Participation of temperate phages LP52 and BL20 in the control of bacitracin synthesis in Bacillus licheniformis

The synthesis of the antibiotic bacitracin in lysogenic and nonlysogenic strains of Bacillus licheniformis 1001 and ATCC10716 has been studied. The antibiotic activity was shown to be about 20% less in lysogens, as compared to nonlysogens. However, the level of bacitracin production was completely restored when temperate bacteriophages BL20 and LP52 were reintroduced into the nonlysogenic strains by virtue of genetic transformation with DNA from lysogenic strains or by transduction with LP52. This may indicate that both phages take part in control of the synthesis of bacitracin. For the time being, the mechanism of regulation is not known. It is likely to be either direct (provided that prophage DNA contains "bacitracin" genes), or indirect.     

Molecular cloning and expression in Escherichia coli of the Bacillus licheniformis bacitracin synthetase 2 gene.

The structural genes for the entire bacitracin synthetase 2 (component II) and for a part of the putative bacitracin synthetase 3 (component III) from Bacillus licheniformis ATCC 10716 were cloned and expressed in Escherichia coli. A cosmid library of B. licheniformis DNA was constructed. The library was screened for the ability to produce bacitracin synthetase by in situ immunoassay using anti-bacitracin synthetase antiserum. A positive clone designated B-15, which has a recombinant plasmid carrying about a 32-kilobase insert of B. licheniformis DNA, was further characterized. Analysis of crude cell extract from B-15 by polyacrylamide gel electrophoresis and Western blotting (immunoblotting) showed that the extract contains two immunoreactant proteins with high molecular weight. One band with a molecular weight of about 240,000 comigrates with bacitracin synthetase 2; the other band is a protein with a molecular weight of about 300,000. Partial purification of the gene products encoded by the recombinant plasmid by gel filtration and hydroxyapatite column chromatography revealed that one gene product catalyzes L-lysine- and L-ornithine-dependent ATP-PPi exchange reactions which are characteristic of bacitracin synthetase 2, and the other product catalyzes L-isoleucine-, L-leucine, L-valine-, and L-histidine-dependent ATP-PPi exchange activities, suggesting the activities of a part of bacitracin synthetase 3. Subcloning experiments indicated that the structural gene for bacitracin synthetase 2 is located near the middle of the insert.     

Enantiotracin

The enantiomer of the antibiotic bacitracin A was prepared by solid-phase total synthesis. ent-Bacitracin A was found to be equally potent to the natural enantiomer in in vitro susceptibility assays. This supports the notion that bacitracin exerts its antibacterial effects through interaction with bactoprenylpyrophosphate, an achiral ligand.     

Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain

The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein-folding catalyst in a variety of molecular pathways. Commercial bacitracin is a mixture of at least 22 structurally related peptides. The inhibitory activity of individual bacitracin analogs on PDI is unknown. For the present study, we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggregation assay. All analogs inhibited PDI, but the activity (IC(50) ) ranged from 20 μm for bacitracin F to 1050 μm for bacitracin B. The mechanism of PDI inhibition by bacitracin is unknown. Here, we show, by MALDI-TOF/TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate-binding domain of PDI.     

Cloning of the genes controlling the biosynthesis of bacitracin in Bacillus licheniformis

The DNA fragment from bacitracin-producing Bacillus licheniformis strain is cloned on pMX39 vector plasmid in Bacillus subtilis cells. Bacillus subtilis cells carrying the cloned fragment inhibit the growth of bacitracin-sensitive tester strain. The observed inhibition of growth is due to the production by Bacillus subtilis of bacteriocin substance that is identified as bacitracin by TLC-chromatography. In contrast to the data published earlier it is shown that Bacillus subtilis can in fact produce the small amounts of bacitracin. Introduction of the cloned Bacillus licheniformis DNA into Bacillus subtilis cells stimulates this bacitracin production. The restriction site map of the Bacillus licheniformis chromosomal region bearing the cloned fragment is constructed.     

