The Streptococcus pneumoniae cia regulon: CiaR target sites and transcription profile analysis

Abstract

The ciaR-ciaH system is one of 13 two-component signal-transducing systems of the human pathogen Streptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by the cia system, the cia regulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing the S. pneumoniae genome. A set of 18 chromosomal regions containing 26 CiaR target sites were detected and proposed to represent the minimal cia regulon. The putative CiaR target loci included genes important for the synthesis and modification of cell wall polymers, peptide pheromone and bacteriocin production, and the htrA-spo0J region. In addition, the transcription profile of cia loss-of-function mutants and those with an apparent activated cia system representing the off and on states of the regulatory system were analyzed. The transcript analysis confirmed the cia-dependent expression of seven putative target loci and revealed three additional cia-regulated loci. Five putative target regions were silent under all conditions, and for the remaining three regions, no cia-dependent expression could be detected. Furthermore, the competence regulon, including the comCDE operon required for induction of competence, was completely repressed by the cia system.

FIG. 1.

Visualization of CiaR target fragments obtained with RsaI-, Sau3A-, and HindIII-restricted chromosomal DNA of S. pneumoniae. DNA fragments obtained after specific elution from bCiaR-coated magnetic beads were separated on a 1% agarose gel, stained with SYBRgreen, and visualized with the help of a fluoroimager. Three different restriction digests were used: Sau3A1, HindIII, and RsaI. Lanes: a, total restricted chromosomal DNA; b, DNA fragments obtained after specific elution in the SPDB assay; c, high-mass DNA ladder marker; d, size ladder marker.

FIG. 2.

Restriction analysis versus microarray hybridization signals of the manLMN region. (A) The numbers in the table indicate fluorescence intensities for each of the probe sets for the manLMN cluster and flanking genes. Positive fluorescent signals are written in boldface. (B) Restriction fragments expected from the map of the S. pneumoniae genome are indicated by lines drawn above the genetic organization of the putative target region. The regions covered by the positive fluorescent probe sets of the microarray are indicated by hatched blocks at the bottom. The vertically hatched area represents the minimal overlapping region of all three deduced restriction fragments. Solid arrows indicate the suggested cia-regulated man operon.

FIG. 3.

Specificity of the SPDB assay. The region upstream of the uppS gene was PCR amplified and restricted with DraI as shown in the diagram below. Left lane, fragments mixed with competitor DNA (molecular size marker); right lane, fragments retained by bCiaR after elution and concentration by precipitation (see Materials and Methods). The two DraI fragments, I and II, are indicated on the right and are marked by open circles in the agarose gel. The sizes of the fragments are indicated on the left.

FIG. 4.

Putative CiaR target regions. The putative CiaR target regions are listed according to their positions on the S. pneumoniae genome (see Table 1 for details). The regions representing the set of reactive probe sets that are present in the different restricted DNA samples are represented by bars above the genetic organization. Putative cia target genes are represented by solid arrows. The boxed segments indicate cia-dependently regulated genes (see Table 2).

FIG. 5.

Genes regulated by the cia system. The intensity scatter graph shows the correlation for the intensities of all transcripts obtained for the loss-of-function cia mutant R6-R1 versus those of R6 in C medium (center) and in THB medium (right). A comparison between two independently grown R6 cultures in C medium is included as a control (left). The two lines flanking the diagonal indicate differences of a factor of 2 and 5, respectively. In the center graph, several genes are downregulated in the mutant. In addition, a large set of genes appears to be upregulated in THB medium (right).

FIG. 6.

β-Galactosidase assay of the cia-dependent activation of the lic promoter (A) and the comC promoter (B). Construction of the lacZ reporter strains is described in Materials and Methods. Cultures were grown at 37°C in THB (A) or C medium (B). Samples were taken at the time points indicated, and the assay of β-galactosidase activity was performed as described in the text. Circles, R6; squares, R6-R1; triangles, R6_TC306; solid symbols, growth (optical density at 560 nm [OD560]); open symbols, LacZ activity (Miller units).