Altering the anaerobic transcription factor FNR confers a hemolytic phenotype on Escherichia coli K12

Abstract

The recent outbreaks of Escherichia coli O157-associated food poisoning have focused attention on the virulence determinants of E. coli. Here, it is reported that single base substitutions in the fnr gene encoding the oxygen-responsive transcription regulator FNR (fumarate and nitrate reduction regulator) are sufficient to confer a hemolytic phenotype on E. coli K12, the widely used laboratory strain. The mechanism involves enhancing the expression of a normally dormant hemolysin gene (hlyE) located in the E. coli chromosome. The mutations direct single amino acid substitutions in the activating regions (AR1 and AR3) of FNR that contact RNA polymerase. It is concluded that altering a resident transcription regulator, or acquisition of a competent heterologous regulator, could generate a pool of hemolytic, and therefore more virulent, strains of E. coli in nature.

Figure 1

Diagrammatic representations of the ternary complexes formed at the two classes of FNR-regulated promoter: (a) wild-type FNR at a class II promoter; (b) correct positioning of RNA polymerase by HlyX and FNR variants at a class I promoter (P_hlyE); and (c) incorrect positioning of RNA polymerase by wild-type FNR at a class I promoter (P_hlyE). The FNR variant proteins favor the formation of productive ternary complexes at the hlyE promoter by either enhancing interaction between AR1 (♦) and α_C or by weakening the interaction between AR3 (★) and σ⁷⁰, thus positioning RNA polymerase correctly with respect to the −10 element.

Figure 2

Predicted structure of an FNR monomer based on that of CRP (14) showing the positions of amino acid substitutions that confer a hemolytic phenotype on E. coli K12. Also shown are: the helix–turn–helix motif (α_E-α_F) in the DNA-binding domain; key amino acid to base pair (protein–DNA) interactions; the essential cysteine ligands of the [4Fe-4S] cluster; and previously identified activating regions, the 73-loop in AR1 (13) and the 85-loop in AR3 (13).