A Mutator Phenotype Promoting the Emergence of Spontaneous Oxidative Stress-Resistant Mutants in Campylobacter jejuni

Abstract

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. As a microaerophilic organism, C. jejuni must be able to defend against oxidative stress encountered both in the host and in the environment. How Campylobacter utilizes a mutation-based mechanism for adaptation to oxidative stress is still unknown. Here we present a previously undescribed phenotypic and genetic mechanism that promotes the emergence of oxidative stress resistant mutants. Specifically, we showed that a naturally occurring mutator phenotype, resulting from a loss of function mutation in the DNA repair enzyme MutY, increased oxidative stress resistance (OX R ) in C. jejuni. We further demonstrated that MutY malfunction didn't directly contribute to the OX R phenotype, but increased the spontaneous mutation rate in the peroxide regulator gene perR, which functions as a repressor for multiple genes involved in oxidative stress resistance. Mutations in PerR resulted in loss of its DNA binding function and derepression of PerR-controlled oxidative stress defense genes, thereby conferring an OX R phenotype and facilitating Campylobacter survival under oxidative stress. These findings reveal a new mechanism that promotes the emergence of spontaneous OX R mutants in bacterial organisms. ABSTRACT Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. As a microaerophilic organism, C. jejuni must be able to defend against oxidative stress encountered both in the host and in the environment. How Campylobacter utilizes a mutation-based mechanism for adaptation to oxidative stress is still unknown. Here we present a previously undescribed phenotypic and genetic mechanism that promotes the emergence of oxidative stress-resistant mutants. Specifically, we showed that a naturally occurring mutator phenotype, resulting from a loss of function mutation in the DNA repair enzyme MutY, increased oxidative stress resistance (OX R ) in C. jejuni. We further demonstrated that MutY malfunction did not directly contribute to the OX R phenotype but increased the spontaneous mutation rate in the peroxide regulator gene perR, which functions as a repressor for multiple genes involved in oxidative stress resistance. Mutations in PerR resulted in loss of its DNA binding function and derepression of PerR-controlled oxidative stress defense genes, thereby conferring an OX R phenotype and facilitating Campylobacter survival under oxidative stress. These findings reveal a new mechanism that promotes the emergence of spontaneous OX R mutants in bacterial organisms.IMPORTANCE Although a mutator phenotype has been shown to promote antibiotic resistance in many bacterial species, little is known about its contribution to the emergence of OX R mutants. This work describes the link between a mutator phenotype and the enhanced emergence of OX R mutants as well as its underlying mechanism involving DNA repair and mutations in PerR. Since DNA repair systems and PerR are well conserved in many bacterial species, especially in Gram positives, t...

Identification of a nth-Like Gene Encoding an Endonuclease III in Campylobacter jejuni

“…To further examine the role of nth in DNA repair, we determined its effect on the spontaneous OX R frequency, which is due to spontaneous loss-of-function mutations in the peroxide regulator perR in C. jejuni (Dai et al, 2017). Previously, we used a promoter-reporter fusion gene P katA - cat to determine the spontaneous OX R mutation frequencies in C. jejuni (Dai et al, 2017). The fusion gene P katA -cat was then inserted into the genome of C. jejuni 11168 and Δ nth strains, generating 11168 P katA -cat and Δ nth P katA -cat strains.…”

“…It was not possible to determine the spontaneous OX R mutation frequency directly by using H 2 O 2 as a selection agent, since this compound is not stable and tends to degrade rapidly in the culture medium. Therefore, a reporter system was developed by fusing the promoter of katA (an oxidative stress response gene) with the chloramphenicol resistance gene cat , generating P katA -cat gene (Dai et al, 2017). This method was utilized in this study to compare spontaneous OX R mutation frequencies of the wild-type and the Δ nth mutant strains.…”

“…Similar to C. jejuni NCTC 11168, both constructs were susceptible to chloramphenicol (MIC = 1–2 μg/mL) as transcription of the P katA - cat gene was inhibited by the oxidative stress regulator PerR. However, spontaneous perR mutations, which compromise the PerR function, would lead to the derepression of the katA promoter of P katA - cat and consequently elevate the MIC of chloramphenicol (16–32 μg/mL) of the PerR mutants from both 11168 P katA - cat and Δ nth P katA - cat (Dai et al, 2017). Therefore, MH agar plates containing 6 μg/mL of chloramphenicol were used in this study to measure spontaneous perR loss-of-function mutations in C. jejuni 11168 P katA - cat and Δ nth P katA - cat .…”

“…A commercially available Nth from E. coli (New England Biolabs) was included as a positive control. An rPerR protein (Dai et al, 2017), which has no endonuclease III activity and was produced and purified from the same E. coli host with the same methods as for rNth and rCj1083c in this study, was used as a negative control. The endonuclease III activity assay was carried out following the manufacturer’s instructions.…”

The Rho-Independent Transcription Terminator for the porA Gene Enhances Expression of the Major Outer Membrane Protein and Campylobacter jejuni Virulence in Abortion Induction

“…C. jejuni isolates were grown on MH plate for 16 h under microaerobic conditions. Total RNA purification from the cultures and subsequent qRT-PCR analysis were carried out as previously described (75). The relative changes (n-fold) in gene transcriptions between different strains were calculated using the Pfaffl method (76).…”