Site-directed mutagenesis of Campylobacter concisus respiratory genes provides insight into the pathogen’s growth requirements

Abstract

Campylobacter concisus is an emerging human pathogen found throughout the entire human oral-gastrointestinal tract. The ability of C. concisus to colonize diverse niches of the human body indicates the pathogen is metabolically versatile. C. concisus is able to grow under both anaerobic conditions and microaerophilic conditions. Hydrogen (H2) has been shown to enhance growth and may even be required. Analysis of several C. concisus genome sequences reveals the presence of two sets of genes encoding for distinct hydrogenases: a H2-uptake-type (“Hyd”) complex and a H2-evolving hydrogenase (“Hyf”). Whole cells hydrogenase assays indicate that the former (H2-uptake) activity is predominant in C. concisus, with activity among the highest we have found for pathogenic bacteria. Attempts to generate site-directed chromosomal mutants were partially successful, as we could disrupt hyfB, but not hydB, suggesting that H2-uptake, but not H2-evolving activity, is an essential respiratory pathway in C. concisus. Furthermore, the tetrathionate reductase ttrA gene was inactivated in various C. concisus genomospecies. Addition of tetrathionate to the medium resulted in a ten-fold increase in cell yield for the WT, while it had no effect on the ttrA mutant growth. To our knowledge, this is the first report of mutants in C. concisus.

Role of Nickel in Microbial Pathogenesis

“…Furthermore, C. concisus has the highest H 2 -uptake hydrogenase activity reported so far among pathogenic bacteria [167]. In agreement with these observations, H 2 was found to be needed for optimal growth under anaerobic conditions, and required for growth under microaerobic conditions, highlighting the importance of the H 2 -uptake hydrogenase in the pathogen's metabolism [76,168].…”

Analyses of energy metabolism and stress defence provide insights into Campylobacter concisus growth and pathogenicity

“…Given this, supplementation of these amino acids and electron donors may potentially increase the growth of C. concisus and the effects of some of these substances on C. concisus growth were examined previously and in this study. H 2 gas has been demonstrated to be a highly effective electron donor for C. concisus growth [51,60]. It has been previously demonstrated that C. concisus had very low growth under anaerobic conditions and no growth under microaerobic conditions without H 2 [42].…”

“…In the presence of H 2 , C. concisus was able to grow under microaerobic conditions and the growth under anaerobic conditions greatly increased [42]. C. concisus has both a H 2 -uptake hydrogenase (Hyd) and a H 2 -generating hydrogenase (Hyf ) [51]. Site-directed mutagenesis demonstrated the critical role of H 2 oxidation for C. concisus growth, with C. concisus additionally found to exhibit extremely high H 2 -uptake hydrogenase activity [51].…”

“…C. concisus has both a H 2 -uptake hydrogenase (Hyd) and a H 2 -generating hydrogenase (Hyf ) [51]. Site-directed mutagenesis demonstrated the critical role of H 2 oxidation for C. concisus growth, with C. concisus additionally found to exhibit extremely high H 2 -uptake hydrogenase activity [51]. Although H 2 evolution has been detected for C. concisus [55], the level of H 2 produced by C. concisus produced only poor growth of C. concisus [60].…”

“…The molecular basis of the non-saccharolytic and hydrogen-dependent nature of C. concisus has not been previously made clear, because few studies have examined the genes and pathways involved in respiration and energy metabolism in C. concisus [51,52]. Recently, more than 200 C. concisus genomes including three complete genomes have become available in public databases, providing the opportunity for us to investigate these pathways using bioinformatics analysis.…”