Response of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A

Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase), fusA (elongation factor G), dnaG (primase), lacD (tagatose 1,6-bisphosphate aldolase), and SACOL0611 (a putative glycosyl transferase). The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability

Insights into the Mechanism of Action of the Two-Peptide Lantibiotic Lacticin 3147

Lacticin 3147 is a two peptide lantibiotc (LtnA1 and LtnA2) that displays nanomolar activity against many Gram-positive bacteria. Lacticin 3147 may exert its antimicrobial effect by several mechanisms. Isothermal titration calorimetry experiments show that only LtnA1 binds to the peptidoglycan precursor lipid II, which could inhibit peptidoglycan biosynthesis. An experimentally supported model of the resulting complex suggests that the key binding partners are the C-terminus of LtnA1 and pyrophosphate of lipid II. A combination of <i>in vivo</i> and <i>in vitro</i> assays indicates that LtnA1 and LtnA2 can induce rapid membrane lysis without the need for lipid II binding. However, the presence of lipid II substantially increases the activity of lacticin 3147. Furthermore, studies with synthetic LtnA2 analogues containing either desmethyl- or oxa-lanthionine rings confirm that the precise geometry of these rings is essential for this synergistic activity

A Rebeccamycin Analog Provides Plasmid-Encoded Niche Defense

Bacterial symbionts of fungus-growing ants occupy a highly specialized ecological niche and face the constant existential threat of displacement by another strain of ant-adapted bacteria. As part of a systematic study of the small molecules underlying this fraternal competition, we discovered an analog of the antitumor agent rebeccamycin, a member of the increasingly important indolocarbazole family. While several gene clusters consistent with this molecule’s newly reported modification had previously been identified in metagenomic studies, the metabolite itself has been cryptic. The biosynthetic gene cluster for 9-methoxyrebeccamycin is encoded on a plasmid in a manner reminiscent of plasmid-derived peptide antimicrobials that commonly mediate antagonism among closely related Gram-negative bacteria

Northern blot analyses for SACOL0678 (A) and SACOL2176 (B) operon.

<p>Total RNA was isolated from <i>S. aureus</i> COL at 0 (t₀), 10 (t₁₀), and 40 (t₄₀) min after the addition of amicoumacin A. 10 µg of total RNA isolated from each culture was separated in a formaldehyde-agarose gel and the RNA-blotted membrane was hybridized with SACOL0678- or SACOL2173(<i>asp23</i>)-specific digoxigenin-labeled probes. The sizes of the transcripts were determined by comparison to an RNA ladder on the same gel. The corresponding stained gels are shown underneath each blot. Schematic views of the gene loci based on NCBI COL genome site are shown with predicted transcripts. σ^B indicates locations of σ^B-controlled promoters. Microarray results of each operon's genes are summarized in the right panel of the corresponding Northern blot gels. Closed squares and open squares show samples taken at t₁₀ and t₄₀, respectively. The average of triplicates and standard deviations are indicated.</p

Serial passage experiments with <i>S. aureus</i> COL selected for increasing resistance to amicoumacin A.

<p>Each circle shows fold-resistance on each day.</p