A Point Mutation in the Transcriptional Repressor PerR Results in a Constitutive Oxidative Stress Response in Clostridioides difficile 630Δ <i>erm</i>

Abstract

The human pathogen <i>Clostridioides difficile</i> has evolved into the leading cause of nosocomial diarrhea. The bacterium is capable of spore formation, which even allows survival of antibiotic treatment. Although <i>C. difficile</i> features an anaerobic lifestyle, we determined a remarkably high oxygen tolerance of the laboratory reference strain 630Δ<i>erm</i> A mutation of a single nucleotide (single nucleotide polymorphism [SNP]) in the DNA sequence (A to G) of the gene encoding the regulatory protein PerR results in an amino acid substitution (Thr to Ala) in one of the helices of the helix-turn-helix DNA binding domain of this transcriptional repressor in <i>C. difficile</i> 630Δ<i>erm</i> PerR is a sensor protein for hydrogen peroxide and controls the expression of genes involved in the oxidative stress response. We show that PerR of <i>C. difficile</i> 630Δ<i>erm</i> has lost its ability to bind the promoter region of PerR-controlled genes. This results in a constitutive derepression of genes encoding oxidative stress proteins such as a rubrerythrin (<i>rbr1</i>) whose mRNA abundance under anaerobic conditions was increased by a factor of about 7 compared to its parental strain <i>C. difficile</i> 630. Rubrerythrin repression in strain 630Δ<i>erm</i> could be restored by the introduction of PerR from strain 630. The permanent oxidative stress response of <i>C. difficile</i> 630Δ<i>erm</i> observed here should be considered in physiological and pathophysiological investigations based on this widely used model strain.<b>IMPORTANCE</b> The intestinal pathogen <i>Clostridioides difficile</i> is one of the major challenges in medical facilities nowadays. In order to better combat the bacterium, detailed knowledge of its physiology is mandatory. <i>C. difficile</i> strain 630Δ<i>erm</i> was generated in a laboratory from the patient-isolated strain <i>C. difficile</i> 630 and represents a reference strain for many researchers in the field, serving as the basis for the construction of insertional gene knockout mutants. In our work, we demonstrate that this strain is characterized by an uncontrolled oxidative stress response as a result of a single-base-pair substitution in the sequence of a transcriptional regulator. <i>C. difficile</i> researchers working with model strain 630Δ<i>erm</i> should be aware of this permanent stress response.