A truncated haemoglobin implicated in oxygen metabolism by the microaerophilic food-borne pathogen Campylobacter jejuni

Abstract

Of the three groups of haemoglobins identified in micro-organisms (single-domain globins, flavohaemoglobins and truncated globins), the last group is the least well understood. The function of the truncated haemoglobin (Ctb) encoded by Cj0465c in the microaerophilic food-borne bacterial pathogen Campylobacter jejuni was investigated by constructing a ctb mutant and characterizing its phenotype. The effects of the ctb mutation on the kinetics of terminal oxidase function in C. jejuni were investigated using oxyleghaemoglobin and oxymyoglobin as sensitive reporters of O 2 consumption. The V max of ctb mutant cells for O 2 , calculated using either globin, was greater than that of wild-type cells at extracellular O 2 concentrations up to~1 mM, suggesting a role for Ctb in moderating O 2 supply for reduction by high-affinity terminal oxidases. However, cells mutated in ctb were disadvantaged when grown under conditions of high aeration, as revealed by measurements of growth yields and rates in batch culture. Furthermore, the rate at which ctb mutant cells consumed O 2 in an O 2 electrode (10-200 mM O 2 ) was approximately half the rate displayed by wild-type cells, reflecting a role for Ctb in respiration at physiologically relevant external O 2 concentrations. However, a lack of sensitivity of the mutant to paraquat or H 2 O 2 indicated that increased oxidative stress under such conditions was not the cause of these phenotypes. O 2 affinities of cells (K m values of approximately 40 nM and 1 mM) were unaffected by mutation of either Ctb or the full-length C. jejuni globin, Cgb. Although the gene encoding Ctb was found to be upregulated by S-nitrosoglutathione (GSNO) and the NO-donating compound S-nitroso-N-acetylpenicillamine (SNAP), a ctb mutant did not display sensitivity to a number of nitrosative stress-generating compounds. The authors conclude that Ctb is involved in moderating O 2 flux within C. jejuni.

Protein structure in the truncated (2/2) hemoglobin family

“…A similar function has been proposed for Mycobacterium tuberculosis where group I HbN would be crucial in protecting the mycobacterium from nitrosative stress in vivo (4,5). In Campylobacter jejuni, a pathogenic agent in gastrointestinal disease, group III HbP may play a role in cell respiration (6,7).…”

Purification and Spectroscopic Characterization of Ctb, a Group III Truncated Hemoglobin Implicated in Oxygen Metabolism in the Food-Borne Pathogen Campylobacter jejuni

“…As for other trHbs, the function of Ctb is ambiguous. On the one hand, a role in oxygen metabolism is indicated by the growth phenotypes of a ctb mutant (19) and the effects of this deletion on the kinetics of oxidase-catalysed respiration. The finding that Ctb is oxygenated in whole E. coli cells indicates that the high affinity of the oxidases for O 2 (47,48) presents no obstruction to Ctb binding O 2 in vivo.…”

“…However, a ctb mutant is not hypersensitive to a range of nitrosative stress-inducing reagents (19). If Ctb were to be involved indirectly in NO detoxification it may, like Hmp, require reduction for sustained NO reductase or "oxygenase" (denitrosylase) activity.…”

“…Its expression is, however, elevated in response to nitrosative stress, a response mediated by the NssR sensor/regulator (10). The microaerophilic nature of C. jejuni implies the presence of mechanisms for high affinity O 2 binding and/or coping with O 2 toxicity and it is possible that Ctb fulfils one of these functions (19). In the present work, we have cloned and over-expressed the C. jejuni Ctb (Cj0465c), characterized it through optical and resonance Raman spectroscopy, and determined its mid-point redox potential.…”

The Single-domain Globin from the Pathogenic Bacterium Campylobacter jejuni

“…The single-domain globin, Cgb, and a truncated globin, Ctb, are both up-regulated by the transcription factor NssR in response to nitrosative stress (21). Cgb has been shown to detoxify NO (2), whereas Ctb is thought to have a role in oxygen metabolism (22) and has been shown to possess a peroxidase-like heme-binding cleft (11). It has been suggested that inhibition of respiration by NO increases the intracellular oxygen tension and that Ctb may deliver oxygen to Cgb for the dioxygenase reaction with NO to produce nitrate (23).…”