Novel ribosomally synthesized peptide antibiotics from microbial genomes
Final Report Abstract
Many bacteria produce gene encoded, ribosomally synthesized peptides, which display antibacterial activity. These peptides often contain post-translational modifications, e.g. thioethers or thiazole/oxazole rings. All modified amino acids are introduced by unique enzymes which are encoded in the vicinity of the structural genes in biosynthetic gene clusters. The focus of the project was the expression of such peptides that are encoded in sequenced bacterial genomes but have not yet been characterized. A number of different gene clusters of sequenced strains was tested for expression, among them also an archeal gene cluster. In the end, we focussed on the most promising peptide that had been idenfied by BLAST searches using the mersacidin biosynthetic enzyme (MrsM) in the NCBI database and derived from a new class II lantibiotic gene cluster in Bacillus pseudomycoides DSM 12442. Mass spectrometric analysis identified a peptide of m/z 2,786 Da in the active fraction of the cell wash extract. The peptide and site directed mutants were expressed in Escherichia coli along with the modifying enzyme. This resulted in the production of a modified peptide with the correct mass, harboring four out of eight possible dehydrations and supported the presence of four thioether and one disulfide bridge. After proteolytic activation by removal of the leader sequence, the core peptide showed antimicrobial activity. A preliminary structure was proposed from MS/MS measurements of the mutant peptides but will have to be confirmed by NMR. Surprisingly, during the purification procedure a second substance was isolated from the producer strain. This substance shows a pronounced synergy with pseudomycoicidin and might be produced by a nearby non-ribosomal peptide biosynthesis gene cluster, which, however has yet to be proven. If it is a non-ribosomal peptide, this would be the first time that such a synergy has been described. Mode of action experiments showed that the mixture of both substances is able to destroy the membrane potential and induce slow efflux of potassium ions. Indicator strains that harbor fusions of promoters reacting to different antibiotic stresses demonstrated that pseudomycoicidin but not the second substance might interfere with cell wall biosynthesis. In conclusion, the current hypothesis is that pseudomycoicidin might bind to lipid II and then form a complex with the second substance, resulting in membrane damage.
Publications
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(2009). Production of the novel two-peptide lantibiotic lichenicidin by Bacillus licheniformis DSM 13. PLoS One. 4(8):e6788
Dischinger J, Josten M, Szekat C, Sahl HG, Bierbaum G
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(2010). Marine myxobacteria as a source of antibiotics--comparison of physiology, polyketide-type genes and antibiotic production of three new isolates of Enhygromyxa salina. Mar Drugs. 8(9):2466-79
Schäberle TF, Goralski E, Neu E, Erol O, Hölzl G, Dörmann P, Bierbaum G, König GM
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(2011). Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42. PLoS One. 6(7):e22389
Herzner AM, Dischinger J, Szekat C, Josten M, Schmitz S, Yakéléba A, Reinartz R, Jansen A, Sahl HG, Piel J, Bierbaum G
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(2013). Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat Prod Rep. 30(1):108-60
Arnison PG, Bibb MJ, Bierbaum G, Bowers AA, Bugni TS, Bulaj G, Camarero JA,Campopiano DJ, Challis GL, Clardy J, Cotter PD, Craik DJ, Dawson M, Dittmann E,Donadio S, Dorrestein PC, Entian KD, Fischbach MA, Garavelli JS, Göransson U,Gruber CW, Haft DH, Hemscheidt TK, Hertweck C, Hill C, Horswill AR, Jaspars M,Kelly WL, Klinman JP, Kuipers OP, Link AJ, Liu W, Marahiel MA, Mitchell DA, Moll GN, Moore BS, Müller R, Nair SK, Nes IF, Norris GE, Olivera BM, Onaka H, Patchett ML, Piel J, Reaney MJ, Rebuffat S, Ross RP, Sahl HG, Schmidt EW, Selsted ME, Severinov K, Shen B, Sivonen K, Smith L, Stein T, Süssmuth RD, Tagg JR, Tang GL, Truman AW, Vederas JC, Walsh CT, Walton JD, Wenzel SC, Willey JM, van der Donk WA
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(2013). Salimabromide: unexpected chemistry from the obligate marine myxobacterium Enhygromxya salina. Chemistry. 19(28):9319-24
Felder S, Dreisigacker S, Kehraus S, Neu E, Bierbaum G, Wright PR, Menche D, Schäberle TF, König GM
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(2013). Salimyxins and enhygrolides: antibiotic, sponge-related metabolites from the obligate marine myxobacterium Enhygromyxa salina. Chembiochem. 14(11):1363-71
Felder S, Kehraus S, Neu E, Bierbaum G, Schäberle TF, König GM
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(2014). Lantibiotics: promising candidates for future applications in health care. Int J Med Microbiol.304(1):51-62
Dischinger J, Basi Chipalu S, Bierbaum
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(2014). The search for new anti-infective drugs: untapped resources and strategies. Int J Med Microbiol. 304(1):1-2
Bierbaum G, Sahl HG
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(2015). Insights into structure-activity relationships of bacterial RNA polymerase inhibiting corallopyronin derivatives. J Nat Prod. 78(10):2505-9
Schäberle TF, Schmitz A, Zocher G, Schiefer A, Kehraus S, Neu E, Roth M, Vassylyev DG, Stehle T, Bierbaum G, Hoerauf A, Pfarr K, König GM
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(2015). Pseudomycoicidin, a class II lantibiotic from Bacillus pseudomycoides. Appl Environ Microbiol. 15;81(10):3419-29
Basi-Chipalu S, Dischinger J, Josten M, Szekat C, Zweynert A, Sahl HG, Bierbaum G
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(2015). Structural variations of the cell wall precursor lipid II and their influence on binding and activity of the lipoglycopeptide antibiotic oritavancin. Antimicrob Agents Chemother. 59(2):772-81
Münch D, Engels I, Müller A, Reder-Christ K, Falkenstein-Paul H, Bierbaum G, Grein F, Bendas G, Sahl HG, Schneider T
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(2016). Activation of the glmS ribozyme confers bacterial growth inhibition. Chembiochem. 18(5):435-440
Mayer G, Schüller A, Matzner D, Lünse C, Wittmann V, Schumacher C, Unsleber S, Brötz-Oesterhelt H, Mayer C, Bierbaum G
