Figure 1.
The lichenicidin gene cluster.
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 B. licheniformis 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.
Figure 2.
Amino acid alignments of two-peptide lantibiotics.
(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.
Figure 3.
Agar well diffusion assays of the B. licheniformis DSM 13 culture supernatant and the isopropanol extract.
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.
Figure 4.
Stability assay: Treatment with proteases.
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 M. luteus while the antimicrobial activity of the culture supernant (green column) was unaffected by these proteases.
Figure 5.
Gene inactivation of the lantibiotic modifications enzyme LicM1 (A) and LicM2 (B) by homologous recombination.
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 B. licheniformis LicM1INT (A) and LicM2INT (B) harbour two copies of the lanM 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.
Figure 6.
MALDI-TOF mass spectra of the B. licheniformis MW3 wild type (A) and its insertion mutans LicM1INT (B) and LicM2INT (C).
The isopropanol extracts of the insertion mutants B. licheniformis LicM1INT and LicM2INT were characterized by the loss of activity against M. luteus. 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.
Figure 7.
HPLC chromatogram of the Bacillus licheniformis isopropanol extract.
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.
Figure 8.
Synergistic effect of fractions containing the Licα and Licβ peptides.
25 µl of HPLC fractions that contained the Licα peptide or the Licβ peptide as seen in mass spectrometry were tested separately and in combination against M. luteus. Only the combination of both peptides showed an antimicrobial activity indicating that Licα and Licβ are required for an optimal effect.
Figure 9.
Co-incubation of the isopropanol cell wash extract and the culture supernatant.
In agar well diffusion assays, the isopropanol extract (25 µl) showed an antimicrobial activity against S. aureus ATCC 33592 (blue bar) while the culture supernantant was inactive (see also Fig. 3). 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.
Table 1.
Oligonucleotides used in this study.