SummaryIt is generally assumed that type A lantibiotics primarily kill bacteria by permeabilization of the cytoplasmic membrane. As previous studies had demonstrated that nisin interacts with the membrane-bound peptidoglycan precursors lipid I and lipid II, we presumed that this interaction could play a role in the pore formation process of lantibiotics. Using a thin-layer chromatography system, we found that only nisin and epidermin, but not Pep5, can form a complex with

The clinical impact of severe infections with yeasts and yeast-like fungi has increased, especially in immunocompromised hosts. In recent years, new antifungal agents with different and partially species-specific activity patterns have become available. Therefore, rapid and reliable species identification is essential for antifungal treatment; however, conventional biochemical methods are time-consuming and require considerable expertise. We

The worldwide spread of antibiotic-resistant bacteria has lent urgency to the search for antibiotics with new modes of action that are devoid of preexisting cross-resistances. We previously described a unique class of acyldepsipeptides (ADEPs) that exerts prominent antibacterial activity against Gram-positive pathogens including streptococci, enterococci, as well as multidrug-resistant Staphylococcus aureus. Here, we report that ADEP prevents cell division in Gram-positive bacteria and induces strong filamentation of rod-shaped Bacillus subtilis and swelling of coccoid S. aureus and Streptococcus pneumoniae. It emerged that ADEP treatment inhibits septum formation at the stage of Z-ring assembly, and that central cell division proteins delocalize from midcell positions. Using in vivo and in vitro studies, we show that the inhibition of Z-ring formation is a consequence of the proteolytic degradation of the essential cell division protein FtsZ. ADEP switches the bacterial ClpP peptidase from a regulated to an uncontrolled protease, and it turned out that FtsZ is particularly prone to degradation by the ADEP-ClpP complex. By preventing cell division, ADEP inhibits a vital cellular process of bacteria that is not targeted by any therapeutically applied antibiotic so far. Their unique multifaceted mechanism of action and antibacterial potency makes them promising lead structures for future antibiotic development.proteolysis | divisome | tubulin | multidrug-resistant Staphylococcus aureus

Friulimicin B is a naturally occurring cyclic lipopeptide, produced by the actinomycete Actinoplanes friuliensis, with excellent activity against gram-positive pathogens, including multidrug-resistant strains. It consists of a macrocyclic decapeptide core and a lipid tail, interlinked by an exocyclic amino acid. Friulimicin is water soluble and amphiphilic, with an overall negative charge. Amphiphilicity is enhanced in the presence of Ca 2؉ , which is also indispensable for antimicrobial activity. Friulimicin shares these physicochemical properties with daptomycin, which is suggested to kill gram-positive bacteria through the formation of pores in the cytoplasmic membrane. In spite of the fact that friulimicin shares features of structure and potency with daptomycin, we found that friulimicin has a unique mode of action and severely affects the cell envelope of gram-positive bacteria, acting via a defined target. We found friulimicin to interrupt the cell wall precursor cycle through the formation of a Ca 2؉ -dependent complex with the bactoprenol phosphate carrier C 55 -P, which is not targeted by any other antibiotic in use. Since C 55 -P also serves as a carrier in teichoic acid biosynthesis and capsule formation, it is likely that friulimicin blocks multiple pathways that are essential for a functional gram-positive cell envelope.

Staphylococcus aureus causes a wide range of hospital infections. Often, these infections involve epidemic methicillin-resistant S. aureus (MRSA) strains that are transferred by health care workers to patients. In order to detect outbreaks that are caused by epidemic strains, the clinical isolates have to be typed. Multilocus sequence typing (MLST) relies on the sequence analysis of housekeeping genes and is used to allocate the strains to sequence types (ST), which can be grouped into clonal complexes (CC). This method has provided a detailed insight into the population structure of MRSA and methicillin-susceptible S. aureus (MSSA) strains (1). Finer discrimination is achieved by pulsed-field gel electrophoresis (PFGE) and spa typing (2). All of these methods need additional experimentation.In matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), different spectra or signatures of cell extracts (3, 4, 5, 6) or whole cells (7,8,9,10,11,12) could be identified for different strains or groups of strains. An increasing number of laboratories use MALDI-TOF MS for the identification of S. aureus. However, the differences in the signatures of the strains have not been evaluated so far, because the discriminatory threshold of the software used in clinical settings is set up to assign the isolate to a species. To this end, more subtle differences are ignored. Another reason is that, so far, MALDI-TOF MS of whole bacterial cells has been employed in a heuristic manner, and for most species, the identities of the compounds that are detected in the measurements are unknown. Thus, the spectra are not well understood, and the variations in the signatures cannot be interpreted.In principle, two spectra might differ by signal intensity, loss of a signal, or by the shift of a signal. Variations in signal intensity are probably caused by expression differences, which might be directly correlated with culture conditions and, therefore, do not give unambiguous information about a genotype of the strain. The loss of a signal is caused by the total failure to express a protein or peptide, which in turn might indicate a mutation causing a frameshift or stop codon, but might also depend on culture conditions, mutations of regulatory factors, or sample preparation. Thus, there is no clear-cut correlation between the loss of a signal and a genotype. In contrast, peak shifts (i.e., loss of a signal coupled to the appearance of a new signal, both of which are correlated to the same peptide) correspond to point mutations in the genes of the peptides detected in the analysis; the mutation leads to an amino acid exchange that alters the molecular weight of the corresponding gene product.In order to characterize the clonal lineages of S. aureus in the MALDI-TOF MS, this work was aimed at the identification of the peptides that are detected in the spectra and that show mass variations between the clonal complexes of S. aureus. To this end, we analyzed the spectra of 401 S. aureus strains, concentrating on...

Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147. In order to further characterize the biochemical nature of the bacteriocin, both peptides were isolated which together are responsible for the antimicrobial activity. The first, LtnA1, is a 3,322 Da 30-amino acid peptide and the second component, LtnA2, is a 29-amino acid peptide with a mass of 2,847 Da. Conventional amino acid analysis revealed that both peptides contain the thioether amino acid, lanthionine, as well as an excess of alanine to that predicted from the genetic sequence of the peptides. Chiral phase gas chromatography coupled with mass spectrometry of amino acid composition indicated that both LtnA1 and LtnA2 contain D-alanine residues and amino acid sequence analysis of LtnA1 confirmed that the D-alanine results from post-translational modification of a serine residue in the primary translation product. Taken together, these results demonstrate that lacticin 3147 is a novel, two-component, D-alanine containing lantibiotic that undergoes extensive post-translational modification which may account for its potent antimicrobial activity against a wide range of Grampositive bacteria.

BackgroundLantibiotics 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 FindingsSequence 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/SignificanceIn 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.

Wall teichoic acid (WTA) or related polyanionic cell wall glycopolymers are produced by most Gram-positive bacterial species and have been implicated in various cellular functions. WTA and the proton gradient across bacterial membranes are known to control the activity of autolysins but the molecular details of these interactions are poorly understood. We demonstrate that WTA contributes substantially to the proton-binding capacity of Staphylococcus aureus cell walls and controls autolysis largely via the major autolysin AtlA whose activity is known to decline at acidic pH values. Compounds that increase or decrease the activity of the respiratory chain, a main source of protons in the cell wall, modulated autolysis rates in WTA-producing cells but did not affect the augmented autolytic activity observed in a WTA-deficient mutant. We propose that WTA represents a cation-exchanger like mesh in the Gram-positive cell envelopes that is required for creating a locally acidified milieu to govern the pH-dependent activity of autolysins.