Expression of the Lantibiotic Mersacidin in Bacillus amyloliquefaciens FZB42

Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent

Production of the novel two-peptide lantibiotic lichenicidin by Bacillus licheniformis DSM 13.

BACKGROUND: Lantibiotics are small microbial peptide antibiotics that are characterized by the presence of the thioether amino acids lanthionine and methyllanthionine. Lantibiotics possess structural genes which encode inactive prepeptides. During maturation, the prepeptide undergoes posttranslational modifications including the introduction of rare amino acids as lanthionine and methyllanthione as well as the proteolytic removal of the leader. The structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG) are organized in biosynthetic gene clusters. METHODOLOGY/PRINCIPAL FINDINGS: Sequence comparisons in the NCBI database showed that Bacillus licheniformis DSM 13 harbours a putative lantibiotic gene cluster which comprises two structural genes (licA1, licA2) and two modification enzymes (licM1, licM2) in addition to 10 ORFs that show sequence similarities to proteins involved in lantibiotic production. A heat labile antimicrobial activity was detected in the culture supernatant and a heat stabile activity was present in the isopropanol cell wash extract of this strain. In agar well diffusion assays both fractions exhibited slightly different activity spectra against Gram-positive bacteria. In order to demonstrate the connection between the lantibiotic gene cluster and one of the antibacterial activities, two Bacillus licheniformis DSM 13 mutant strains harbouring insertions in the structural genes of the modification enzymes licM1 and licM2 were constructed. These strains were characterized by a loss of activity in the isopropanol extract and substractive MALDI-TOF predicted masses of 3020.6 Da and 3250.6 Da for the active peptides. CONCLUSIONS/SIGNIFICANCE: In conclusion, B. licheniformis DSM 13 produces an antimicrobial substance that represents the two-peptide lantibiotic lichenicidin and that shows activity against a wide range of Gram-positive bacteria including methicillin resistant Staphylococcus aureus strains

MALDI-TOF mass spectra of the <i>B. licheniformis</i> MW3 wild type (A) and its insertion mutans LicM1INT (B) and LicM2INT (C).

<p>The isopropanol extracts of the insertion mutants <i>B. licheniformis</i> LicM1INT and LicM2INT were characterized by the loss of activity against <i>M. luteus</i>. MALDI-TOF spectra of these isopropanol extracts in comparison to the wildtype (A) showed the loss of a peak at 3251 Da in the case of the LicM1 insertion mutant (B) indicating that this peak represents the protonated form of the active Licα peptide. The insertion of a plasmid into the gene of the modification enzyme LicM2 inactivated this enzyme and most probably exerted a polar effect on the downstream genes, thus affecting production of both peptides. This mutant did not produce the Licα peptide and is further characterized by the absence of a 3021 Da peak, which might represent the protonated form of the mature Licβ peptide, harboring 12 dehydrated residues (C). In some cultivations a further peak of 3039 Da was observed, which probably denotes a Licβ peptide with only 11 of 15 possible dehydrations.</p

Agar well diffusion assays of the <i>B. licheniformis</i> DSM 13 culture supernatant and the isopropanol extract.

<p>The culture supernatant (green bars) as well as the isopropanol extract (blue bars) were active against Gram-positive bacteria but showed slightly different spectra of activity, indicating that supernatant and isopropanol cell wash extract contained different antibacterial compounds.</p

Co-incubation of the isopropanol cell wash extract and the culture supernatant.

<p>In agar well diffusion assays, the isopropanol extract (25 µl) showed an antimicrobial activity against <i>S. aureus</i> ATCC 33592 (blue bar) while the culture supernantant was inactive (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006788#pone-0006788-g003" target="_blank">Fig. 3</a>). In order to test protease stability of the isopropanol extract, 25 µl of extract were mixed with 25 µl of culture supernatant (blue-green patterned bar), incubated for 2 h and then tested by agar diffusion. The co-incubation of both extracts had no effect on the activity of the isopropanol extract, indicating that the antimicrobial substance is stable against the proteases excreted by the producer strain.</p

HPLC chromatogram of the <i>Bacillus licheniformis</i> isopropanol extract.

<p>The isopropanol extract was applied to a POROS RP-HPLC column and eluted in a gradient of 20% to 55% acetronitrile (containing 0.1% TFA). Maldi-TOF analysis of active fractions [+ medium activity, ++ strong activity and (+) poor activity] showed the presence of masses representing the Licα peptide or the Licβ peptide.</p

Gene inactivation of the lantibiotic modifications enzyme LicM1 (A) and LicM2 (B) by homologous recombination.

<p>Gene inactivations of the modifications enzymes LicM1 and LicM2 were performed by plasmid integration using the recombinant plasmids pMADDelLicM1AC and pMADLicM2AC. The resulting insertion mutants <i>B. licheniformis</i> LicM1INT (A) and LicM2INT (B) harbour two copies of the <i>lanM</i> genes within the lantibiotic gene cluster: a copy that is transcribed but is truncated and a second copy that does not dispose of promoter, Shine Dalgarno sequence and start codon. Genes that derive from the plasmid pMADDelLicM1AC are marked in green colours and those deriving from pMADLicM2AC are marked in orange and red.</p

Amino acid alignments of two-peptide lantibiotics.

<p>(A) Amino acid sequence alignment of the LicA1 propeptide with the LanA1 propeptides of the two-peptide lantibiotics plantaricin (PlwA1, AAG02567), staphylococcin C55 (SacA1, BAB78438), lacticin 3147 (LtnA1, O87236), haloduracin (HalA1, BAB04173), BHT (BhtA1, AAZ76603) and Smb (SmbA1, BAD72777) and, for comparison, the propeptide of mersacidin (MrsA, Z47559). Amino acid identities of the LanA1 propeptides are highlighted in green (100%), pink (75%) and blue (35%). The thioether bridging pattern represents that of the Halα and Plwα peptide. (B) Amino acid sequence alignment of the LicA2 propeptide with the Lanβ/LanA2 propeptides of the two-peptide lantibiotics plantaricin (PlwA2, AAG02566), staphylococcin C55 (SacA2, BAB78439), lacticin 3147 (LtnA2, O87237), haloduracin (HalA2, BAB04172), BHT (BhtA2, AAZ76602) and Smb (SmbA2, BAD72776). Amino acid identities are highlighted in green (100%), pink (75%) and blue (35%). The thioether bridging pattern represents that of the Halβ and Plwβ peptides.</p

Stability assay: Treatment with proteases.

<p>No loss of both antimicrobial activities was detected after treatment with trypsin and chymotrypsin for 4 h for both extracts. However, incubation of the isopropanol cell wash extract (blue bar) with proteinase K and pronase E resulted in a total loss of activity against <i>M. luteus</i> while the antimicrobial activity of the culture supernant (green column) was unaffected by these proteases.</p

The lichenicidin gene cluster.

<p>The genes of the prepeptides are black; the genes of the two modification enzymes are grey. The other genes are marked with the following patterns: processing transporter, vertical stripes; the peptidase gene, checkerboard pattern; the putative regulator gene, horizontal stripes; genes that might be involved in immunity and encode proteins that have similarity to transporters, dots. The numbers give the last two digits of the locus tags in the annotation of the <i>B. licheniformis</i> DSM 13 isolate. For the small white orfs, no functions could be assigned so far, however similar orfs are encoded in the vicinity of the haloduracin gene cluster.</p