Enhancement of edeine production in Brevibacillus brevis X23 via in situ promoter engineering.

Edeines, a group of cationic antimicrobial peptides produced by the soil bacterium Brevibacillus, have broad biological effects, such as antimicrobial, anticancer and immunosuppressive activities. However, the yield of edeines in wild-type (WT) Brevibacillus is extremely low, and chemical synthesis of edeines is a time-consuming process. Genetic engineering has proven to be an effective approach to produce antibiotics with high yield. In this study, the edeine biosynthetic gene cluster (ede BGC), which is involved in edeine production, was identified and characterized in Brevibacillus brevis X23. To improve edeine production in B. brevis X23, the ede BGC promoter was replaced with six different promoters, Pmwp , Pspc , PxylA , Pshuttle-09 , Pgrac or P43 , through double-crossover homologous recombination. The new promoters significantly increased the expression of the ede BGC as well as edeine production by 2.9 ± 0.4 to 20.5 ± 1.2-fold and 3.6 ± 0.1to 8.7 ± 0.7-fold respectively. The highest yield of edeines (83.6 mg l-1 ) was obtained in B. brevis X23 with the Pmwp promoter. This study provides a practical approach for producing high yields of edeines in B. brevis.

Brevibacillus fortis NRS-1210 produces edeines that inhibit the in vitro growth of conidia and chlamydospores of the onion pathogen Fusarium oxysporum f. sp. cepae.

Onions can be damaged by Fusarium basal rot caused by the soilborne fungus Fusarium oxysporum f. sp. cepae (FOC). Control of this pathogen is challenging since there is limited genetic resistance in onion. The identification of molecules that inhibit this pathogen is needed. Antagonism screening showed Brevibacillus fortis NRS-1210 secreted antifungal compounds into growth medium. The spent growth medium, diluted 1:1, inhibited growth of FOC conidia after seven hours and killed 67-91% of conidia after 11 h. The spent medium also inhibited growth of propagules from F. graminearum, F. proliferatum, F. verticillioides and Galactomyces citri-aurantii. Full strength spent growth medium did not effectively kill FOC conidia and chlamydospores inoculated into a sand cornmeal mixture. In silico analysis of the B. fortis NRS-1210 genome indicated the biosynthetic clusters of several antibiotics. Fractionation of spent medium followed by reverse-phase liquid chromatography with tandem mass spectrometry analysis found that fractions with the most antifungal activity contained a combination of edeines A, B and F and no other recognized antibiotics. 1H NMR signals of the active fraction corresponded to edeine, a pentapeptide with broad spectrum antimicrobial activity which blocks translation in both prokaryotes and eukaryotes. Comparative genomics of Brevibacillus genomes shows edeine producers form a clade which consists of: Brevibacillus brevis, Brevibacillus formosus, 'Brevibacillus antibioticus', Brevibacillus schisleri, Brevibacillus fortis, and Brevibacillus porteri. This observation suggests edeine played an important role in the evolution and speciation of the Brevibacillus genus.

Self resistance to the atypical cationic antimicrobial peptide edeine of Brevibacillus brevis Vm4 by the N-acetyltransferase EdeQ.

Edeines are atypical cationic peptides produced by Brevibacillus brevis Vm4 with broad-spectrum antimicrobial activity. These linear nonribosomal peptides bind to the 30S ribosomal subunit and block t-RNA binding to the P-site. To identify the mechanism of high-level self-resistance in the producing organism, the B. brevis Vm4 genome was sequenced and the edeine biosynthetic cluster discovered. A potential edeine-modifying enzyme, EdeQ, showed similarity to spermidine N-acetyltransferases. EdeQ was purified and shown to convert edeine to N-acetyledeine, which is inactive against cells in vivo and against cell-free extracts. Unexpectedly, tandem mass spectroscopy and nuclear magnetic resonance demonstrate that N-acylation occurs on the free amine of the internal diaminopropionic acid rather than the N-terminal spermidine polyamine. Acetylation of edeine by EdeQ abolishes its ability to inhibit translation, thus conferring resistance to the antibiotic in the producing organism.

The immunoregulatory effects of edeine analogues in mice.

The edeines analogs were tested in several in vitro and in vivo assays using the mouse model, with edeine B (peptide W1) and cyclosporine A as reference compounds. The peptides displayed moderate, stimulatory effects on concanavalin A-induced (ConA-induced) splenocyte proliferation, whereas their effects on pokeweed mitogen-induced (PWM-induced) splenocyte proliferation were inhibitory. The peptides inhibited lipopolysacharide-induced (LPS-induced) tumor necrosis factor alpha production but had little effect on interleukin 6 production. In the model of the humoral immune response in vitro to sheep red blood cells, peptide 1 was distinctly stimulatory in the investigated concentrations (1-100 microg/ml), whereas peptides 3 and 4 only stimulated the number of antibody-forming cells at the highest concentration (100 microg/ml). In the model of the delayed type hypersensitivity in vivo to ovalbumin, the peptides were moderately suppressive (3 being the most active). The reference peptide W1 stimulated ConA-induced cell proliferation at 1-10 microg/ml but was inhibitory at 100 microg/ml. It also inhibited PWM-induced cell proliferation in a dose-dependent manner. This peptide had no effect on the humoral immune response in vitro or on cytokine production, but inhibited DTH reaction in vivo. The relationship between structure and activity, and a possible mode of action of the peptides, is discussed in this paper.

Structure activity relationship studies on the antimicrobial activity of novel edeine A and D analogues.

Edeines are pentapeptide amide antibiotics composed of four nonprotein amino acids, glycine, and polyamine. They exhibit antimicrobial and immunosuppressive activities and are universal inhibitors of translation. Moreover, it was proven that the free ionizable carboxy group in the (2R, 6S, 7R)-2,6-diamino-7-hydroxyazelaic acid moiety is not essential for biological activity of these compounds. In this paper we describe the synthesis of four novel edeine A and D analogues in which the above-mentioned acid residue was replaced with the (3R, 4S)- or (3S, 4S)-4,5-diamino-3-hydroxypentanoic acid moiety. In one compound we also introduced into molecule the 3-N,N-dimethyl derivative of (S)-2,3-diaminopropanoic acid to prevent the transpeptidation process, which results in the loss of biological activity of alpha-isomers of edeines. All peptides were synthesized applying the active ester and azide methods and on the basis of the coupling of suitable N-terminal tripeptides with proper C-terminal dipeptide amides. The activities of the newly obtained edeine analogues against selected strains of bacteria and fungi are also presented.

Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3.

The small ribosomal subunit is responsible for the decoding of genetic information and plays a key role in the initiation of protein synthesis. We analyzed by X-ray crystallography the structures of three different complexes of the small ribosomal subunit of Thermus thermophilus with the A-site inhibitor tetracycline, the universal initiation inhibitor edeine and the C-terminal domain of the translation initiation factor IF3. The crystal structure analysis of the complex with tetracycline revealed the functionally important site responsible for the blockage of the A-site. Five additional tetracycline sites resolve most of the controversial biochemical data on the location of tetracycline. The interaction of edeine with the small subunit indicates its role in inhibiting initiation and shows its involvement with P-site tRNA. The location of the C-terminal domain of IF3, at the solvent side of the platform, sheds light on the formation of the initiation complex, and implies that the anti-association activity of IF3 is due to its influence on the conformational dynamics of the small ribosomal subunit.