Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level β-lactam resistance (oxacillin MIC 2-4 μg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC �256 μg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit β) or rpoC (RNA polymerase subunit β') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve.

SummaryAntibiotic-resistant bacterial pathogens pose an urgent healthcare threat, prompting a demand for new medicines. We report the mode of action of the natural ansamycin antibiotic kanglemycin A (KglA). KglA binds bacterial RNA polymerase at the rifampicin-binding pocket but maintains potency against RNA polymerases containing rifampicin-resistant mutations. KglA has antibiotic activity against rifampicin-resistant Gram-positive bacteria and multidrug-resistant Mycobacterium tuberculosis (MDR-M. tuberculosis). The X-ray crystal structures of KglA with the Escherichia coli RNA polymerase holoenzyme and Thermus thermophilus RNA polymerase-promoter complex reveal an altered—compared with rifampicin—conformation of KglA within the rifampicin-binding pocket. Unique deoxysugar and succinate ansa bridge substituents make additional contacts with a separate, hydrophobic pocket of RNA polymerase and preclude the formation of initial dinucleotides, respectively. Previous ansa-chain modifications in the rifamycin series have proven unsuccessful. Thus, KglA represents a key starting point for the development of a new class of ansa-chain derivatized ansamycins to tackle rifampicin resistance.

The madurastatins are pentapeptide siderophores originally described as containing an unusual salicylate-capped N-terminal aziridine ring. Isolation of madurastatin C1 (1) (also designated MBJ-0034), from Actinomadura sp. DEM31376 (itself isolated from a deep sea sediment), prompted structural reevaluation of the madurastatin siderophores, in line with the recent work of Thorson and Shaaban. NMR spectroscopy in combination with partial synthesis allowed confirmation of the structure of madurastatin C1 (1) as containing an N-terminal 2-(2-hydroxyphenyl)oxazoline in place of the originally postulated aziridine, while absolute stereochemistry was determined via Harada's advanced Marfey's method. Therefore, this work further supports Thorson and Shaaban's proposed structural revision of the madurastatin class of siderophores (madurastatins A1 (2), B1 (3), C1 (1), and MBJ-0036 (4)) as N-terminal 2-(2-hydroxyphenyl)oxazolines.

Transcription, the first phase of gene expression, is performed by the multi-subunit RNA polymerase (RNAP). Bacterial RNAP is a validated target for clinical antibiotics. Many natural and synthetic compounds are now known to target RNAP, inhibiting various stages of the transcription cycle. However, very few RNAP inhibitors are used clinically. A detailed knowledge of inhibitors and their mechanisms of action (MOA) is vital for the future development of efficacious antibiotics. Moreover, inhibitors of RNAP are often useful tools with which to dissect RNAP function. Here, we review the MOA of antimicrobial transcription inhibitors.

RNA polymerases (RNAPs) accomplish the first step of gene expression in all living organisms. However, the sequence divergence between bacterial and human RNAPs makes the bacterial RNAP a promising target for antibiotic development. The most clinically important and extensively studied class of antibiotics known to inhibit bacterial RNAP are the rifamycins. For example, rifamycins are a vital element of the current combination therapy for treatment of tuberculosis. Here, we provide an overview of the history of the discovery of rifamycins, their mechanisms of action, the mechanisms of bacterial resistance against them, and progress in their further development.