ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) O157:H7 strain EDL933 harbors multiple prophage-associated open reading frames (ORFs) in its genome which are highly homologous to the chromosomalnanSgene. The latter is part of thenanCMSoperon, which is present in mostE. colistrains and encodes an esterase which is responsible for the monodeacetylation of 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2). Whereas one prophage-borne ORF (z1466) has been characterized in previous studies, the functions of the othernanS-homologous ORFs are unknown. In the current study, thenanS-homologous ORFs of EDL933 were initially studiedin silico. Due to their homology to the chromosomalnanSgene and their location in prophage genomes, we designated themnanS-p and numbered the differentnanS-p alleles consecutively from 1 to 10. The two allelesnanS-p2 andnanS-p4 were selected for production of recombinant proteins, their enzymatic activities were investigated, and differences in their temperature optima were found. Furthermore, a function of these enzymes in substrate utilization could be demonstrated using anE. coliC600ΔnanSmutant in a growth medium with Neu5,9Ac2as the carbon source and supplementation with the different recombinant NanS-p proteins. Moreover, generation of sequential deletions of allnanS-p alleles in strain EDL933 and subsequent growth experiments demonstrated a gene dose effect on the utilization of Neu5,9Ac2. Since Neu5,9Ac2is an important component of human and animal gut mucus and since the nutrient availability in the large intestine is limited, we hypothesize that the presence of multiple Neu5,9Ac2esterases provides them a nutrient supply under certain conditions in the large intestine, even if particular prophages are lost.IMPORTANCEIn this study, a group of homologous prophage-bornenanS-p alleles and two of the corresponding enzymes of enterohemorrhagicE. coli(EHEC) O157:H7 strain EDL933 that may be important to provide alternative genes for substrate utilization were characterized.
Comparative analysis of mRNA transcripts of HT-29 cell line expressed in identical quantities for pathogenic E. coli strains UM146 and UM147 with control Escherichia coli Nissle 1917