A novel toxin regulator, the CPE1446-CPE1447 protein heteromeric complex, controls toxin genes in Clostridium perfringens

Abstract

Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium that is widespread in environmental soil and sewage, as well as in animal intestines. It is also a causative agent of diseases in humans and other animals, and it produces numerous extracellular enzymes and toxins. Although these toxins have been characterized in detail, regulators of toxin genes are less well understood. The present study identified CPE1447 and CPE1446 as novel regulators of toxin gene expression. CPE1447 and CPE1446 are cotranscribed as an operon, and the encoded proteins have a helix-turn-helix (HTH) motif at the N termini of their amino acid sequences, suggesting that CPE1447 and CPE1446 control the target genes as transcriptional regulators. The expression of several genes encoding toxins was changed in both a CPE1446 mutant and a CPE1447-CPE1446 deletion mutant. Complementation of CPE1446 and CPE1447 revealed that CPE1447 and CPE1446 coordinately regulate their target genes. CPE1447 protein was coprecipitated with His-tagged CPE1446 protein, indicating that the CPE1447 and CPE1446 proteins form a stable complex in C. perfringens under their native conditions. Although the small RNA that regulates several genes under the VirR/VirS two-component system (VR-RNA) positively affected CPE1447-CPE1446 mRNA expression, it did not control expression of the CPE1447-CPE1446 regulon, demonstrating that CPE1447 and CPE1446 regulate a different set of toxin genes from the VirR/VirS-VR-RNA cascade.

Fig. 1.

Expression of CPE1447-CPE1446 mRNA. (A) Schematic drawing of the CPE1447-CPE1446 locus in the C. perfringensgenome. The transcriptional start site and terminator are indicated by a bent arrow and a stem-loop structure, respectively. Previously annotated CPE1448 and CPE1447 genes are indicated by dashed arrows. (B) Northern blots of strains 13 (VR-RNA+) and TS140 (VR-RNA−) harboring the pJIR418 plasmid vector. Total RNA was isolated from cultures at the indicated time points, and 2 μg of total RNA was subjected to Northern blot analysis using a probe specific for the CPE1446 gene. The 16S rRNA on the blot detected by methylene blue staining is indicated at the bottom. The arrowhead indicates polycistronic mRNA encoding CPE1447 and CPE1446.

Fig. 2.

Determination of the transcriptional start site located upstream of CPE1447. (A) Primer extension analysis proceeded using ³²P-labeled primer and total RNA isolated from the cultures of strains 13, TS140, and NO16 at the mid-exponential phase. The sequence around the transcriptional start site for CPE1447 was determined by sequence reaction using the same primer and is indicated on the left. (B) DNA sequence around the CPE1447 promoter. The transcriptional start site of CPE1447 is indicated by a bent arrow. Putative −35 and −10 sequence regions are boxed. The ribosome binding site (RBS) and start codon of CPE1447 are shown in boldface.

Fig. 3.

CPE1447 and CPE1446 regulate expression of several toxin genes. (A) Schematic drawing of suicide plasmid pNO13 integrated into the CPE1446 gene of C. perfringensstrain 13. The transcriptional start site and terminator are indicated by a bent arrow and a stem-loop structure, respectively. (B) Schematic drawing of a DNA fragment used to delete the CPE1447 and CPE1446 genes in C. perfringensstrain 13. The fragment contains upstream and downstream sequences of CPE1447 and CPE1446, respectively, and an erythromycin resistance gene. (C) Northern blots of strains 13, NO13, and NO16. Total RNA (1 μg) isolated from culture at the mid-exponential phase was resolved on 1% agarose gels containing 2% formaldehyde and then subjected to Northern blot analysis using toxin gene-specific probes.

Fig. 4.

CPE1447 and CPE1446 function in a coordinated manner. C. perfringensstrains 13, NO13, and NO16 harboring the CPE1446 or CPE1447 and CPE1446 expression vector were grown at 37°C to the mid-exponential phase. Total RNA (1 μg) isolated from the culture was used for Northern blot analysis with the indicated gene-specific probes. The 23S and 16S rRNAs on the blot detected by methylene blue staining are indicated at the bottom.

Fig. 5.

CPE1447 and CPE1446 form a stable complex in vivo. (A) Coprecipitation of coexpressed CPE1447 and CPE1446-His proteins in E. colicells. Coprecipitated His-tagged proteins (20 ng) were detected by Western blot analysis using anti-His tag antibody (left). Purified proteins (2 μg) were resolved by SDS-PAGE and stained with Coomassie brilliant blue (CBB) (right). (B) SDS-PAGE (top) and Western blots (bottom) of proteins coprecipitated with CPE1446-His protein from C. perfringenscells. The protein complexes were purified from extracts of C. perfringensNO16 cells harboring plasmids expressing CPE1447-CPE1446 or CPE1447 and His-tagged CPE1446. The SDS-PAGE gel was stained with Coomassie brilliant blue. The Western blots were probed with anti-His tag antibody.

Fig. 6.

VR-RNA influences the amounts of CPE1447-GST and CPE1446-His proteins. (A) Western blots of strain 13 (VR-RNA+) or TS140 (VR-RNA−) harboring pCPE43. Each lane was loaded with 0.02 A₂₈₀unit of protein obtained from cells at the indicated time points. A GST fusion protein and DnaK were probed with anti-GST and anti-DnaK antibody, respectively. (B) Western blots of strain 13 or TS140 harboring pCPE6. Each lane was loaded with 0.02 A₂₈₀unit of protein from cells at the indicated time points. A His tag fusion protein and DnaK were probed with anti-His tag and anti-DnaK antibody, respectively.

Fig. 7.

CPE1447-CPE1446 and VR-RNA independently regulate the target genes. Northern blot analysis of strains 13, NO16, and TS140 was performed with the indicated gene-specific probes. C. perfringenswas grown and harvested at the indicated time points. Total RNA (1 μg) isolated from cultures was subjected to Northern blot analysis. The 16S and 23S rRNAs on the blot detected by methylene blue staining are indicated at the bottom.

Fig. 8.

Determination of transcriptional start sites of nagI, nagK, and nanI. (A) Primer extension analysis of nagI, nagK, and nanIusing ³²P-labeled primer and total RNA isolated from cultures of strains 13 and NO16 at the mid-exponential phase. The sequence around the transcriptional start site of each gene was determined by sequence reaction using the same primer. The transcriptional start sites are shown on the left. (B) Promoter sequences of hyaluronidase and sialidase genes in C. perfringensstrain 13 (1). The dashed boxes indicate −10 and −35 boxes. The ranscriptional start site is indicated by +1′. The length of the 5′ UTR of each gene is also indicated.