Iron restriction induces preferential down-regulation of H2-consuming over H2-evolving reactions during fermentative growth of Escherichia coli

Abstract

BackgroundEscherichia coli synthesizes three anaerobically inducible [NiFe]-hydrogenases (Hyd). All three enzymes have a [NiFe]-cofactor in the large subunit and each enzyme also has an iron-sulfur-containing small subunit that is required for electron transfer. In order to synthesize functionally active Hyd enzymes iron must be supplied to the maturation pathways for both the large and small subunits. The focus of this study was the analysis of the iron uptake systems required for synthesis of active Hyd-1, Hyd-2 and Hyd-3 during fermentative growth.ResultsA transposon-insertion mutant impaired in hydrogenase enzyme activity was isolated. The mutation was in the feoB gene encoding the ferrous iron transport system. The levels of both hydrogen-oxidizing enzymes Hyd-1 and Hyd-2 as determined by specific in-gel activity staining were reduced at least 10-fold in the mutant after anaerobic fermentative growth in minimal medium, while the hydrogen-evolving Hyd-3 activity was less severely affected. Supplementation of the growth medium with ferric iron, which is taken up by e.g. the siderophore enterobactin, resulted in phenotypic complementation of the feoB mutant. Growth in rich medium demonstrated that a mutant lacking both the ferrous iron transport system and enterobactin biosynthesis (entC) was devoid of Hyd-1 and Hyd-2 activity but retained some hydrogen-evolving Hyd-3 activity. Analysis of crude extracts derived from the feoB entC double null mutant revealed that the large subunits of the hydrogen-oxidizing enzymes Hyd-1 and Hyd-2 were absent. Analysis of lacZ fusions demonstrated, however, that expression of the hya, hyb and hyc operons was reduced only by maximally 50% in the mutants compared with the wild type.ConclusionsOur findings demonstrate that the ferrous iron transport system is the principal route of iron uptake for anaerobic hydrogenase biosynthesis, with a contribution from the ferric-enterobactin system. Hydrogen-oxidizing enzyme function was abolished in a feoB entC double mutant and this appears to be due to post-translational effects. The retention of residual hydrogen-evolving activity, even in the feoB entC double null mutant suggests that sufficient iron can be scavenged to synthesize this key fermentative enzyme complex in preference to the hydrogen-uptake enzymes.