Amylocyclicin,a Novel Circular Bacteriocin Produced by Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 is a Gram-positive plant growth-promoting bacterium with an impressive capacity to synthesize nonribosomal secondary metabolites with antimicrobial activity. Here we report on a novel circular bacteriocin which is ribosomally synthesized by FZB42. The compound displayed high antibacterial activity against closely related Gram-positive bacteria. Transposon mutagenesis and subsequent site-specific mutagenesis combined with matrix-assisted laser desorption ionization–time of flight mass spectroscopy revealed that a cluster of six genes covering 4,490 bp was responsible for the production, modification, and export of and immunity to an antibacterial compound, here designated amylocyclicin, with a molecular mass of 6,381 Da. Peptide sequencing of the fragments obtained after tryptic digestion of the purified peptide revealed posttranslational cleavage of an N-terminal extension and head-to-tail circularization of the novel bacteriocin. Homology to other putative circular bacteriocins in related bacteria let us assume that this type of peptide is widespread among the Bacillus/Paenibacillus taxon.     

Properties of Bac W42, a bacteriocin produced by Bacillus subtilis W42 isolated from Cheonggukjang

Ten Bacillus strains with antimicrobial activities were isolated from Cheonggukjang produced at different parts in Korea. They all inhibited Listeria monocytogenes ATCC 19111 and nine inhibited Bacillus cereus ATCC 14579. Four isolates (W42, H27, SKE 12, and K21) showing strong inhibiting activities were identified as B. subtilis. B. subtilis W42 was the most inhibiting strain. The antimicrobial activity of culture supernatant from B. subtilis W42 was destroyed completely by proteinase K treatment, indicating that a bacteriocin was the responsible agent. The bacteriocin, Bac W42, was most stable at pH 7 and stable between pH 3-6 and 8-9. Bac W42 was stable up to 80°C. BHI (brain heart infusion) and TSB (tryptic soy broth) were the best media for the activity (320 AU/ml) followed by LB (160 AU/ml). Bac W42 was partially purified by column chromatographies. The specific activity was increased from 1,151.2 AU/ml to 9,043.5 AU/ml and the final yield was 26.3%. Bac W42 was 5.4 kDa in size as determined by SDS-PAGE. Bac W42 showed bactericidal activity against L. monocytogenes ATCC 19111.     

Antibacterial activities of cryptotanshinone and dihydrotanshinone I from a medicinal herb, Salvia miltiorrhiza Bunge

Cryptotanshinone and dihydrotanshinone I, constituents of a medicinal plant, Salvia miltiorrhiza Bunge, had antibacterial activity against a broad range of Gram positive bacteria. These compounds generated superoxide radicals in Bacillus subtilis lysates. A recombination-deficient mutant strain of B. subtilis was 2- to 8-fold more sensitive than a wild strain, and this hypersensitivity was reduced in the presence of dithiothreitol as an antioxidant. DNA, RNA, and protein syntheses in B. subtilis were non-selectively inhibited by these compounds. These results suggest that superoxide radicals are important in the antibacterial actions of the agents.     

Identification and characterization of leucocyclicin Q, a novel cyclic bacteriocin produced by Leuconostoc mesenteroides TK41401

The culture supernatant of Leuconostoc mesenteroides TK41401, isolated from Japanese pickles, possessed antimicrobial activity against broad range of a bacterial genera and particularly strong activity against Bacillus coagulans, the major contaminant of pickles. An antimicrobial peptide was purified in three chromatographic steps, and its molecular mass was determined to be 6,115.59 Da by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). The primary structure of this peptide was determined by amino acid and DNA sequencing, and these analyses revealed that it was translated as a 63-residue precursor. This precursor showed high similarity to the precursor of lactocyclicin Q, a cyclic bacteriocin produced by Lactococcus sp. strain QU 12. The molecular weight calculated after cyclization, which was presumed to involve the same process as in lactocyclicin Q (between L3 and W63), agreed with that estimated by ESI-TOF MS. This peptide was proved to be a novel cyclic bacteriocin, and it was termed leucocyclicin Q. The antimicrobial spectrum of this bacteriocin clearly differed from that of lactocyclicin Q, even though their primary structures were quite similar. This is the first report of a cyclic bacteriocin produced by a strain of the genus Leuconostoc.     

Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains

Aims:  The identification of a new compound active against Agrobacterium tumefaciens .
Methods and Results:  The culture conditions of a newly isolated Bacillus subtilis strain, designed 14B, were optimized, as a first step, to produce its bacteriocin (termed Bac 14B) for the biocontrol of Agrobacterium spp., the causal agents of the crown gall disease. Bac 14B was then partially purified and biochemically characterized. Bacillus subtilis 14B was observed to produce an antibacterial compound having a protinaceous nature. As estimated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), the semi-purified bacteriocin substance was found to be a monomeric protein with a molecular weight of 21 kDa. While the latter's antimicrobial activity was completely stable during exposure to a temperature range of up to 100°C for 2 h, its initial activity was totally lost at 121°C for 20 min. The maximum bacteriocin production (4096 AU ml⁻¹) was recorded after 96 h-incubation in an optimized Luria Bertani medium supplemented with 10 g l⁻¹ glucose, 15 g l⁻¹ K₂HPO₄ and 5 g l⁻¹ MgSO₄ 7H₂O at 30°C in a shaking flask culture. Interestingly, the B. subtilis 14B culture supernatant that contained the bacteriocin under study was proved efficient in reducing both the percentage of galled plants and the number of galls in tomato.
Conclusion:  The findings revealed that B. subtilis 14B and its bacteriocin are efficient in reducing the percentage of infections in plants caused by Ag. tumefaciens .
Significance and Impact of the Study:  The results could be useful for the nurserymen who are particularly interested in the biocontrol of the crown gall disease.     

Partial characterization of polyfermenticin SCD, a newly identified bacteriocin of Bacillus polyfermenticus

AIMS: To characterize polyfermenticin SCD, a newly identified bacteriocin of Bacillus polyfermenticus SCD. METHODS AND RESULTS: Bacillus polyfermenticus SCD was identified as a bacteriocin producer with a bactericidal activity against Bacillus subtilis IFO 12113. Polyfermenticin SCD, named tentatively as the bacteriocin produced by B. polyfermenticus SCD, showed a narrow spectrum of activity against Gram-positive and Gram-negative bacteria, a yeast and moulds. Production of polyfermenticin SCD in a 5 l jar fermenter followed typical kinetics of primary metabolite synthesis. The antibacterial activity of polyfermenticin SCD on sensitive indicator cells disappeared completely by treatment with proteinase K, which indicates its proteinaceous nature. Polyfermenticin SCD seemed to be very stable throughout the pH range of 2.0 to 9.0, and it was relatively heat labile compared with other bacteriocins. Direct detection of polyfermenticin SCD activity on SDS-PAGE suggested that it had an apparent molecular mass of about 14.3 kDa. CONCLUSIONS: Bacillus polyfermenticus SCD produced relatively heat-labile polyfermenticin SCD with a narrow spectrum of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus polyfermenticus SCD is a commercial probiotic which has been used for the treatment of long-term intestinal disorders. New findings on polyfermenticin SCD will be valuable in the evaluation of commercial probiotics. Polyfermenticin SCD can be used to control Bacillus spoilage organisms as a biological control agent.     

Characterization of putative class II bacteriocins identified from a non-bacteriocin-producing strain Lactobacillus casei ATCC 334

Several putative class II bacteriocin-like genes were identified in Lactobacillus casei ATCC 334, all of which might encode peptides with a double-glycine leader. Six peptides encoded by these genes were heterologously expressed in Escherichia coli and then partially purified in order to test their bacteriocin activity. The results revealed that the mature LSEI_2163 peptide was a class IId bacteriocin that exhibited antimicrobial activity against some lactobacilli and several Listeria species. Similarly, mature LSEI_2386 was a putative pheromone peptide that also had significant bacteriocin activity against several Listeria species. The activities of both peptides tolerated 121°C for 30 min but not treatment with proteinase K or trypsin. The two Cys residues located at positions 4 and 24 in the mature LSEI_2163 peptide were shown by mass spectrometry to form a disulfide bridge, which was required for optimal antibacterial activity. However, replacement of one or both Cys with Ser would cause significant reduction of the antibacterial activity, the reduction being greater when only one of the Cys residues (C4S) was replaced than when both (C4S/C24S) were replaced.     

