The responses of Haemophilus influenzae to DNA gyrase inhibitors were analyzed at the transcriptional and the translational level. High-density microarrays based on the genomic sequence were used to monitor the expression levels of >80% of the genes in this bacterium. In parallel the proteins were analyzed by two-dimensional electrophoresis. DNA gyrase inhibitors of two different functional classes were used. Novobiocin, as a representative of one class, inhibits the ATPase activity of the enzyme, thereby indirectly changing the degree of DNA supercoiling. Ciprofloxacin, a representative of the second class, obstructs supercoiling by inhibiting the DNA cleavage-resealing reaction. Our results clearly show that different responses can be observed. Treatment with the ATPase inhibitor Novobiocin changed the expression rates of many genes, reflecting the fact that the initiation of transcription for many genes is sensitive to DNA supercoiling. Ciprofloxacin mainly stimulated the expression of DNA repair systems as a response to the DNA damage caused by the stable ternary complexes. In addition, changed expression levels were also observed for some genes coding for proteins either annotated as "unknown function" or "hypothetical" or for proteins not directly involved in DNA topology or repair.

. Pyrimido[1,6-a]benzimidazoles have been found to represent a new class of potent DNA gyrase inhibitors which also act at the A subunit. To determine alterations in the DNA sequence specificity of DNA gyrase for cleavage sites in the presence of inhibitors of both classes or in the presence of Ca 2؉ , we used DNA restriction fragments of 164, 85, and 71 bp from the pBR322 plasmid as model substrates. Each contained, at a different position, the 20-bp pBR322 sequence around position 990, where DNA gyrase preferentially cleaves in the presence of quinolones. Our results show that pyrimido[1,6-a]benzimidazoles have a mode of action similar to that of quinolones; they inhibit the resealing step and influence the DNA sequence specificity of DNA gyrase in the same way. Differences between inhibitors of both classes could be observed only in the preferences of DNA gyrase for these cleavage sites. The 20-bp sequence appeared to have some properties that induced DNA gyrase to cleave all three DNA fragments in the presence of inhibitors within this sequence, whereas cleavage in the presence of Ca 2؉ was in addition dependent on the length of the DNA fragments.The prokaryotic DNA gyrase (EC 5.99.1.3) belongs to the class of topoisomerase II proteins and is able to catalyze the ATP-dependent negative supercoiling of double-stranded closed circular DNA (23). The active enzyme is a tetramer consisting of two A subunits and two B subunits. It is thought that in the initial stage of the supercoiling process the enzyme binds to double-stranded DNA and that about 120 bp is wrapped around the tetrameric protein in a single positive supercoil. There have been several proposals for the existence of specific sites for the interaction of DNA gyrase with DNA. In Escherichia coli and Salmonella typhimurium, there is a family of repetitive extragenic palindromic sequences, and there is evidence that DNA gyrase binds preferentially to these sites (30). It has been proposed that the par locus in the pSC101 plasmid represents a DNA gyrase binding site (29), and in bacteriophage Mu a sequence that is required for efficient replicative transposition is proposed as a strong DNA gyrase binding site (20). After binding, DNA gyrase cleaves each strand at sites separated by 4 bp and transiently forms a covalent phosphotyrosine bond between the 5Ј-phosphate groups of the cleaved DNA and Tyr-122 of the A subunits (8, 28). A segment of DNA is translocated through the break and presumably through at least part of the protein complex. There are data indicating that DNA gyrase binding to DNA is sufficiently stable to allow processive supercoiling before the enzyme dissociates from the DNA (16). At some point in the reaction, an ATP molecule binds to each B subunit and is hydrolyzed. Binding of ATP promotes a conformational change of the tetramer, and it is thought that this change brings the DNA segment to be translocated close to the doublestranded DNA break (21). The hydrolysis of ATP is required for further catalytic cycles.For the DNA supercoiling ...

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