniformis can therefore be considered non-pathogenic to humans in general. Food-borne illness caused by possible B. licheniformis toxins have been reported, but only in a very few cases and only in connection with consumption of inappropriately prepared food. Considerable experience concerning the industrial use of recombinant B. licheniformis strains has now accumulated and authorities in the United States, Europe and Japan have approved production with and products from recombinant B. licheniformis strains. We conclude that B. licheniformis is a safe host for the production of harmless, industrial products. Correspondence to: B. Diderichsen