Synthesis and antibacterial activity of peptide deformylase inhibitors

Peptide deformylase catalyzes the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria. Its essential character in bacterial cells makes it an attractive target for antibacterial drug design. In this work, we have rationally designed and synthesized a series of peptide thiols that act as potent, reversible inhibitors of purified recombinant peptide deformylase from Escherichia coli and Bacillus subtilis. The most potent inhibitor has a K(I) value of 11 nM toward the B. subtilis enzyme. These inhibitors showed antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations (MIC) as low as 5 microM ( approximately 2 microg/mL). The PDF inhibitors induce bacterial cell lysis and are bactericidal toward all four bacterial strains that have been tested, B. subtilis, Staphylococcus epidermidis, Enterococcus faecalis, and E. coli. Resistance evaluation of one of the inhibitors (1b) against B. subtilis showed that no resistant clone could be found from >1 x 10(9) cells. Quantitative analysis using a set of inhibitors designed to possess varying potencies against the deformylase enzyme revealed a linear correlation between the MIC values and the K(I) values. These results suggest that peptide deformylase is the likely molecular target responsible for the antibacterial activity of these inhibitors and is therefore a viable target for antibacterial drug design.     

Identification of Bacillus subtilis sigma-dependent genes that provide intrinsic resistance to antimicrobial compounds produced by Bacilli

Bacillus subtilis produces many antibiotics of varying structures and specificity. Here we identify a prominent role for sigma(W), an extracytoplasmic function (ECF) sigma factor, in providing intrinsic resistance to antimicrobial compounds produced by other Bacilli. By using a panel of B. subtilis mutants disrupted for each of the 30 known sigma(W)-dependent operons we identified resistance genes for at least three different antimicrobial compounds. The ydbST and fosB genes contribute to resistance to antimicrobial compound(s) produced by B. amyloliquefaciens FZB42, the yqeZyqfAB operon provides resistance to the SPbeta prophage-encoded bacteriocin sublancin, and the yknWXYZ operon and yfhL provide resistance to the antimicrobial peptide SdpC. YfhL encodes a paralogue of SdpI, a membrane protein that provides immunity to SdpC. In competition experiments, we identify sigma(W) as a key factor in allowing B. subtilis to resist antibiotic killing and encroachment by competing strains. Together with the previous observation that sigma(W) provides inducible resistance against the Streptomyces antibiotic fosfomycin, these studies support the notion that sigma(W) controls an antibiosis regulon important in the microbial ecology of soil bacteria.     

Characterization of an antibacterial peptide produced by Brevibacterium linens

AIMS: The aim of this research was to investigate the antimicrobial activity produced by Brevibacterium linens ATCC 9175. METHODS AND RESULTS: A bacteriocin produced by the red smear cheese bacterium B. linens ATCC 9175 was identified. The antimicrobial activity was first produced at the exponential growth phase. A crude bacteriocin obtained from the culture supernatant fluid was inhibitory to some indicator strains. It inhibited the growth of Listeria monocytogenes ATCC 7644, B. linens ATCC 9172 and Corynebacterium fimi NCTC 7547, but was inactive against the Gram-negative bacteria and yeast tested. The bacteriocin was stable at 30 degrees C but the activity was lost when the temperature reached 50 degrees C. It was sensitive to the proteolytic action of trypsin, papain and pronase E and was active between pH 6.0 and 9.0. The bacteriocin was bactericidal to L. monocytogenes at 40 AU ml(-1). Bacteriostasis was observed for a low dose of bacteriocin (20 AU ml(-1)). CONCLUSIONS: An antibacterial peptide produced by B. linens was characterized, presenting potential for use as a biopreservative in food systems. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of a novel bacteriocin active against L. monocytogenes addresses an important aspect of food protection against pathogens and spoilage micro-organisms.     

Identification of downstream target genes of latent membrane protein 1 in nasopharyngeal carcinoma cells by suppression subtractive hybridization

Proteolytic digestion of bovine beta-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15-20), AASDISLLDAQSAPLR (residues 25-40), IPAVFK (residues 78-83) and VLVLDTDYK (residues 92-100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55-64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of beta-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of beta-lactoglobulin could be useful to increase its antimicrobial function.     

