SummaryTo address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.

In all multicellular organisms growth and morphogenesis must be coordinated, but for higher plants, this is of particular importance because the timing of organogenesis is not fixed but occurs in response to environmental constraints. One particularly dramatic developmental juncture is the response of dicotyledonous seedlings to light. The det3 mutant of Arabidopsis develops morphologically as a light-grown plant even when it is grown in the dark. In addition, it shows organ-specific defects in cell elongation and has a reduced response to brassinosteroids (BRs). We have isolated the DET3 gene by positional cloning and provide functional and biochemical evidence that it encodes subunit C of the vacuolar H + -ATPase (V-ATPase). We show that the hypocotyl elongation defect in the det3 mutant is conditional and provide evidence that this is due to an alternative mechanism of V-ATPase assembly. Together with the expression pattern of the DET3 gene revealed by GFP fluorescence, our data provide in vivo evidence for a role for the V-ATPase in the control of cell elongation and in the regulation of meristem activity. During the development of multicellular organisms, an intricate coordination of cell division and cell enlargement is necessary to achieve both morphogenesis and growth. In contrast to our rapidly growing knowledge of pattern formation and morphogenesis in a variety of model organisms, relatively little is known about the mechanisms that control cell and organ growth and integrate it with morphogenesis. Because plants are sessile, such mechanisms are of pivotal importance as their postembryonic development takes place under a multitude of environmental constraints, including the quality and quantity of light and the availability of water and nutrients. To compensate for their lack of mobility, plants have achieved a unique plasticity of development, which allows them to adapt to their environment. Both the initiation of organs by the apical meristems, and their subsequent growth through further cell divisions and cell expansion, continue throughout the plant life cycle. Therefore, growth and morphogenesis are not only coordinated with each other, but must provide the flexibility for adaptation to suboptimal environmental conditions.One of the most striking examples for developmental plasticity in response to an environmental cue is found during early seedling development. When dicotyledenous seedlings germinate in the absence of light, morphogenesis is inhibited and growth is achieved mostly by organ-specific cell expansion. Hypocotyl cells elongate Ն100-fold of their embryonic length to position the shoot apical meristem into an environment providing light necessary to establish photoautotrophic growth. The closed cotyledons and the formation of the apical hook protect the largely inactive shoot apical meristem. Once this so-called etiolated seedling reaches the light, however, it switches to the photomorphogenetic program in which new organs develop and growth is achieved by both cell division and cell e...

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.

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During a 6-week period, 10 patients were admitted to a hospital for treatment of knee or shoulder joint infections due to Serratia species. Isolates from eight patients were identified as Serratia marcescens with identical biochemical characteristics and antibiotic susceptibility patterns. Before the onset of infections, all patients had been treated by two orthopedic surgeons who shared an office. Studies revealed that infections were associated with previous joint injections (P = 4.44 x 10-5) of methylprednisolone and lidocaine. Environmental cultures revealed that a canister of cotton balls soaked in aqueous benzalkonium chloride and two multiple-dose vials of methylprednisolone previously used by office personnel were contaminated with the epidemic strain of S. marcescens. The canister may have served as a potential reservoir for contamination of sterile solutions and equipment used for joint injections, of skin at the injection site, and of hands of personnel. No further cases occurred after the use of aqueous benzalkonium chloride was discontinued.