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. 2011 Jan;193(1):215-24.
doi: 10.1128/JB.00784-10. Epub 2010 Oct 22.

Plantazolicin, a novel microcin B17/streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42

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Plantazolicin, a novel microcin B17/streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42

Romy Scholz et al. J Bacteriol. 2011 Jan.

Abstract

Here we report on a novel thiazole/oxazole-modified microcin (TOMM) from Bacillus amyloliquefaciens FZB42, a Gram-positive soil bacterium. This organism is well known for stimulating plant growth and biosynthesizing complex small molecules that suppress the growth of bacterial and fungal plant pathogens. Like microcin B17 and streptolysin S, the TOMM from B. amyloliquefaciens FZB42 undergoes extensive posttranslational modification to become a bioactive natural product. Our data show that the modified peptide bears a molecular mass of 1,335 Da and displays antibacterial activity toward closely related Gram-positive bacteria. A cluster of 12 genes that covers ∼10 kb is essential for the production, modification, export, and self-immunity of this natural product. We have named this compound plantazolicin (PZN), based on the association of several producing organisms with plants and the incorporation of azole heterocycles, which derive from Cys, Ser, and Thr residues of the precursor peptide.

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Figures

FIG. 1.
FIG. 1.
Thiazole/oxazole-modified microcin (TOMM) biosynthesis. Through the action of a trimeric “BCD” complex, consisting of a cyclodehydratase (C, green), dehydrogenase (B, yellow), and docking/scaffolding protein (D, blue), thiazoles and (methyl)oxazoles are incorporated onto a peptidic scaffold (black). These heterocycles are synthesized from serine/threonine (X = O; R = H/CH3) and cysteine (X = S; R = H) residues of the inactive precursor peptide and yield a bioactive natural product. The chemical transformations carried out by the cyclodehydratase and the dehydrogenase are shown, along with the corresponding mass change from the parent peptide in daltons.
FIG. 2.
FIG. 2.
MALDI-TOF mass spectra of B. amyloliquefaciens surface extracts. The samples were prepared from FZB42 (A) and RS6 (B) cells. Surface extracts were prepared and measured as described in Materials and Methods. Peaks at 1,336.8 [M + H]+, 1,354.8 [M + H2O + H]+, 1,358.8 [M + Na]+, and 1,374.8 [M + K]+ Da indicate the presence of PZN in the wild-type strain (A) and in the Δsfp Δbac mutant strain RS6 (B).
FIG. 3.
FIG. 3.
Effect of mutations in the pzn gene cluster on biological activity. (A) Extracted ion chromatogram of PZN ([M + H]+ 1,336 Da) of FZB42 and mutant strains. This compound (m/z 1,336.6 Da) (left inset) is synthesized by wild-type FZB42 and by the mutant strains RS6 (Δsfp Δbac), RS27, and RS29. RSpMarA2 (ΔdegU) overproduces PZN. Strains RS26, RS28, RS31, and RS32 were deficient in PZN production. Strain RS33 (right inset) produced a compound with m/z 1,308.5 Da, suggesting the loss of two methyl groups (−H2 + C2H6, −28 Da). (B) Hemolytic activity of 100 μl extract from RSpMarA2 and RS26 on blood agar plates. (C) Antibacterial activity of PZN. (Left) HPLC-purified PZN (10 μl of 100 μg/μl suspended in water) was spotted onto an agar plate of B. subtilis HB0042 (sigW null) and incubated for 16 h to assess growth inhibition. (Right) Water (10 μl; negative control). (D) Growth curves of strains RS6 and pznF mutant RS29. After approximately 20 h of growth, the RS29 culture density is 9 log units lower than that of RS6.
FIG. 4.
FIG. 4.
Characterization of cell surface extract. Samples were prepared from RSpMarA2 and separated by 18% Tris-Tricine-SDS-PAGE either stained with Coomassie EZBlue (A) or double-stained with Coomassie EZBlue and Schiff's reagent (B). Lane M, molecular size markers; lanes 1 to 5, 1 μl, 2 μl, 5 μl, 10 μl, and 20 μl surface extract. (C) MALDI-TOF mass spectrum of the hydrated form of PZN (m/z 1,336 + 18 = 1,354 Da) obtained by excising the peptide band from the Tris-Tricine-SDS-PAGE gel.
FIG. 5.
FIG. 5.
Plantazolicin gene cluster. (A) FZB42 PZN gene cluster (9,892 bp) and amino acid sequence of the precursor peptide. -, putative leader peptide processing site. (B) Proposed functions of individual PZN genes. Upon deletion of pznF and pznI, cpd1335 (PZN) was detected by mass spectrometry. Deletion of pznL resulted in desmethyl PZN (m/z 1,308 Da), while individual inactivation of all other tested genes (pznABCJ) did not produce PZN. The functions of pznF, pznI, and pznJ require further exploration, but preliminary data suggest that pznF plays a role in immunity, pznI encodes a pentapeptide repeat protein, and pznJ is required for PZN maturation.
FIG. 6.
FIG. 6.
RT-PCR reveals polycistronic mRNAs. Reverse transcriptase (RT) PCR was performed using 1 μg of FZB42 RNA isolated from stationary phase using a commercially available kit. PCR products were analyzed in comparison to a negative control lacking reverse transcriptase (−RT). (A) All genes in the putative PZN cluster are transcribed under the culturing conditions employed. Cells (1 ml at an OD600 of 1.0) were removed from a stationary culture growing at 37°C 24 h after inoculation. Total RNA was isolated and converted to cDNA by RT-PCR. cDNA (500 ng), excluding pznE and pznL (750 ng each), was added to each reaction mixture. Gene fragments were then amplified using specific primers and PCR. Amplicons were assessed by separation on 1.1% agarose gels containing ethidium bromide and visualized by UV illumination. (B) Amplification of adjacent pzn genes reveals polycistronic mRNA. Junction I-J did not reveal a significant band; junction E-L was visible under extreme contrast (data not shown). All amplicons migrate on the basis of their expected sizes. Numbers at left represent DNA size standards.

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