Effect of hydrogen peroxide on antibacterial activities of Canadian honeys

Honey is recognized as an efficacious topical antimicrobial agent in the treatment of burns and wounds. The antimicrobial activity in some honeys depends on the endogenous hydrogen peroxide content. This study was aimed to determine whether honey's hydrogen peroxide level could serve as a honey-specific, activity-associated biomarker that would allow predicting and assessing the therapeutic effects of honey. Using a broth microdilution assay, I analyzed antibacterial activities of 42 Canadian honeys against two bacterial strains: Escherichia coli (ATCC 14948) and Bacillus subtilis (ATCC 6633). The MIC90 and MIC50 were established from the dose-response relationship between antibacterial activities and honey concentrations. The impact of H2O2 on antibacterial activity was determined (i) by measuring the levels of H2O2 before and after its removal by catalase and (ii) by correlating the results with levels of antibacterial activities. Canadian honeys demonstrated moderate to high antibacterial activity against both bacterial species. Both MIC90 and MIC50 revealed that the honeys exhibited a selective growth inhibitory activity against E. coli, and this activity was strongly influenced by endogenous H2O2 concentrations. Bacillus subtilis activity was marginally significantly correlated with H2O2 content. The removal of H2O2 by catalase reduced the honeys' antibacterial activity, but the enzyme was unable to completely decompose endogenous H2O2. The 25%-30% H2O2 "leftover" was significantly correlated with the honeys' residual antibacterial activity against E. coli. These data indicate that all Canadian honeys exhibited antibacterial activity, with higher selectivity against E. coli than B. subtilis, and that these antibacterial activities were correlated with hydrogen peroxide production in honeys. Hydrogen peroxide levels in honey, therefore, is a strong predictor of the honey's antibacterial activity.     

Isolation and characterization of a transposon mutant of Pseudomonas fluorescens AU63 deficient in antifungal activity against Pythium ultimum

Tn5 transposon mutagenesis via electroporation of Pseudomonas fluorescens AU63 was used to generate mutants deficient in antifungal activity against the phytopathogenic fungi Pythium ultimum and Thielaviopsis basicola. Mutant C-45 was obtained by an initial screen for the loss of antibacterial activity against Bacillus subtilis and a subsequent screen of mutants obtained for the loss of antifungal activity against pathogenic fungi. A single chromosomal insertion of Tn5 in the chromosome of Ps. fluorescens C-45 was confirmed by Southern blot hybridization. A metabolite responsible for the observed antibacterial and antifungal activities was identified using thin layer chromatography. The antimicrobial activities of the partially purified substance present in the parental strain and missing in the C-45 mutant were not affected by protease, high temperature, acid or alkali treatment. These results provide the basis for a structural analysis of this new antimicrobial substance and the genetic elucidation of its biosynthesis.     

Design, NMR characterization and activity of a 21-residue peptide fragment of bacteriocin AS-48 containing its putative membrane interacting region.

Bacteriocin AS-48 is a 70-residue cyclic polypeptide from Enterococcus faecalis that shows a broad antimicrobial spectrum against both Gram-positive and Gram-negative bacteria. The structure of bacteriocin AS-48 consists of a globular arrangement of five helices with a high positive electrostatic potential in the region comprising helix 4, the turn linking helix 4 and 5, and the N-terminus of helix 5. This region has been considered to participate in its biological activity and in particular in membrane permeation. To understand the mechanism of the antibacterial activity of AS-48 and to discriminate the several mechanisms proposed, a simplified bacteriocin was designed consisting of 21 residues and containing the high positively charged region. A disulfide bridge was introduced at an appropriate position to stabilize the peptide and to conserve the helix-turn-helix arrangement in the parent molecule. According to (1)H and (13)C NMR data, the designed simplified bacteriocin fragment adopts a significant population of a native-like helical hairpin conformation in aqueous solution, which is further stabilized in 30% TFE. The designed peptide does not show any antibacterial activity, though it is shown to compete with the intact native bacteriocin AS-48. These results suggest that the mechanism of membrane disruption by bacteriocin is not as simple as being driven by a deposition of positively charged molecules on the plane of the bacterial membrane. Some other regions of the protein must be present such as, for instance, hydrophobic regions so as to enhance the accumulation of the peptide and favour membrane permeation.     

Detection of antimicrobial activities and bacteriocin structural genes in faecal enterococci of wild animals

The production of antimicrobial activities as well as the presence of bacteriocin structural genes (entA, entB, entP, entQ, cylL, entAS-48, bac31, and entL50A/B) were studied in 140 non-selected faecal enterococcal isolates recovered from wild animals. Eight different indicator strains (including Listeria monocytogenes, Pediococcus pentosaceus, and different enterococcal species) were used for antimicrobial activity detection. Twenty-five of the 140 enterococci (18%) showed antimicrobial activity against L. monocytogenes and 33 additional isolates (24%) showed antimicrobial activity against other indicator strains, but Listeria. At least one bacteriocin structural gene was detected in 17 of the 25 enterococci with antimicrobial activity against L. monocytogenes and different combinations of entA, entB, entP, entQ, entL50A/B, and cylL genes were detected; entA and entB were the most prevalent detected genes, and they were generally associated. Bacteriocin structural genes were detected in 10 of 33 isolates with antimicrobial activity against indicator strains other than Listeria, and the cylL gene was the most prevalent one, especially in E. faecalis isolates.     

