comCDE (Competence) Operon Is Regulated by CcpA in Streptococcus pneumoniae D39

Abstract

Natural transformation plays an important role in the formation of drug-resistant bacteria. Exploring the regulatory mechanism of natural transformation can aid the discovery of new antibacterial targets and reduce the emergence of drug-resistant bacteria. Competence is a prerequisite of natural transformation in Streptococcus pneumoniae, in which comCDE operon is the core regulator of competence. To date, only ComE has been shown to directly regulate comCDE transcription. In this study, a transcriptional regulator, the catabolite control protein A (CcpA), was identified that directly regulated comCDE transcription. We confirmed that CcpA binds to the cis-acting catabolite response elements (cre) in the comCDE promoter region to regulate comCDE transcription and transformation. Moreover, CcpA can coregulate comCDE transcription with phosphorylated and dephosphorylated ComE. Regulation of comCDE transcription and transformation by CcpA was also affected by carbon source signals. Together, these insights demonstrate the versatility of CcpA and provide a theoretical basis for reducing the emergence of drug-resistant bacteria. IMPORTANCE Streptococcus pneumoniae is a major cause of bacterial infections in humans, such as pneumonia, bacteremia, meningitis, otitis media, and sinusitis. Like most streptococci, S. pneumoniae is naturally competent and employs this ability to augment its adaptive evolution. The current study illustrates CcpA, a carbon catabolite regulator, can participate in the competence process by regulating comCDE transcription, and this process is regulated by different carbon source signals. These hidden abilities are likely critical for adaptation and colonization in the environment.

Keywords: CcpA; Streptococcus pneumoniae D39; competence; transformation.

FIG 1

Effects of ccpA on S. pneumoniae transformation. (A) The ccpA mRNA of strains D39s, D39ΔccpA, and D39ΔccpA::ccpA were determined by real-time quantitative PCR (qPCR). qPCR was conducted in triplicate. (B) Use of Western blot to probe the expression of CcpA in D39s, D39ΔccpA, and D39ΔccpA::ccpA. A representative result for three independent experiments is displayed. (C, D) Effects of ccpA on the transformation of encapsulated S. pneumoniae using genomic DNA (gDNA) encoding an erythromycin resistance gene (C) and plasmids as DNA donors (D). Three replicates, averages, and standard errors of the mean (SEMs) are plotted. *, P < 0.05; **, P < 0.01; ***, P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG 2

Dosage effects of CcpA on transformation. (A) Genetic organization of CEPlac-ccpA expression platform. The Cm gene is constitutively expressed via the P-_Cm promoter and confers chloramphenicol resistance. The ccpA gene is expressed via Plac, the sequence of which is shown. (B) Western blot comparing expression levels of CEPlac-dprA in various isopropyl β-d-1-thiogalactoside (IPTG) concentrations to those of wild-type, ΔccpA (ccpA^–), and ΔccpA::ccpA (ccpA⁺) cells. (C) qPCR measurements of ccpA mRNA levels from strain ΔccpA::CEPlac-ccpA at the indicated IPTG concentrations. (D) Western blot comparing ComE expression levels of ΔccpA::CEPlac-ccpA strain at the indicated IPTG concentrations. (E) Transformation efficiencies of ΔccpA::CEPlac-ccpA at the indicated IPTG concentrations. All strains were grown in C+Y medium containing different concentrations of IPTG. At an optical density at 600 nm (OD₆₀₀) of ~0.10, CSP was treated for 10 min, and then bacterial precipitate was collected for Western blotting (WB) or qPCR. Average of three replicates and SEMs are plotted. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. RBS, ribosome-binding site; Start, start codon (ATG).

