Here, we determined the transcriptional response to the DNA relaxing agent novobiocin in S. aureus. Only a distinctive set of operons were found to be sensitive to supercoiling. In total, 11% of the genes were influenced by novobiocin. This is in good agreement with results observed in E. coli, in which 7% of the genome was affected .
We were able to show that recA transcription in S. aureus was sensitive to novobiocin treatment. This indicates that the recA promoter is highly dependent on DNA supercoiling imposed by active gyrase. Novobiocin had no impact on recA transcription in a strain with a mutation in gyrB (nov142). Thus secondary effects of novobiocin on other potential targets do not play a role in this regard. Of note, the novobiocin effect was independent of LexA because a similar effect of novobiocin on recA transcription was observed in an artificial recA promoter lacking the LexA binding motif. Thus, S. aureus is able to sense supercoiling to modulate the SOS response by adjusting the RecA level in the cell. In this way, aminocoumarins can counteract SOS-inducing conditions and their consequences, such as those imposed by ciprofloxacin .
In S. aureus, the gyrA and gyrB subunits are co-transcribed with recF, and the whole operon was severely upregulated after novobiocin treatment. GyrB is widely used as reference gene in qRT-PCR because it was shown that the expression of gyrB (and/or recF) is not influenced by major virulence regulators or different growth conditions . In addition, the expression of the operon was also found to be insensitive towards ciprofloxacin (Figure 1). In many other organisms, including S. pneumoniae and E. coli, gyrA and gyrB are distantly located, and the expression of these genes is independently regulated by several factors, including nucleoid-associated proteins. In S. aureus, the promoter preceding recF-gyrB-gyrA has presumably evolved to directly measure supercoiling imbalance, leading to upregulation of gyrase under relaxed conditions. Thus far, the environmental conditions that can impose such changes in S. aureus remain unclear.
Microarray analysis further reveals that several additional genes are influenced by novobiocin treatment. Some of these genes are presumably indirectly affected through secondary regulatory mechanisms. In this regard, the profound inhibition of the arlRS operon by novobiocin was of special interest. By searching microarray databases , this operon was not described to be differentially expressed by other regulatory mechanisms in S. aureus, indicating that the arlRS promoter itself is sensitive to supercoiling. Interestingly, the arlRS system was previously described to be involved in regulating the supercoiling level in S. aureus. Deletion of arlRS resulted in an increased level of supercoiling, an effect opposite to that of novobiocin treatment . Thus, downregulation of arlRS under novobiocin treatment can be viewed as a compensatory mechanism. In agreement with this assumption is the observation that, for some virulence factors, the impact of arlRS mutation is opposite to that of novobiocin treatment, and several of the genes described to be under the control of arlRS were also found to be influenced by novobiocin. However, analysis of a selected set of genes showed that the effect of novobiocin observed in the arl mutant strain (Figure 3) is similar to that in the wild type, indicating that novobiocin affects gene transcription independent of arlRS.
It is well recognised that the level of supercoiling is highly dynamic and affects gene expression directly and distinctly. However, the reason why different genes have different sensitivities towards supercoiling is under debate. In E. coli, a crosstalk between DNA supercoiling and nucleoid-associated proteins is involved in coordinated gene expression. The spatial ordering of genes along the chromosome corresponds to an inferred gradient of superhelical density [30, 31]. S. pneumoniae, like S. aureus, lacks many of the major nucleoid-associated proteins. In this organism, genes responding to changes in the level of supercoiling were found to be organised in chromosomal clusters. According to these topology-related models, the localisation of a given gene would dictate whether it is positively or negatively regulated by changes in supercoiling. In our analysis, we were unable to confirm such an association by mapping the responsive genes along the chromosome (Figure 4). In our analysis 11% of genes of S. aureus responded to novobiocin. By choosing different concentration of the drug and incubation time, the amount of responsive genes may vary and might impact the outcome of cluster analysis. Also apart from supercoiling, the folded nucleoid also forms tertiary structures, which might form clustered regions which might be missed using a simple mapping of genes along the chromosome.
Nevertheless, dislocation of three different promoters showed that the responsiveness of a given gene is determined by the promoter region and probably independent of the chromosomal localisation. These results are similar to analyses of the E. coli gyrA and gyrB promoters, both of which are activated under relaxing conditions : a reporter gene fused with gyrA or gyrB sequence was inducible by an aminocoumarin, also suggesting that only a small region of DNA is necessary for supercoiling sensitivity. In E. coli and Streptococcus pneumonia supercoiling sensitive genes were characterized by a different composition of nucleotides, with a higher AT content in upregulated genes. However, in S. aureus the AT content of up versus downregulated promoters were found not to differ (unpublished observation).
Our findings may be in line with a previous assumption that the supercoiling responsiveness of a gene may be correlated with the length of the spacer region between the -35 and -10 regions [33, 34]. According to this model, expression should be higher at a low level of supercoiling for genes with short spacers (less than the optimal 17 bp) and higher at elevated levels of supercoiling for genes with long spacers (greater than 17 bp). Spacing was shown to influence supercoiling sensitivity in E. coli and Helicobacter pylori. Our results also indicate that the length of the spacer might play a role in the supercoiling sensitivity of a given gene. For example, the highly upregulated gene gyrA has a relative short spacer, whereas the down regulated gene recA has a longer spacer.