Growth kinetics of S. pneumoniae R6 after Nov addition. (A) Exponentially growing cultures in AGCH at OD_{620 nm} = 0.4 were treated with the indicated Nov concentrations. Relative values of two independent replicates are indicated. (B) Relative values (mean ± SEM) are indicated, being the initial one 1.53 × 10⁸ colony forming U/ml. (C) Left column, phase contrast images; right column, DAPI staining.

Transcriptional response to relaxation measured by qRT–PCR of DNA topoisomerases genes. Cultures of R6 and R6GyrB were grown as described in Figure 1. Total RNA was isolated, cDNA was synthesized and subjected to qRT–PCR. Data were normalized both to time 0 min and to that obtained from the amplification of rpoB and 16SrDNA genes. Relative values (mean of three independent replicates ±SD) are represented. A culture of R6GyrB (as R6, GyrBS127L, Nov MIC = 32 µg/ml) was treated with 16 µg/ml (0.5× MIC) and cultures of R6 (Nov MIC = 1 µg/ml) with 1, 10, 20 and 40 µg/ml (1× to 40× MIC). Genes used for the qRT–PCR with fragments amplified represented by black boxes above the corresponding gene are showed in different colours.

Localization of relaxation responsive genes in the S. pneumoniae chromosome. (A) Cultures were grown as described in Figure 1 and treated with different Nov concentrations. Samples were taken before the addition of the drug (time 0 min), and at indicated times, total RNA was isolated, double-stranded cDNA was synthesized, labelled with Cy3 and hybridized on high-density arrays. The relative fold variation of each gene is represented against the 3′ location of each open reading frame in the S. pneumoniae R6 chromosome (bases 1 to 2 038 615). Boxes indicate the transcriptional clusters: up-regulated in red, down-regulated in blue. (B) Analysis of percentage of AT content calculated at intervals of 100 bp along the whole R6 genome applying a window of 10 kb. Boxes indicate transcriptional clusters defined in (A). Flanking regions are shown in grey.

Characteristics of S. pneumoniae R6 genes in relation to its transcriptional relaxation response. (A) Plot of the first two axes generated by correspondence analysis for codon usage, synonymous codon usage, and residue composition in 2236 S. pneumoniae R6 protein-coding genes. Up-regulated (red), down-regulated (blue), flanking (green) and unaltered (grey) gene values corresponding to the two most relevant axes derived from correspondence analysis for general codon, and residue composition are shown. (B) COG functional classes representation in domains. COG functional classes with a significantly increase (red), decrease (blue) or unaltered (grey) number of members. The ratio of the number of expected and observed members, together with the significance of the change through P-value assessed by chi-squared test (P ≤ 1E-1) are shown in parentheses below each functional class. AA, amino acid transport and metabolism; CH, carbohydrate transport and metabolism; CO, coenzyme transport and metabolism; CW, cell wall/membrane/envelope biogenesis; DM, defence mechanisms; E, energy production and conversion; II, inorganic ion transport and metabolism; NT, nucleotide transport and metabolism; ST, signal transduction; T, transcription; TR, translation, ribosome structure and biogenesis.

pLS1 topoisomers distribution after novobiocin treatment. (A) Map of pLS1 (GenBank accession number M29725) showing genes location and direction of transcription: replication (A, repA and B, repB), truncated mobilization protein (D, orfD) and a tetracycline pump (tet). (B) Cartoon illustrating pLS1 topoisomer distribution after two-dimensional electrophoresis in agarose gels run in the presence of 1 and 2 µg/ml chloroquine in the first and second dimensions, respectively. Arrows at the top left corner indicate running direction during first and second dimensions, respectively. OC, open circle, L, linear forms. Negative supercoiled topoisomers are in white and positive supercoiled in black. It was previously determined that 2 µg/ml chloroquine introduces 14 positive supercoils. A black arrowhead indicates the topoisomer that migrated with ΔLk = 0 in the second dimension. Therefore, this topoisomer migrated with a ΔWr = −14 during the first dimension. An empty arrowhead indicates the most abundant topoisomer. (C) pLS1 topoisomer distribution after different Nov treatments (indicated to the left). Cultures were grown as described in ‘Material and Methods’ section. Samples were taken before the addition of the drug (time 0 min) and at the times indicated on top. The corresponding supercoiling density (σ) value is indicated below each autoradiogram.

Gene expression analysis and validation of microarray data by qRT–PCR. (A) Venn diagram with three circles, each one corresponding to one time interval in each condition, showing the differentially expressed genes in six conditions. The area is proportional to the number of genes. A complete list of the genes can be found in Supplementary Table S1. (B) Classification of the responsive genes in the six conditions. (C) Correlation of the changes in RNA transcript levels using data from microarray hybridizations and qRT–PCR. Values can be found in Supplemental Table S2.

Representation of the R6 genome showing responsive genes at five conditions and clusters location. Conditions considered were 0.5× MIC (5 and 15 min) and 40× MIC (5, 15 and 30 min). Genes are showed as arrows which head indicates the direction of transcription (as provided in http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?database=ntsp02). Red, up-regulated genes; blue, down-regulated genes. Values can be found in Supplementary Table S1. Boxes shown the clusters defined in Table 1. Flanking regions are shown in grey stripped boxes.