Pseudomycoicidin, a Class II Lantibiotic from Bacillus pseudomycoides.

Lantibiotics are ribosomally synthesized antimicrobial peptides with substantial posttranslational modifications. They are characterized by the unique amino acids lanthionine and methyllanthionine, which are introduced by dehydration of Ser/Thr residues and linkage of the resulting dehydrated amino acids with Cys residues. BLAST searches using the mersacidin biosynthetic enzyme (MrsM) in the NCBI database revealed a new class II lantibiotic gene cluster in Bacillus pseudomycoides DSM 12442. Production of an antimicrobial substance with activity against Gram-positive bacteria was detectable in a cell wash extract of this strain. The substance was partially purified, and mass spectrometric analysis predicted a peptide of 2,786 Da in the active fraction. In order to characterize the putative lantibiotic further, heterologous expression of the predicted biosynthetic genes was performed in Escherichia coli. Coexpression of the prepeptide (PseA) along with the corresponding modification enzyme (PseM) resulted in the production of a modified peptide with the corresponding mass, carrying four out of eight possible dehydrations and supporting the presence of four thioether and one disulfide bridge. After the proteolytic removal of the leader, the core peptide exhibited antimicrobial activity. In conclusion, pseudomycoicidin is a novel lantibiotic with antimicrobial activity that was heterologously produced in E. coli.

Lantibiotics: promising candidates for future applications in health care.

The immense potential of bacteria for production of antimicrobials represents an inexhaustible source of new antibiotics. An emerging class of natural products is constituted by ribosomally synthesized and posttranslationally modified peptides (RiPPs). "Lantibiotics" (lanthionine and/or methyl-lanthionine containing antibiotics) belong to the earliest members of this class. The characteristic thioether amino acids are introduced into the precursor peptides by enzyme-mediated posttranslational modifications. The encouraging antimicrobial activity of lantibiotics against multiresistant clinical pathogens, their stability against proteases, heat and oxidation make lantibiotics interesting candidates for novel antimicrobial applications in many areas of the healthcare sector and associated industries. In addition to applications as alternatives to classical antibiotics, lantibiotics can be used as probiotics, prophylactics or additives. Furthermore, the in vitro activity of the lantibiotic modification machinery opens the possibility to generate either improved synthetic lantibiotic peptides or to introduce thioether cross-links into existing therapeutics.

Identification of agr-positive methicillin-resistant Staphylococcus aureus harbouring the class A mec complex by MALDI-TOF mass spectrometry.

A small peptide called PSM-mec is encoded on the type II, III and VIII SCCmec cassettes present in the genomes of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains. This peptide is excreted by agr-positive strains, which represent about 89% of the strains of our collection and can be identified by the presence of delta toxin in mass spectrometry. The presence of the peptide in the MALDI-TOF MS spectra of whole cells was proved by a knock-down experiment employing a clone that expressed antisense RNA to psm-mec. Furthermore, evaluation of a collection of clinical agr-positive MRSA and MSSA isolates and type strains showed that, using a detection window of m/z 2411-2419, the PSM-mec is detected by mass spectrometry of whole cells with a sensitivity of 0.95 and a specificity of 1, thereby enabling rapid identification of a subgroup of MRSA with a method that is used during routine identification procedures.

Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42.

UNLABELLED: 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. METHODOLOGY/PRINCIPAL FINDINGS: The aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens. CONCLUSIONS/SIGNIFICANCE: The lantibiotic mersacidin can be produced in B. amyloliquefaciens FZB42, which is closely related to the wild type producer strain of mersacidin. The new mersacidin producer strain enables us to use the full potential of the biosynthetic gene cluster for genetic manipulation and downstream modification approaches.

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.