The Campylobacter jejuni RacRS system regulates fumarate utilization in a low oxygen environment

Abstract

The natural environment of the human pathogen Campylobacter jejuni is the gastrointestinal tract of warm-blooded animals. In the gut, the availability of oxygen is limited; therefore, less efficient electron acceptors such as nitrate or fumarate are used by C. jejuni. The molecular mechanisms that regulate the activity of the highly branched respiratory chain of C. jejuni are still a mystery mainly because C. jejuni lacks homologues of transcription factors known to regulate energy metabolism in other bacteria. Here we demonstrate that dependent on the available electron acceptors the two-component system RacRS controls the production of fumarate from aspartate, as well as its transport and reduction to succinate. Transcription profiling, DNAse protection and functional assays showed that phosphorylated RacR binds to and represses at least five promoter elements located in front of genes involved in the uptake and synthesis of fumarate. The RacRS system is active in the presence of nitrate and trimethyl-amine-N-oxide under oxygen-limited conditions when fumarate is less preferred as an alternative electron acceptor. In the inactive state, RacRS allows utilization of fumarate for respiration. The unique C. jejuni RacRS regulatory system illustrates the disparate evolution of Campylobacter and aids the survival of this pathogen.

Analysis of the Activity and Regulon of the Two-Component Regulatory System Composed by Cjj81176_1484 and Cjj81176_1483 of Campylobacter jejuni

“…A recent study of the RacRS TCS of C. jejuni revealed that chuC, Cjj0210-Cjj0211, and Cjj1385-Cjj1386 expression levels were all decreased in a racR mutant (27). However, expression of the Cjj1484-Cjj1483 TCS, which is the subject of this work, was significantly higher in the racR mutant (27). Further investigation was unable to demonstrate that RacR interacted specifically with the promoters for chuC, Cjj0210-Cjj0211, and Cjj1385-Cjj1386, creating some speculation about whether the RacRS TCS may only indirectly regulate expression of these genes.…”

“…For Cjj0210-Cjj0211, chuC, exbB2, and exbD2, evidence exists that the peroxide regulator (PerR) also influences expression of these genes (55). A recent study of the RacRS TCS of C. jejuni revealed that chuC, Cjj0210-Cjj0211, and Cjj1385-Cjj1386 expression levels were all decreased in a racR mutant (27). However, expression of the Cjj1484-Cjj1483 TCS, which is the subject of this work, was significantly higher in the racR mutant (27).…”

“…Whereas DccRS directly influences expression of genes encoding colonization determinants (22), CprRS is required for expression of genes for biofilm formation (24). RacRS is required for expression of genes encoding proteins for the heat shock response and fumarate metabolism, depending on the strain and growth conditions used for analysis (25)(26)(27). Additionally, the PhosSR TCS is required for expression of 12 genes, 5 of which encode proteins required for phosphate acquisition (28).…”

Catabolite repression in Campylobacter jejuni correlates with intracellular succinate levels

“…However, we were unable to show that acetylphosphate can phosphorylate RacR in in vitro experiments, nor did we observe contrasting phenotypes in pta and ackA mutant strains, which produce low or high levels of acetylphosphate respectively (data not shown). Another possibility is that unphosphorylated RacR has residual transcription inducing activity, as it still binds to its target promotors in the unphosphorylated state in vitro (van der Stel et al, 2015a). The cj1000 mutant strain showed an increase in GGT activity in the exponential growth phase but no change in GOGAT activity (Fig.…”

“…After 3, 6, 9 or 15 h cultures were harvested and RNA was isolated immediately using the RNA-Bee kit. RT-qPCR was performed as before (van der Stel et al, 2015a). In brief, DNAse treated RNA (10 ng) was used to determine the amount of specific transcripts using the Takyon No Rox qPCR-kit with Euroscript II reverse transcriptase (Eurogentec, Seraing, Belgium), according to the manufacture's protocol in a Lightcycler 480 machine (Roche, Penzberg, Germany) using custom DNA-oligo's (Supporting Information Table S2).…”