Engineering an Escherichia coli Strain to Characterize Heme Metabolism

Iron is necessary for growth of bacteria. In this context, iron restriction is one of the mechanisms used in humans to fight bacterial infection. Iron restriction also includes limited access to the host heme that may constitute a potential abundant source of iron for invading bacteria. Most bacteria including laboratory Escherichia coli do not have the genes necessary to import heme from the environment and to degrade this porphyrin and release iron. However, several human pathogens possess a set of genes (chu) that allow the import and processing of heme, including pathogenic E. coli strains such as O157:H7 that causes severe food infection. The chu gene cluster is presumably involved in capturing, internalizing and metabolizing heme to release iron which would be used for growth under iron restricted condition (1). The chu gene cluster contains 8 genes some of which have no clearly defined function.  

To facilitate the characterization of the function of the individual chu genes, all 8 genes (chuASTUVWXY) were introduced in a single copy the genome of a non-pathogenic E. coli strain in which the entE gene encoding the siderophore enterobactin is inactivated to exacerbate the effect of iron restriction on growth. The chu genes were first cloned as sub-clusters in two pOSIP vectors (2) that allow the easy integration in the E. coli genome from the plasmid-encoded integrases. The chu genes were then integrated in the genome of the E. coli JW0586-1 (DentE) strain of the Keio collection (3). Gene expression of the chu genes was regulated from their natural promoters. In vivo assays revealed that this engineered E. coli/chuASTUVWXY grows well on minimal medium depleted of iron when supplemented with various sources of heme (free heme, myoglobin and hemoglobin). As expected, growth was strictly dependant on the inclusion of the gene encoding the outer membrane heme receptor chuA (4). On the other hand, several chu genes including those encoding the periplasmic transporters appear to contribute to the optimal growth of the engineered strain depending of the growth conditions. This work reports the building of the E. coli/chuASTUVWXY engineered strain that is an important tool to fully characterize the function of the chu genes under various growth conditions, environmental stresses and the presence of competing strains form the community.

1-      Ouellet, Y. H. et al. 2016. J. Inorg. Biochem. 154 : 103-113

2-      St-Pierre, F. et al. 2014. ACS Synt. Biol. 2 : 537-541

3-      Baba, T. et al. 2006. Mol. Syst. Biol. 2006.0008

4-      Hagan, E. C. and Mobley, H. L. 2009. Mol. Microbiol. 71: 79-91