An increase in negative supercoiling in bacteria reveals topology-reacting gene clusters and a homeostatic response mediated by the DNA topoisomerase I gene.

We studied the transcriptional response to an increase in DNA supercoiling in Streptococcus pneumoniae by using seconeolitsine, a new topoisomerase I inhibitor. A homeostatic response allowing recovery of supercoiling was observed in cells treated with subinhibitory seconeolitsine concentrations. Supercoiling increases of 40.7% (6 µM) and 72.9% (8 µM) were lowered to 8.5% and 44.1%, respectively. Likewise, drug removal facilitated the recovery of cell viability and DNA-supercoiling. Transcription of topoisomerase I depended on the supercoiling level. Also specific binding of topoisomerase I to the gyrase A gene promoter was detected by chromatin-immunoprecipitation. The transcriptomic response to 8 µM seconeolitsine had two stages. An early stage, associated to an increase in supercoiling, affected 10% of the genome. A late stage, manifested by supercoiling recovery, affected 2% of the genome. Nearly 25% of the early responsive genes formed 12 clusters with a coordinated transcription. Clusters were 6.7-31.4 kb in length and included 9-22 responsive genes. These clusters partially overlapped with those observed under DNA relaxation, suggesting that bacteria manage supercoiling stress using pathways with common components. This is the first report of a coordinated global transcriptomic response that is triggered by an increase in DNA supercoiling in bacteria.

Role of global and local topology in the regulation of gene expression in Streptococcus pneumoniae.

The most basic level of transcription regulation in Streptococcus pneumoniae is the organization of its chromosome in topological domains. In response to drugs that caused DNA-relaxation, a global transcriptional response was observed. Several chromosomal domains were identified based on the transcriptional response of their genes: up-regulated (U), down-regulated (D), non-regulated (N), and flanking (F). We show that these distinct domains have different expression and conservation characteristics. Microarray fluorescence units under non-relaxation conditions were used as a measure of gene transcriptional level. Fluorescence units were significantly lower in F genes than in the other domains with a similar AT content. The transcriptional level of the domains categorized them was D>U>F. In addition, a comparison of 12 S. pneumoniae genome sequences showed a conservation of gene composition within U and D domains, and an extensive gene interchange in F domains. We tested the organization of chromosomal domains by measuring the relaxation-mediated transcription of eight insertions of a heterologous Ptccat cassette, two in each type of domain, showing that transcription depended on their chromosomal location. Moreover, transcription from the four promoters directing the five genes involved in supercoiling homeostasis, located either in U (gyrB), D (topA), or N (gyrA and parEC) domains was analyzed both in their chromosomal locations and in a replicating plasmid. Although expression from the chromosomal PgyrB and PtopA showed the expected domain regulation, their expression was down-regulated in the plasmid, which behaved as a D domain. However, both PparE and PgyrA carried their own regulatory signals, their topology-dependent expression being equivalent in the plasmid or in the chromosome. In PgyrA a DNA bend acted as a DNA supercoiling sensor. These results revealed that DNA topology functions as a general transcriptional regulator, superimposed upon other more specific regulatory mechanisms.