Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide

Abstract

We have identified in the Streptococcus pneumoniae genome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (blpAB) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC. The putative translation product of blpC, a small gene located downstream of blpAB, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of the blp genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended -10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.

FIG. 1

Genomic organization of the blp regulon. Genes induced by BlpC* are clustered and were found on two contigs (A and B). Physical locations of these two contigs on the S. pneumoniae chromosome remain unknown. Genes as predicted by the GeneMark software are shown as thick arrows above the bold line. Patterned arrows were used for clarity. Genes encoding peptides with a leader of the double-glycine type are indicated with dg (superscript), and blp genes of unknown function are marked with u (superscript). Potential promoter regions with a conserved regulatory element comprised of two direct repeats upstream of extended −10 elements are shown as waved arrows and numbered P1 to P7. Promoter numbering is the same as in Fig. 5. The hairpin-like symbols indicate positions of inverted repeats that have the potential to form transcriptional terminators. Light arrows below the bold line indicate the transcriptional map with transcript sizes as measured by Northern blotting. blpA# indicates that the blpA gene exists in at least two allelic forms.

FIG. 2

Genes specifically induced by BlpC*. (A) Strain KNR.7/87 was grown to an optical density at 600 nm of 0.3, and the synthetic peptide BlpC*_KNR.7/87 was added to a final concentration of 250 ng/ml to half of the culture. Cell pellets were collected after 30 min, RNA was extracted, and cDNA was labeled and hybridized to the microarray as described in Materials and Methods. The scatter graph shows the correlation for the intensities of all transcripts obtained for KNR.7/87 (x axis) versus KNR.7/87 30 min after addition of BlpC*_KNR.7/87 (y axis). These values are the average of two independent experiments. The two solid lines flanking the diagonal indicate a difference of a factor of 2. Relevant changes involved 16 genes (circled). Due to the frameshift in blpA sequenced from strain KNR.7/87, this gene is represented by two independent probe sets on the microarray. (B) Same intensity scatter graph with KNR.7/87 after addition of BlpC*_R6. KNR.7/87 (x axis) versus KNR.7/87 30 min after addition of BlpC*_R6 (y axis). No genes were found to be differentially expressed. (C) Bar graph showing the normalized average intensity differences for a selection of genes in KNR.7/87 (dark bars) and KNR.7/87 30 min after addition of BlpC*_KNR.7/87 (light bars). Genes are sorted as they appear on the contigs as depicted in Fig. 1. All 16 genes of the blp regulon plus the TCS operon (blpSRH) and a control ribosomal protein gene (rpsI) are shown (blpA again represented twice as two independent probe sets). The y axis shows average intensity differences calculated as described in Materials and Methods and is representative of the relative transcript abundance.

FIG. 3

Sequence alignments for BlpC and BlpH. (A) Amino acid sequences of the various forms of the ComC (Csp) and BlpC signal peptide precursors. Amino acid residues in the leader peptides that match a reported consensus sequence (10) are shown in bold. Shading indicates amino acid similarity within the predicted mature signal peptide. (B) Comparison of two regions of the sensor domain of the BlpH histidine kinase from six S. pneumoniae strains. Significant substitutions are indicated in bold. Additional significant nonconservative amino acid substitutions are shaded. Boxes enclose long stretches of significant amino acid substitutions of the BlpH kinase from S. pneumoniae D39 and R6. A line above the sequence indicates stretches predicted to form TM segments II and V.

FIG. 4

Northern blot transcript analysis of KNR.7/87. Total RNA was isolated as described for microarray analysis. RNA probes were prepared and labeled with [³²P]UTP using Lig'nScribe (Ambion). Probes correspond to the coding sequences of the genes indicated above the lanes as described in Materials and Methods. (A) Each probe was hybridized against two samples, KNR.7/87 and the same strain 30 min after addition, or in the absence, of synthetic BlpC*, indicated as + or − BlpC* above each lane. The size of the main transcript measured with each probe is indicated below each pair of lanes. The thick vertical lines indicate positions of 23S and 16S rRNAs. (B) An additional lane (stat) containing the same amount of total RNA isolated from stationary-phase KNR.7/87 culture was added. A blpA probe was used in the hybridization. The faint bands revealed by the blpA, blpJ, and blpL probes might correspond to transcript forms resulting from additional promoters, transcriptional readthrough, regulatory mRNA processing, cross-hybridization, or interference with rRNA.

FIG. 5

Alignment of potential regulatory sequence elements identified upstream of operons induced upon entry into stationary phase or by external application of the synthetic pheromone peptide BlpC*. Two direct imperfect repeats (11 bp) separated by an extremely conserved spacer element of 10 bp are located 19 bp upstream of an extended −10 hexamer. The most conserved nucleotide positions are shaded. A pattern deduced from these elements is indicated using the GCG code for the degenerated nucleotide positions.

FIG. 6

Amino acid comparison for the predicted Blp precursors with a typical Gly-Gly motif. Identical amino acid residues are shown in bold, while less conserved residues are shaded within the leader peptide. Note the high content of glycine and alanine residues of the expected processed forms. Below BlpJ and BlpI are compared with the components of the two-component thermophilin 13, a pore-forming bacteriocin produced by S. thermophilus (25). Conserved residues are shaded in gray.