Identification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilis.

Hemolymph from partially fed virgin Dermacentor variabilis females was collected following Borrelia burgdorferi challenge and assayed for antimicrobial activity against Bacillus subtilis and B. burgdorferi. A small inducible cationic peptide was identified by SDS-PAGE in the hemolymph of these ticks as early as 1h post challenge. Following purification by a three-step procedure involving sequential SepPak elution, reversed phase high performance liquid chromatography (RP-HPLC) and gel electrophoresis, the yield of the active peptide was approximately 0.1% of the total protein in the hemolymph plasma. The molecular weight, 4.2kDa, was determined by MALDI-TOF mass spectrometry. N-terminal sequencing by the Edman degradation method gave a sequence for the first 30 amino acids as: G-F-G-C-P-L-N-Q-G-A-C-H-N-H-C-R-S-I-(R)-(R)-(R)-G-G-Y-C-S-Q-I-I-K. A computer search of databases showed that the peptide had 83% similarity to a defensin found in a scorpion. This is the first report of a defensin from a tick. The peptide was stable at least up to 70 degrees C. Although the tick defensin alone was not immediately effective against B. burgdorferi, tick defensin plus lysozyme killed more than 65% of cultured B. burgdorferi within 1h.     

Antimicrobial and antiviral screening of novel indole carboxamide and propanamide derivatives

A few series of indole derivatives were screened for antimicrobial, antifungal and anti-HBV activities. The compounds were tested for their in vitro antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and for their antifungal activity against Candida albicans using a disc diffusion method, which measures the diameter of the inhibition zone around a paper disc soaked in a solution of the test compounds. The antimicrobial activity results showed that all compounds are as a active as the standard compound ampicillin against Staphylococcus aureus. It was also found that indole carboxamide derivatives, substituted at 3-position with several benzyl groups, showed better inhibition of Bacillus subtilis than their congeners substituted at 2-position. Activity patterns of the compounds against Escherichia coli and Staphylococcus aureus were found slightly different by the same method. In this case, there was no correlation between structure and activity of the compounds. The antifungal activity of carboxamide derivatives was found higher compared to that of the propanamide derivatives. The minimum inhibitory concentration (MIC) values of some indole derivatives were also determined by the tube dilution technique. The MIC values of the compounds were found nearly 20- to 100-fold smaller compared to the standard compounds ciprofloxacin and ampicillin (1.56-3.13 microg/ml and 1.56-12.5 microg/ml, respectively) against Staphylococcus aureus, Bacillus subtilis and Escherichia coli. The MIC values of the tested compounds showed that these are better inhibitors for Candida albicans. Indole derivatives were screened by the anti-HBV susceptibility test. No compound showed good inhibition against the HBV virus.     

Macrolactin N, a new peptide deformylase inhibitor produced by Bacillus subtilis

A new 24-membered ring lactone, macrolactin N, was isolated from a culture broth of Bacillus subtilis and its structure was established by various spectral analysis. Macrolactin N inhibited Staphylococcus aureus peptide deformylase with an IC50 value of 7.5 microM and also showed antibacterial activity against Escherichia coli and S. aureus.     

Antimicrobial activity of hydrophobic xanthones from Cudrania cochinchinensis against Bacillus subtilis and methicillin-resistant Staphylococcus aureus

Ten xanthones with one or two isoprenoid groups and a prenylated benzophenone isolated from roots of Cudrania cochinchinensis (Moraceae) were tested for their antimicrobial activities against Bacillus subtilis and methicillin-resistant Staphylococcus aureus (MRSA). Among these compounds, gerontoxanthone H exhibited considerable antibacterial activity against B. subtilis (MIC = 1.56 microg/ml). Four xanthones, gerontoxanthone I, toxyloxanthone C, cudraxanthone S, and 1,3,7-trihydroxy-2-prenylxanthone, showed weak antibacterial activity against the bacterium (MICs = 3.13-6.25 microg/ml). These compounds also exhibited similar MIC values against methicillin-sensitive S. aureus, MRSAs, and Micrococcus luteus.     

An In Silico Evaluation of Molecular Interaction Between Antimicrobial Peptide Subtilosin A of Bacillus subtilis with Virulent Proteins of Aeromonas hydrophila

International Journal of Peptide Research and Therapeutics - Subtilosin A, a cyclic peptide from Bacillus subtilis is known for its antimicrobial activity against a diverse range of bacteria....