The hmp gene encoding the NO-inducible flavohaemoglobin in Escherichia coli confers a protective advantage in resisting killing within macrophages, but not in vitro: Links with swarming motility

Escherichia coli flavohaemoglobin (Hmp) is the best-understood nitric oxide (NO) detoxifying protein and exhibits a robust dioxygenase activity, converting NO to nitrate ion with oxygen as co-substrate. Synthesis of Hmp via transcriptional regulation of hmp gene expression is an adaptive response to NO and related nitrosative stresses since Hmp levels are greatly elevated on exposure in vitro to these agents. Here we show that expression of hmp is greatly enhanced by NO but not by other haem ligands (azide, cyanide and carbon monoxide). Flavohaemoglobins of other pathogenic bacteria have been implicated in conferring resistance to NO in vitro and in macrophage-like cells but the role of the E. coli flavohaemoglobin has not been studied in macrophages. We therefore compared survival of wild-type K-12 E. coli cells and an isogenic hmp mutant after internalisation by human macrophages. Wild-type bacteria survived significantly better than the hmp mutant after incubation with macrophages, despite binding and internalisation rates being similar for both strains. Unexpectedly, however, when grown in MOPS minimal medium, in mixed cultures, more hmp mutant cells were recovered than wild-type. Significantly, an hmp mutant failed to exhibit swarming motility on soft agar and this phenotype was rescued by a plasmid-borne copy of the wild-type hmp+ gene. Thus, although Hmp constitutes an important mechanism of protection from NO-mediated killing by human macrophages in the model E. coli strain K-12, and probably contributes to the survival of enteropathogenic E. coli during the intestinal inflammatory response, synthesis of Hmp in vitro may represent a selective disadvantage. The lack of swarming motility of the hmp mutant and its aflagellate state suggest that Hmp synthesis is a metabolic burden in the absence of NO-related stresses.