Inactivation of alternative sigma factor 54 (RpoN) leads to increased acid resistance, and alters locus of enterocyte effacement (LEE) expression in Escherichia coli O157 : H7

Abstract

Alternative sigma factor 54 (RpoN) is an important regulator of stress resistance and virulence genes in many bacterial species. In this study, we report on the gene expression alterations that follow rpoN inactivation in Escherichia coli O157 : H7 strain Sakai (Sakai rpoN : : kan), and the influence of RpoN on the acid resistance phenotype. Microarray gene expression profiling revealed the differential expression of 103 genes in SakairpoN : : kan relative to Sakai. This included the growth-phase-dependent upregulation of genes required for glutamate-dependent acid resistance (GDAR) ( gadA, gadB, gadC and gadE), and the downregulation of locus of enterocyte effacement (LEE) genes, which encode a type III secretion system. Upregulation of gad genes in SakairpoN : : kan during exponential growth correlated with increased GDAR and survival in a model stomach system. Complementation of SakairpoN : : kan with a cloned version of rpoN restored acid susceptibility. Genes involved in GDAR regulation, including rpoS (sigma factor 38) and gadE (acid-responsive regulator), were shown to be required for the survival of SakairpoN : : kan by the GDAR mechanism. This study describes the contribution of rpoN to acid resistance and GDAR gene regulation, and reveals RpoN to be an important regulator of stress resistance and virulence genes in E. coli O157 : H7.

Fig. 1.

Differential expression of LEE genes by GSEA analysis in SakairpoN : : kan compared to Sakai. The fold change in gene expression is plotted against gene or locus ID (ECs no.) for all five LEE operons. Filled symbols represent genes determined to be significantly altered in expression by GSEA. All genes significant by SAM were also significant by GSEA. For orientation, names for selected LEE genes are provided to the immediate right of their respective plots.

Fig. 2.

Growth-phase analysis of gad genes, rpoS and hns expression by qRT-PCR. ○, Sakai; △, SakairpoN : : kan. Error bars represent sem (n=3), and the asterisks denote significance by Welch's t-test (P<0.05).