Role for dithiolopyrrolones in disrupting bacterial metal homeostasis

Antibiotic resistance is a rising health threat worldwide, against which novel strategies are urgently needed. We have taken a systems approach to examine a potent and underexplored class of broad-spectrum antibiotics, the dithiolopyrrolones (DTPs). Our results indicate that DTPs disrupt cellular processes by high-affinity chelation of essential metal ions and inhibition of a subset of metalloenzymes. This mode of action is unique amongst antibiotics and may be further explored for treatment of multidrug-resistant infections. Our study also highlights chemical genomics as a powerful approach for the identification of antimicrobial mechanisms of action

Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis.

The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr use a shared radical-mediated mechanism. In addition to the radical SAM CX(3)CX(2)C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function, putatively to form (cysteine 355 in RlmN) and resolve (cysteine 118 in RlmN) a covalent intermediate needed to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further clarify the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species between RlmN and its rRNA substrate and well as the roles of the conserved cysteine residues in catalysis

Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers

Single nucleotide polymorphism (SNP) discovery and genotyping are essential to genetic mapping. There remains a need for a simple, inexpensive platform that allows high-density SNP discovery and genotyping in large populations. Here we describe the sequencing of restriction-site associated DNA (RAD) tags, which identified more than 13,000 SNPs, and mapped three traits in two model organisms, using less than half the capacity of one Illumina sequencing run. We demonstrated that different marker densities can be attained by choice of restriction enzyme. Furthermore, we developed a barcoding system for sample multiplexing and fine mapped the genetic basis of lateral plate armor loss in threespine stickleback by identifying recombinant breakpoints in F2 individuals. Barcoding also facilitated mapping of a second trait, a reduction of pelvic structure, by in silico re-sorting of individuals. To further demonstrate the ease of the RAD sequencing approach we identified polymorphic markers and mapped an induced mutation in Neurospora crassa. Sequencing of RAD markers is an integrated platform for SNP discovery and genotyping. This approach should be widely applicable to genetic mapping in a variety of organisms

DNA Methylation and Normal Chromosome Behavior in Neurospora Depend on Five Components of a Histone Methyltransferase Complex, DCDC

Methylation of DNA and of Lysine 9 on histone H3 (H3K9) is associated with gene silencing in many animals, plants, and fungi. In Neurospora crassa, methylation of H3K9 by DIM-5 directs cytosine methylation by recruiting a complex containing Heterochromatin Protein-1 (HP1) and the DIM-2 DNA methyltransferase. We report genetic, proteomic, and biochemical investigations into how DIM-5 is controlled. These studies revealed DCDC, a previously unknown protein complex including DIM-5, DIM-7, DIM-9, CUL4, and DDB1. Components of DCDC are required for H3K9me3, proper chromosome segregation, and DNA methylation. DCDC-defective strains, but not HP1-defective strains, are hypersensitive to MMS, revealing an HP1-independent function of H3K9 methylation. In addition to DDB1, DIM-7, and the WD40 domain protein DIM-9, other presumptive DCAFs (DDB1/CUL4 associated factors) co-purified with CUL4, suggesting that CUL4/DDB1 forms multiple complexes with distinct functions. This conclusion was supported by results of drug sensitivity tests. CUL4, DDB1, and DIM-9 are not required for localization of DIM-5 to incipient heterochromatin domains, indicating that recruitment of DIM-5 to chromatin is not sufficient to direct H3K9me3. DIM-7 is required for DIM-5 localization and mediates interaction of DIM-5 with DDB1/CUL4 through DIM-9. These data support a two-step mechanism for H3K9 methylation in Neurospora

Input Metadata for Statistical Analysis

These files include the metadata for conditions and arrays used as input for the statistical analysis and generation of the fitness score

A Chemical-Genomic Screen of Neglected Antibiotics Reveals Illicit Transport of Kasugamycin and Blasticidin S.

Fighting antibiotic resistance requires a deeper understanding of the genetic factors that determine the antibiotic susceptibility of bacteria. Here we describe a chemical-genomic screen in Escherichia coli K-12 that was designed to discover new aspects of antibiotic resistance by focusing on a set of 26 antibiotics and other stresses with poorly characterized mode-of-action and determinants of resistance. We show that the screen identifies new resistance determinants for these antibiotics including a common signature from two antimicrobials, kasugamycin and blasticidin S, used to treat crop diseases like rice blast and fire blight. Following this signature, we further investigated the mechanistic basis for susceptibility to kasugamycin and blasticidin S in E. coli using both genetic and biochemical approaches. We provide evidence that these compounds hijack an overlapping set of peptide ABC-importers to enter the bacterial cell. Loss of uptake may be an underappreciated mechanism for the development of kasugamycin resistance in bacterial plant pathogens

High-Density Detection of Restriction-Site-Associated DNA Markers for Rapid Mapping of Mutated Loci in Neurospora

The wealth of sequence information available for Neurospora crassa and other fungi has greatly facilitated evolutionary and molecular analyses of this group. Although “reverse” genetics, in which genes are first identified by their sequence rather than by their mutant phenotypes, serves as a valuable new approach for elucidating biological processes, classical “forward” genetic analysis is still extremely useful. Unfortunately, mapping mutations and identifying the corresponding genes has typically been slow and laborious. To facilitate forward genetics in Neurospora, we have adapted microarray-based restriction-site-associated DNA (RAD) mapping for use with N. crassa oligonucleotide microarrays. This technique was used to simultaneously detect an unprecedented number of genomewide restriction site polymorphisms from two N. crassa strains: Mauriceville and Oak Ridge. Furthermore, RAD mapping was used to quickly map a previously unknown gene, defective in methylation-7 (dim-7)

Metadata for the chemical genomics plates.

Metadata for the plates used in the chemical genomics screen, including uid, image analysis filename, condition and concentration of antibiotic, source plate, flags, and plate opacity IQR