Introduction

Escherichia coli (E. coli) O104:H4 caused in 2011 in Germany the enterohemorrhagic E. coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome worldwide. We recently described an E. coli O104:H4 isolate that had acquired a single nucleotide polymorphism in the start codon (ATG>ATA) of rpoS, coding for the alternative RNA polymerase sigma factor S (RpoS), which resulted in enhanced virulence gene expression and drastically altered metabolism.

Goal

The goal of this work was to monitor the effect of rpoS deletion (ΔrpoS) on E. coli O104:H4 carbon source utilization and fitness.

Material and Methods

The rpoS gene was deleted by homologous recombineering in E. coli O104:H4 Δstx2. The effect of ΔrpoS on carbon source utilization was analysed by phenotype microarrays using the BIOLOG PM1 plate. E. coli O104:H4 Δstx2 and Δstx2 ΔrpoS were co-cultured for 24 hours in minimal M9 medium supplemented with 0.2% single carbon source and the bacterial cell numbers were determined by counting colony forming units.

Results

Biolog PM1 phenotype microarrays revealed a strong correlation between the effect of rpoS ATG>ATA and ΔrpoS on E. coli O104:H4 carbon source utilization. Namely, the strains carrying an intact rpoS allele assimilated more efficiently sugars, e.g. arabinose, galactose and fucose, whereas both rpoS ATG>ATA allele and ΔrpoS resulted in stronger metabolic activity with amino acids, carboxylic and dicarboxylic acids (TCA cycle substrates). Interestingly, competition experiments with E. coli O104:H4 Δstx2 and Δstx2 ΔrpoS in M9 supplemented with single carbon sources revealed a dramatic competitive advantage of the ΔrpoS strain with the tested amino acids, i.e. L-alanine, L-glutamine and L-aspartic acid, however, no and only moderate advantage of the rpoS wild type strain with galactose and arabinose, respectively.

Summary

We could show that rpoS deletion profoundly alters the carbon source utilization and fitness of E. coli O104:H4. Thus, this work provides additional evidence that RpoS acts as a global regulator of E. coli O104:H4 metabolism and further emphasizes its importance in EHEC colonization and virulence.