RpoS- and OxyR-independent induction of HPI catalase at stationary phase in Escherichia coli and identification of rpoS mutations in common laboratory strains

Abstract

A rapid spectrophotometric assay to determine the activities of HPI and HPII catalases in Escherichia coli extracts has been developed. This assay is based upon the differential heat stabilities of the two enzymes and offers significant advantages over previous methods for quantitation of their activities. Measurement of catalase activities in extracts of various mutant strains confirmed the ability of this method to accurately distinguish the two activities. Contrary to previously published results, HPI catalase activity was observed to increase at stationary phase in strains lacking the stationary-phase sigma factor s (RpoS). This increase was independent of OxyR and also occurred in a strain lacking the HPII structural gene, katE. These results suggest a potential novel pathway for HPI induction in response to increased oxidative stress in the absence of HPII. Measurement of HPII activity in strains carrying mutations in pcm (encoding the L-isoaspartyl protein methyltransferase) and surE led to the finding that these strains also have an amber mutation in rpoS; sequencing demonstrated the presence of this mutation in several commonly used laboratory strains of E. coli, including AB1157, W1485, and JC7623.Escherichia coli has two catalase enzymes, HPI and HPII, which catalyze the dismutation of hydrogen peroxide to water and oxygen (24). HPI, the product of the katG gene, is transcriptionally induced during logarithmic growth in response to low concentrations of hydrogen peroxide. This induction requires the positive activator OxyR, which directly senses oxidative stress and is activated by conformational change upon oxidation occurring at a key cysteine residue (37). HPII, on the other hand, is not peroxide inducible; its gene, katE, is transcribed at the transition from exponential growth to stationary phase by RNA polymerase containing the alternative sigma subunit s , the product of the rpoS (or katF) gene and a key player in survival of stationary phase and other stresses (25,30).Interest has been focused on these two catalases not only because of their important roles in protecting cells against the effects of oxidative stress (6) but also because of their suitability as reporter enzymes in the study of global regulatory systems. Quantitation of HPII activity provides a convenient means of monitoring the level of s (45). Similarly, HPI can serve as a measure of the activation of the OxyR stress response regulon by peroxide or other agents (12).Several biochemical assays for catalase activity have been developed in order to facilitate the study of these enzymes and their regulatory systems. Qualitatively, catalase can be demonstrated by simply dropping peroxide on a bacterial colony and observing bubbles arising from the evolution of oxygen (15). Quantitation of catalase activity can be accomplished either by using a spectrophotometer to measure reduction in absorbance at 240 nm as hydrogen peroxide (H 2 O 2 ) is consumed (2) or by measurement of oxygen evolution with an oxygen electrode (33). However, ne...

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“…4). These results were consistent with previous reports 20,21 . No change in catalase II activity was observed.…”

The role of oxidative stress genes and effect of pH on methylene blue sensitized photooxidation ofEscherichia coli

“…This gene is controlled by rpoS. rpoS provides the control of katE that plays a role in oxidative stress as in many cases of E. coli at stationary phases in starvation stress as in phosphate buffer [19]. The importance of this gene can be related to the exposed photooxidative stress at stationary phase.…”

The respiratory chain is the cell's Achilles' heel during UVA inactivation in Escherichia coli

“…Specific activities were measured in wholecell assays. The activity of the two enzymes can be separated by a heat inactivation treatment step because HPII is heat-stable whereas HPI is heat-labile (Visick & Clarke, 1997). Cells were washed and diluted in PBS to an OD 546 of 1.25 and then exposed to UVA irradiation.…”