RelA/DTD-mediated regulation of spore formation and toxin production by Clostridium perfringens type A strain SM101

Abstract

RelA is a global regulator for stationary phase development in the model bacterium Bacillus subtilis. The relA gene forms a bicistronic operon with the downstream dtd gene. In this study, we evaluated the significance of RelA and DTD proteins in spore formation and toxin production by an important gastrointestinal pathogen Clostridium perfringens. Our β-glucuronidase assay showed that in C. perfringens strain SM101, relA forms a bicistronic operon with its downstream dtd gene, and the relA promoter is expressed during both vegetative and sporulation conditions. By constructing double relA dtd and single dtd mutants in C. perfringens SM101, we found that: (1) RelA is required for maintaining the efficient growth capacity of SM101 cells during vegetative conditions; (2) both RelA and DTD are required for spore formation and enterotoxin (CPE) production by SM101; (3) RelA/DTD activate CodY, which is known to activate spore formation and CPE production in SM101 by activating a key sporulation-specific σ factor F; (4) as expected, RelA/DTD activate sporulation-specific σ factors (σ, σ, σ and σ) by positively regulating Spo0A production; and finally (5) RelA, but not DTD, negatively regulates phospholipase C (PLC) production by repressing plc gene expression. Collectively, our results demonstrate that RelA modulates cellular physiology such as growth, spore formation and toxin production by C. perfringens type A strain SM101, although DTD also plays a role in these pleiotropic functions in coordination with RelA during sporulation. These findings have implications for the understanding of the mechanisms involved in the infectious cycle of C. perfringens.

The (p)ppGpp Synthetase RSH Mediates Stationary-Phase Onset and Antibiotic Stress Survival in Clostridioides difficile

“…1B). rsh also shares an operon with the predicted D-aminoacyl-tRNA deacylase dtd; in Clostridium perfringens, the rsh homolog and dtd are coexpressed and function together to regulate sporulation (83). A previous microarray analysis of the C. difficile strain 630 transcriptional response to various stresses has shown a 2-fold upregulation of relQ after exposure to ampicillin and clindamycin, further suggesting that (p)ppGpp signaling could play a role in C. difficile antibiotic survival (84).…”

Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringens

“…For instance, adk has been experimentally tested in 3 different organisms ( Bacillus cereus group strains 33 , C. difficile 10 , and C. botulinum 12 ), yet it was only identified as stably expressed in C. difficile . Three of the 10 genes investigated ( gyrA , rrs, and rpoA ) have been used previously as an endogenous control for C. perfringens, although validity for these genes was not documented 7 , 17 , 18 , 20 , 34 .…”

“…In the Abildgaard study 17 , alpha toxin levels and expression were investigated using TaqMan analysis using gyrA (encoding DNA gyrase) or rplL (encoding the ribosomal protein L7/L12) as the reference genes for normalization. Both the Saito 18 and Kawarizadeh 20 studies utilized the 16S rRNA gene as the reference gene. In the Xiao study 19 , the genes dnaE and sigH were used to normalize expression values, whereas in the Yu 7 study, rpoA was used for normalization.…”

“…The transcription level of 16S rRNA gene in this clostridial species, on the other hand, was found to be the least stable when assessed under these tested growth conditions 11 . There have been several studies using RT-qPCR to assess changes in relative gene expression in C. perfringens that used different reference genes for the normalization step 7,[17][18][19][20] . In the Abildgaard study 17 , alpha toxin levels and expression were investigated using TaqMan analysis using gyrA (encoding DNA gyrase) or rplL (encoding the ribosomal protein L7/L12) as the reference genes for normalization.…”