Molecular analysis of BcrR, a membrane-bound bacitracin sensor and DNA-binding protein from Enterococcus faecalis

BcrR has been identified as a novel regulatory protein of high level bacitracin resistance encoded by the bcrABD operon in Enterococcus faecalis. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins, and topological modeling predicts that the C-terminal domain contains four transmembrane alpha-helices. These data have led to the hypothesis that BcrR functions as both a membrane-bound sensor and transducer of bacitracin availability to regulate bcrABD expression. To characterize the bcrABD promoter and identify the promoter elements to which BcrR binds, a series of bcrA-lacZ fusions were constructed. A 69-bp region was identified that was essential for bacitracin-dependent bcrA-lacZ expression. Mutations that targeted this region were used to identify two inverted repeat sequences, each with the sequence 5'-GACA(N)(7)TGTC-3', on the bcrABD promoter that were required for bcrA-lacZ expression. To study BcrR binding to this region, we over-produced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-beta-d-maltoside, and subsequently purified it via Ni(2+)-nitrilotriacetic acid and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes, and BcrR binding to bcrABD promoter DNA was analyzed using electrophoretic mobility shift assays. Both inverted repeat sequences were required for BcrR binding, both in the presence and absence of bacitracin. These data demonstrate that membrane-bound BcrR binds specifically to the bcrABD promoter, irrespective of bacitracin concentration. We therefore propose that bacitracin-dependent induction of bcrABD expression by BcrR occurs after DNA binding.     

Isolation and characterization of Tn917-generated bacitracin deficient mutants of Bacillus licheniformis

Abstract Two Tn917-generated bacitracin deficient mutants of Bacillus licheniformis were isolated. Southern blot analysis of chromosomal DNA extracted from both insertional mutants showed that Tn917 inserted in the vicinity of the gene coding for the enzyme BA2 of the bacitracin synthetase enzyme complex. Measurements of bacitracin synthetase activity in cell-free extracts and positive hybridization signals in the vicinity of the BA2 gene indicate that in both bacitracin deficient mutants Tn917 could be inserted in the BA1 gene or in segments involved in regulation. Thus, it could be possible that the genes for bacitracin synthetase are clustered in the B. licheniformis genome.     

Sporulation of a bacitracin-sensitive mutant of Bacillus licheniformis is self-inhibited by bacitracin

A mutant of Bacillus licheniformis (BLU166) sensitive to its own antibiotic bacitracin was isolated and the mutation bcr-l was mapped close to the bacitracin synthetase genes. The sensitivity was shown to be specific for bacitracin. Two further bacitracin-sensitive strains were constructed, one (BLU171) with normal ability to synthesize bacitracin, and one (BLU170) a bacitracin non-producer. In addition to an increased sensitivity of growing cells to bacitracin, sporulation of the mutant strain BLU171 was self-inhibited by bacitracin. It is concluded that (1) there might exist at least two levels of resistance to bacitracin; (2) mutation bcr-1 affects a 'structural' component, which may protect the sensitive reaction of cell-wall biosynthesis; (3) sporulation is affected to a greater extent by bacitracin than vegetative growth; and (4) synthesis of bacitracin is independent of the presence of this resistance mechanism since the sensitive mutant produces similar amounts of the antibiotic to the wild-type strain.     

Antibiotic bacitracin induces hydrolytic degradation of nucleic acids

Background

Bacitracin is a polypeptide antibiotic active against Gram-positive bacterial strains. Its mechanism of action postulates disturbing the cell wall synthesis by inhibiting dephosphorylation of the lipid carrier. We have discovered that bacitracin induces degradation of nucleic acids, being particularly active against RNA.

Methods

In the examination of the nucleolytic activity of bacitracin several model RNA and DNA oligomers were used. The oligomers were labeled at their 5′ ends with ³²P radioisotope and following treatment with bacitracin the cleavage sites and efficiency were determined.

Results and conclusions

Bacitracin induces degradation of RNA at guanosine residues, preferentially in single-stranded RNA regions. Bacitracin is also able to degrade DNA to some extent but comparable effects to those observed with RNA require its 10-fold higher concentration. The sites of degradation in DNA are very infrequent and preferentially occur near cytidine residues. Free radicals are not involved in the reaction, and which probably proceeds via a hydrolytic mechanism. The phosphate groups at the cleavage sites are present at the 3' ends of RNA products and at the 5' ends of DNA fragments. Importantly, the presence of EDTA does not influence RNA degradation but completely inhibits the degradation of DNA. For DNA degradation divalent metal ions like Mg^2 +, Mn^2 + or Zn^2 + are absolutely necessary.

General significance

The ability of bacitracin to degrade nucleic acids via a hydrolytic mechanism was a surprising observation, and it is of interest whether these properties can contribute to its mechanisms of action during antibiotic treatment.     

