Staphylococcus aureus TargetArray: comprehensive differential essential gene expression as a mechanistic tool to profile antibacterials.

The widespread emergence of antibiotic-resistant bacteria and a lack of new pharmaceutical development have catalyzed a need for new and innovative approaches for antibiotic drug discovery. One bottleneck in antibiotic discovery is the lack of a rapid and comprehensive method to identify compound mode of action (MOA). Since a hallmark of antibiotic action is as an inhibitor of essential cellular targets and processes, we identify a set of 308 essential genes in the clinically important pathogen Staphylococcus aureus. A total of 446 strains differentially expressing these genes were constructed in a comprehensive platform of sensitized and resistant strains. A subset of strains allows either target underexpression or target overexpression by heterologous promoter replacements with a suite of tetracycline-regulatable promoters. A further subset of 236 antisense RNA-expressing clones allows knockdown expression of cognate targets. Knockdown expression confers selective antibiotic hypersensitivity, while target overexpression confers resistance. The antisense strains were configured into a TargetArray in which pools of sensitized strains were challenged in fitness tests. A rapid detection method measures strain responses toward antibiotics. The TargetArray antibiotic fitness test results show mechanistically informative biological fingerprints that allow MOA elucidation.

Comparison of the essential cellular functions of the two murA genes of Bacillus anthracis.

Targeted antisense and gene replacement mutagenesis experiments demonstrate that only the murA1 gene and not the murA2 gene is required for the normal cellular growth of Bacillus anthracis. Antisense-based modulation of murA1 gene expression hypersensitizes cells to the MurA-specific antibiotic fosfomycin despite the normally high resistance of B. anthracis to this drug.

Evaluation of the metS and murB loci for antibiotic discovery using targeted antisense RNA expression analysis in Bacillus anthracis.

The biowarfare-relevant bacterial pathogen Bacillus anthracis contains two paralogs each of the metS and murB genes, which encode the important antibiotic target functions methionyl-tRNA synthetase and UDP-N-acetylenolpyruvoylglucosamine reductase, respectively. Empirical screens were conducted to detect and characterize gene fragments of each of these four genes that could cause growth reduction of B. anthracis when inducibly expressed from a plasmid-borne promoter. Numerous such gene fragments that were overwhelmingly in the antisense orientation were identified for the metS1 and murB2 alleles, while no such orientation bias was seen for the metS2 and murB1 alleles. Gene replacement mutagenesis was used to confirm the essentiality of the metS1 and murB2 alleles, and the nonessentiality of the metS2 and murB1 alleles, for vegetative growth. Induced transcription of RNA from metS1 and murB2 antisense-oriented gene fragments resulted in specific reduction of mRNA of their cognate genes. Attenuation of MetS1 enzyme expression hypersensitized B. anthracis cells to a MetS-specific antimicrobial compound but not to other antibiotics that affect cell wall assembly, fatty acid biosynthesis, protein translation, or DNA replication. Antisense-dependent reduction of MurB2 enzyme expression caused hypersensitivity to beta-lactam antibiotics, a synergistic response that has also been noted for the MurA-specific antibiotic fosfomycin. These experiments form the basis of mode-of-action detection assays that can be used in the discovery of novel MetS- or MurB-specific antibiotic drugs that are effective against B. anthracis or other gram-positive bacterial pathogens.

The synchrony phenotype persists after elimination of multiple GATC sites from the dnaA promoter of Escherichia coli.

To examine whether methylation of the GATC sites present in the dnaA promoter region is responsible for the strict temporal coordination of initiation events at oriC as measured by the synchrony of initiation, we introduced point mutations eliminating three (TGW1) and five (TGW2) of the six GATC sites present in the dnaA promoter region. All of the strains containing these mutations, including the one with five GATC sites eliminated, initiated chromosomal replication synchronously.

Role of DNA methylation at GATC sites in the dnaA promoter, dnaAp2.

DnaA protein is required for the initiation of DNA replication at the bacterial chromosomal origin, oriC, and at the origins of many plasmids. The concentration of DnaA protein is an important factor in determining when initiation occurs during the cell cycle. Methylation of GATC sites in the dnaAp2 promoter, two of which are in the -35 and -10 sequences, has been predicted to play an important role in regulating dnaA gene expression during the cell cycle because the promoter is sequestered from methylation immediately following replication. Mutations that eliminate these two GATC sites but do not substantially change the activity of the promoter were introduced into a reporter gene fusion and into the chromosome. The chromosomal mutants are able to initiate DNA replication synchronously at both moderately slow and fast growth rates, demonstrating that GATC methylation at these two sites is not directly involved in providing the necessary amount of DnaA for precise timing of initiation during the cell cycle. Either sequestration does not involve these GATC sites, or cell cycle control of DnaA expression is not required to supply the concentration necessary for correct timing of initiation.