FIG 3

Transcription of comCDE and ssbB in S. pneumoniae. (A) (Panel a) Diagram of the genomic organization of tRNA^Arg-comCDE in a wild-type strain. (Panel b) Construction of the luciferase reporter strain for comCDE expression. (Panel c) Diagrams of the genomic organization of ssbB in a wild-type strain. (Panel d) Construction of the luciferase reporter strain for ssbB gene expression. (B) Transcription profiles of the comCDE operon in wild-type strain and ccpA deletion mutant during growth were determined using luciferase reporter strains WT-Pcom-luc and ΔccpA-Pcom-luc. comCDE transcription was reported by luciferase activity (relative light units [RLU] per OD₆₀₀) of WT-Pcom-luc (red solid lines) and D39ΔccpA-Pcom-luc strains (blue solid line), respectively. Overnight cultures of the luciferase reporter strains were each diluted into fresh C+Y medium and cultured at 37°C. Luciferase activity and optical density at 600 nm (OD₆₀₀) were measured at 30-min intervals. comCDE is expressed as RLU per OD₆₀₀. (C) Luciferase activities and growth curves of the WT-PssbB-luc (red line with circles) or D39ΔccpA-PssbB-luc (blue line with squares) strains. Luciferase activity and OD₆₀₀ were measured at 30-min intervals. ssbB is expressed as RLU per OD₆₀₀. (D, E) Transcription profiles of comCDE (D) and ssbB (E) during the early exponential growth in wild-type and the ccpA deletion mutant. At an OD₆₀₀ of ~0.10, luciferase activity and OD₆₀₀ were measured at 10-min intervals. The comCDE and ssbB expressions are expressed as RLU/OD₆₀₀. (F) The luciferase gene was fused downstream of PcomCDE of ΔccpA::CEPlac-ccpA strain to measure comCDE transcription. All strains were grown in C+Y medium. At an OD₆₀₀ of ~0.10, CSP was treated for 10 min, and then luciferase activity was measured. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

Kinetics of transformation efficiency and gene expression during competence. (A) Transformation efficiency at six time points immediately before or after exposure to exogenous CSP in strains D39s and ΔccpA. The D39s and ΔccpA strains were grown on C+Y medium. At an OD₆₀₀ of ~0.10, CSP was added, and the transformation efficiency was measured at the indicated time points after CSP addition. (B) The relative abundance of early competence genes with different CSP treatment times. (C) The relative abundance of late competence genes with different CSP treatment times. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 5

Electrophoretic mobility shift assay (EMSA) determines CcpA binding to the PcomCDE. (A) Schematic diagram depicting tRNA^Arg and the downstream intergenic region between tRNA^Arg and PcomCDE. (B) Binding of CcpA to the P240 fragment. Binding reactions to 1 ng P240 were conducted with CcpA protein concentrations from 0 to 2.0 μg (lanes 1 to 6), and 100 ng of non-biotin-labeled P240 (Np240) (lanes 7 and 8) were added to compete with the labeled probe. (C) DNase I footprinting assay. (D) Binding of CcpA to the P179 fragment. Binding reactions to 1 ng P179 were conducted with CcpA protein concentrations from 0 to 5 μg (lanes 1 to 5). (E) Binding of CcpA to the P54 fragment. Binding reactions to 1 ng P54 were conducted with CcpA protein concentrations from 0 to 5.0 μg (lanes 1 to 5), and 100 ng of non-biotin-labeled P54 (Np240) (lane 6) were added to compete with the labeled probe. (F) EMSA was conducted to determine CcpA binding to Mut cre1 (lanes 1 to 4) or Mut cre2 (lanes 5 to 8). Gray and black arrows specify the free probes and protein-DNA complexes, respectively. nt, nucleotide. Cre, catabolite response elements.

FIG 6

Effects of CcpA binding to cre at the PcomCDE on S. pneumoniae D39 transformation. (A) Genetic organization of the PcomCDE in different cre strains. The panel shows the DNA sequence of the tRNA^Arg-comCDE region containing the tRNA^Arg terminator, ComE binding sites, the promoter region of comCDE (−10 and −35 sequences), and the start codon of comC gene. The bold red letters represent the four cre regions that CcpA binds to the PcomCDE. D39s represents the genetic organization of the PcomCDE in WT; cre1, cre1(*), cre2, cre3, and cre4 represent the genetic organization of the PcomCDE in different cre strains. The dashed lines mean bases that are deleted. (B to E) The effects of cre1 (B), cre2 (C), cre3 (D), and cre4 (E) on S. pneumoniae transformation. (Panel i) Transformation efficiency. (Panel ii) Detection of CcpA and ComE expression by Western blotting. (Panel iii) Transcription profiles of the ssbB. A series of strains containing only single cre sequences in a strain ΔccpA::CEPlac-ccpA background was constructed. All strains were grown in C+Y. At an OD₆₀₀ of ~0.10, CSP was treated for 10 min, and then ComE protein and luciferase activity were measured. *, P < 0.05; **, P < 0.01; ***, P < 0.001. RLU, relative light units.

