Kari Ohlsen

A genome-wide strategy for the identification of essential genes in Staphylococcus aureus

To 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.

A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis.

The Spo0JA and Spo0JB proteins of Bacillus subtilis are similar to the ParA and ParB plasmid‐partitioning proteins, respectively, and mutation of spo0JB prevents the expression of stage II genes of sporulation. This phenotype is a consequence of Spo0JA activity in the absence of Spo0JB, and its basis was unknown. In the studies reported here, Spo0JA was found specifically to dissociate transcription initiation complexes formed in vitro by the phosphorylated sporulation transcription factor Spo0A and RNA polymerase with the spoIIG promoter. This repressor‐like activity is likely to be the basis for preventing the onset of differentiation in vivo. Spo0JB is known to neutralize Spo0JA activity in vivo and also to interact with a mitotic‐like apparatus responsible for chromosome partitioning. These data suggest that Spo0JA and Spo0JB form a regulatory link between chromosome partition and development gene expression.