Conditioning of uropathogenic Escherichia coli to nitrosative stress

While in transit within and between hosts, uropathogenic E. coli (UPEC) encounter multiple stresses, including substantial levels of nitric oxide and reactive nitrogen intermediates. Strains of UPEC become conditioned to high concentrations of acidified sodium nitrite (ASN), a model system used to generate nitrosative stress. We used microarrays to define the expression profile of UPEC that have been conditioned for growth in ASN. Separate colonies of the UPEC isolate UTI89 grown on LB agar plates from a -80°C freezer stock were used to start overnight shaking cultures in MES-buffered LB broth (MES-LB, pH 5.0). Each culture was then diluted 1:100 into 7 ml MES-LB with or without 3 mM ASN in loosely capped 20-by-150-mm borosilicate glass tubes and grown with shaking at 37°C. Growth was monitored by optical density until OD600 = 1.5, at which point cultures were spun down, and pellets frozen at -80°C for at least 12 h. RNA was extracted with hot phenol-chloroform and purified by CsCl centrifugation. cDNA was synthesized, fragmented, and labeled for hybridization to Affymetrix GeneChip® E. coli Genome 2.0 Arrays.