FIG 7

Regulation of comCDE transcription by CcpA, ComE^D58E, or ComE^D58A. (A) Competitive EMSA was conducted to determine CcpA, wild-type ComE, and ComE^D58E binding to the PcomCDE. (B) Interaction of CcpA with ComE was verified by COIP. (C) ComE protein of ComE^D58E and ComE^D58A mutants were determined by Western blotting. All strains were grown in C+Y medium containing different concentrations of IPTG. At an OD₆₀₀ of ~0.10, CSP was treated for 10 min, and then bacterial precipitate was collected by centrifugation for WB. (D, E) Transcription profiles of comCDE of ComE^D58E (D) and ComE^D58A mutants (E). At an OD₆₀₀ of ~0.10, luciferase activity was measured at the indicated time points. The comCDE expression was expressed as RLU because luciferase was significantly different among the groups, and bacterial growth (OD₆₀₀) over a short period of time did not change the results. RLU, relative light units.

FIG 8

Effects of carbohydrates on CcpA regulatory activity. (A) Transformation efficiencies of D39s and D39ΔccpA in fresh C+Y medium containing either 0.2% glucose (Glu) or 0.2% galactose (Gal). (B) Effects of Glu and Gal on comCDE transcription. (C) Transformation efficiencies of D39s and D39ΔccpA strains in fresh C+Y medium containing different concentrations of glucose. (D) comCDE transcription in D39s-Pcom-luc and D39ΔccpA-Pcom-luc strains in fresh C+Y medium containing different concentrations of glucose. (E) Transformation efficiencies of D39s, D39ΔccpA, HPr S46D, and HPr S46D-CcpA strains in fresh C+Y medium. (F) comCDE transcription of D39s, D39ΔccpA, HPr S46D, and HPr S46D-CcpA strains in fresh C+Y medium. When the strains were grown in C+Y to an OD₆₀₀ of ~0.10, CSP was treated for 10 min, and then luciferase activity was measured. The comCDE expression was expressed as RLU because luciferase was significantly different among the groups, and bacterial growth (OD₆₀₀) over a short period of time did not change the results. *, P < 0.05; **, P < 0.01; ***, P < 0.001. RLU, relative light units.

FIG 9

A working model of CcpA-regulated competence in S. pneumoniae D39. The comCDE operon is the core regulatory element of competence. S. pneumoniae controls competence development through basal and autoregulatory transcription of the comCDE. There are four CcpA-bound cre sites on PcomCDE, and except for cre1, CcpA participates in comCDE basal and autoregulatory transcription via binding to by binding to these sites. PcomCDE initiates the autoregulated transcription that is enhanced by phosphorylated ComE but inhibited by dephosphorylated ComE. CcpA can cooperate with phosphorylated and dephosphorylated ComE to bind PcomCDE to maintain the optimal comCDE transcription. Remarkably, acting as a global regulator of carbohydrate metabolism genes, CcpA can regulate comCDE transcription by responding to the availability of carbohydrates. PTS delivers the phosphoryl group provided by PEP to HPr, enzyme I (EI), and enzyme II (EII) in turn and mediates carbohydrate transport across the cell membrane. In the presence of preferred carbohydrate, HPr can be phosphorylated at Ser-46, and P-Ser-HPr binds to the CcpA protein. The complex of CcpA and P-Ser-HPr binds to cre sites on the DNA and thereby represses the transcription of catabolic genes (CCR effects). This process can affect the binding of CcpA to cre in PcomCDE, thus regulating the comCDE transcription. However, in the absence of preferred carbohydrate, HPr can be phosphorylated at His-15, and P-His-HPr phosphorylates contribute to the activation of catabolic operons. Activation of the nonpreferred carbohydrate metabolism can inhibit the transcription of comCDE. Finally, CcpA affects competence by regulating comCDE transcription. CCR, carbohydrate catabolite repression.Cre, catabolite response elements.