The degradation of [125I]iodoinsulin by cytosol from rat liver and the formation of high-molecular-weight products. A study by gel chromatography

Degradation of [125I]iodoinsulin by cytosol from rat liver (as judged by the appearance of perchloric acid soluble counts) occurs rapidly and generates both high- and low-molecular-weight labelled products on chromatography on AcA54 or AcA202. One of the low-molecular-weight products can be converted to a high-molecular-weight product by further incubation with fresh cytosol. Degradation is not inhibited by chloroquine, but is affected by bacitracin. In the presence of bacitracin most of the radioactivity elutes in the void volume and possibly represents interaction of undegraded insulin with other cytoplasmic components.     

代谢工程改造L-半胱氨酸供给模块促地衣芽胞杆菌 高效合成杆菌肽

杆菌肽是一种主要由芽胞杆菌产生的广谱性抗生素,目前作为兽药广泛应用于畜禽养殖领域。前体氨基酸供应不足可能是限制微生物发酵高产杆菌肽的重要因素。文中以杆菌肽工业生产菌株——地衣芽胞杆菌Bacillus licheniformis DW2为出发菌株,研究l-半胱氨酸供给模块强化对杆菌肽合成的影响。首先,构建了l-半胱氨酸合成酶基因cysK强化表达菌株,杆菌肽效价相比于对照菌株提高了9.47%。接着,为提高l-半胱氨酸合成前体供给,对l-丝氨酸乙酰转移酶基因cysE和硫代硫酸盐/硫酸盐胞内转运蛋白基因cysP进行强化,杆菌肽产量分别提高了7.23%和8.52%。随后,结果表明转运蛋白TcyP负责从胞外向胞内转运胱氨酸,强化表达TcyP后胞内l-半胱氨酸浓度和杆菌肽效价分别提高了29.19%和7.79%。通过组合代谢工程育种,在整合表达了基因cysK基础上,利用强启动子PbacA分别替换基因cysP、cysE和tcyP原始启动子,得到工程菌株CYS4 (DW2::cysK-PbacA(cysP)- PbacA(cysE)-PbacA(tcyP)),杆菌肽效价达到910.02 U/mL,相比于出发菌株DW2 (747.71 U/mL) 提高了21.10%。最后,通过3 L发酵罐小试实验,进一步证实了强化l-半胱氨酸有利于杆菌肽合成。研究表明,强化胞内l-半胱氨酸供给水平是提高地衣芽胞杆菌中杆菌肽产量的有效策略,为杆菌肽工业生产提供了一株具有良好应用前景的菌株。     

Effect and control of glucose feeding on bacitracin production by fed-batch culture of Bacillus licheniformis

The effect of glucose feeding on bacitracin production was investigated by fed-batch culture of Bacillus licheniformis. In batch culture, bacitracin secretion was induced after the glucose initially contained in the medium was completely consumed. The concentration of bacitracin, however, increased to no more than 340 units·ml⁻¹ in the batch cultivations. Therefore, additional glucose was supplied after exhaustion of the initial glucose. The effect of glucose feeding on bacitracin biosynthesss was investigated in two ways, the pH-stat modal feeding method and the CO₂-dependent feeding method. A kinetic study of bacitracin production found that some glucose was necessary, even during the bacitracin production phase. Excessive feeding of glucose, however, caused a reduction in bacitracin biosynthetic activity. When 50 g·l⁻¹ of defatted soy bean meal (SBM) was used, the bacitracin concentration reached 670 units·ml⁻¹ with the pH-stat modal feeding method and 610 units·ml⁻¹ with the CO₂-dependent feeding method, respectively. The yield of bacitracin from consumed glucose was better for the pH-stat method. Using this control strategy, the highest concentration of bacitracin (940 units·ml⁻¹) was obtained with 150 g·l⁻¹ of SBM.     

Effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broiler chickens

Aims: Determining the effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broilers. Methods and Results: 16S rRNA gene-based polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR–DGGE) profiling, DNA sequencing and real-time quantitative PCR techniques were used. The richness of both ileal and caecal microbiota increased with chicken age. The microbiota from those birds of the same age demonstrated relatively similar PCR–DGGE profiles and tended to form closely related clusters in the relatedness analyses. Dietary treatment with bacitracin (50 mg kg⁻¹) and access to range did not change the richness but altered the composition of the microbiota. The impact of bacitracin was particularly obvious in 3-day-old chicks. Lactobacilli were abundant in the caecal microbiota of 3-day-old chicks regardless of the dietary treatment with bacitracin. The access to range enriched Bifidobacterium in both the ileum and caeca. Conclusions: Bacitracin, bird age and access to range all influenced bacterial microbiota in the ileum and caeca of broilers, with bird age having the greatest apparent effect. Significance and Impact of the Study: Providing useful information for the development of antibiotic replacement therapy for poultry production.     

Degradation by eco R1 endonuclease of DNA isolated from phages infectingBacillus licheniformis andBacillus thuringiensis

DNA was isolated from lytic phages of two strains, Bacillus licheniformis, a producer of bacitracin, and Bacillus thuringiensis forming protein paracrystals with pronounced insecticidal effects. Its sensitivity to Eco R1 restriction endonuclease was determined. It was the aim of the work to find out whether these phages could serve as vectors in the transfer and possible amplification of genes of the two important industrial species of bacilli. Approximate values of the molecular weight of DNA of the two phages were determined after degradation of the phage DNA by Eco R1, followed by comparison of electrophoretic mobility of individual fragments with that of the Eco R1-degraded DNA of phage lambdab2.     

Characterization of the Mechanism of the Staphylococcus aureus Cell Envelope by Bacitracin and Bacitracin-Metal Ions

Bacitracin is a metal-dependent dodecapeptide antipeptide produced by Bacillus species. Microcalorimetry was used to study the antimicrobial activity of bacitracin and bacitracin-metal ion complexation inhibited on Staphylococcus aureus at 37 degrees C. The affinity of metal ions binding to bacitracin was investigated by isothermal titration calorimetry and was as follows: Cu(II) >or= Ni(II) > Co(II) > Zn(II) >or= Mn(II). The metal ion binding affinity is not relative to the antimicrobial activity of bacitracin-metal complexation. Atomic force microscopic images revealed that the surface of S. aureus treated by bacitracin-Zn(II) was rather rough compared to that treated by bacitracin only. The central cell surface displayed small depressed grooves around the septal annulus at the onset of division. Bacitracin mainly inhibited the splitting system within the thick cross walls as seen by transmission electron microscopy (TEM). The inhibition mechanism of bacitracin may be relative to the assistance of Zn(II) coordination with the cell surface as seen by TEM. We can put forward that the activity of bacitracin only inhibited growth and division initially from the synthesis of the cell wall, especially the cell wall of the septal annulus. The divalent metal ions function to increase the adsorption of bacitracin onto the cell surface.     

Comparison of checkerboard and time-kill methods for the analysis of two antibiotics combined

Activity interaction analysis of two antibiotics by two methods: checkerboard and "time-kill" was compared during this study. Combinations of procaine penicillin, polymyxin B and bacitracin with neomycin and procaine penicillin with dihydrostreptomycin were examined. Checkerboard method is the most widely used technique for antimicrobials interactions analyses. The "time-kill" method, performed by the broth macrodilution technique, provides a dynamic picture of antimicrobial action and interaction over time (based on serial colony counts). Differences of "time-kill" method and the checkerboard technique, allow single visual examination (after 16 to 24 hours of incubation). Additive and inhibition effects were observed in combinations of neomycin with beta-lactam antibiotic (procaine penicillin) and peptide antibiotics (bacitracin and polymyxin B) on clinical strain S. Enteritidis IL 35 "Time-kill" method also confirmed observations mentioned above. In combinations of procaine penicillin with dihydrostreptomycin on strains E. coli IL 531 and E. coli IL 256 synergy effects on checkerboard technique were noticed. Such observation was not confirmed by the "time-kill" method. The methodologies and definitions of synergism are variable and not standardized. This situation should be improved, because comparison of the results obtained by different methods becomes a very difficult task.     

Safety of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> ST8Sh and Its Bacteriocin

Total DNA extracted from Lb. plantarum ST8Sh was screened for the presence of more than 50 genes related to production of biogenic amines (histidine decarboxylase, tyrosine decarboxylase, and ornithine decarboxylase), virulence factors (sex pheromones, gelatinase, cytolysin, hyaluronidase, aggregation substance, enterococcal surface protein, endocarditis antigen, adhesion of collagen, integration factors), and antibiotic resistance (vancomycin, tetracycline, erythromycin, gentamicin, chloramphenicol, bacitracin). Lb. plantarum ST8Sh showed a low presence of virulence genes. Only 13 genes were detected (related to sex pheromones, aggregation substance, adhesion of collagen, tetracycline, gentamicin, chloramphenicol, erythromycin, but not to vancomycin, and bacitracin) and may be considered as indication of safety for application in fermented food products. In addition, interaction between Lb. plantarum ST8Sh and drugs from different groups were determined in order to establish possible application of the strain in combination with commercial drugs. Cytotoxicity of the semi-purified bacteriocins produced by Lb. plantarum ST8Sh was depended on applied concentration—highly cytotoxic when applied at 25 μg/mL and no cytotoxicity at 5 μg/mL.