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...

SummaryProteins, like DNA, are subject to various forms of damage that can render them non-functional. Conformational changes and covalent chemical alterations occur spontaneously, and the rates of these reactions can be increased by environmental stresses such as heat, oxidative agents, or changes in pH or osmotic conditions. Although affected proteins can tie replaced by de novo biosynthesis, cells -especially those subjected to stress or nutrient limitation -have developed mechanisms which can either restore damaged polypeptides to an active state or remove them. Such mechanisms can spare the biosynthetic capacity of the cell and ensure that the presence of non-functional molecules does not disrupt cell physiology. Three major mechanisms, which operate in bacteria as well as eukaryotic organisms, have been descritied. First, chaperones not only assist in proper de novo folding of proteins but also provide an Important means of restoring activity to conformationally damaged proteins. Second, enzymatic 'repair' systems exist to directly reverse certain forms of protein damage, including proline isomerization, methionine oxidation and the formation of isoaspartyl residues. Finally, proteotysis provides a 'last-resort' means of dealing with abnormal proteins which cannot be repaired. Protein maintenance and repair may be of special importance for bacteria preparing to survive extended periods In stationary phase: both constitutive and induced mechanisms are utilized to permit survival despite greatly reduced protein synthesis.

The 27-kilodalton (kDa) mosquitocidal protein gene from Bacillus thuringiensis subsp. israelensis has been cloned as a 10-kilobase (kb) HindIII fragment from plasmid DNA; efficient expression in Escherichia coli KM1 depends on a region of DNA located approximately 4 kb upstream (K. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987). We have cloned the upstream DNA region and show that it contains a complete open reading frame (ORF) encoding a protein with a molecular mass of 19,584 Da. Sequencing of adjacent stretches of DNA revealed two partial ORFs: one has 55.2% identity in an overlap of 319 amino acids to the putative transposase of IS231 of B. thuringiensis subsp. thuringiensis, and the other, a 78-codon partial ORF, may be the carboxyl terminus of the 67-kDa protein previously observed in maxicells of strain KM1. A 0.8-kb fragment containing only the 20-kDa protein gene greatly enhanced the expression of the 27-kDa protein in E. coli. The introduction of nonsense codons into the 20-kDa protein gene ORF abolished this effect, indicating that the gene product, not the mRNA or DNA, is required for the enhancement. The effect of the 20-kDa protein gene on various fusions of lacZ to the 27-kDa protein gene suggests that the 20-kDa protein acts after the initiation of translation of the 27-kDa protein gene.

No abstract

Like its homologs throughout the biological world, thel-isoaspartyl protein repair methyltransferase ofEscherichia coli, encoded by the pcm gene, can convert abnormal l-isoaspartyl residues in proteins (which form spontaneously from asparaginyl or aspartyl residues) to normal aspartyl residues. Mutations in pcm were reported to greatly reduce survival in stationary phase and when cells were subjected to heat or osmotic stresses (C. Li and S. Clarke, Proc. Natl. Acad. Sci. USA 89:9885–9889, 1992). However, we subsequently demonstrated that those strains had a secondary mutation inrpoS, which encodes a stationary-phase-specific sigma factor (J. E. Visick and S. Clarke, J. Bacteriol. 179:4158–4163, 1997). We now show that the rpoS mutation, resulting in a 90% decrease in HPII catalase activity, can account for the previously observed phenotypes. We further demonstrate that a new pcmmutant lacks these phenotypes. Interestingly, the newly constructedpcm mutant, when maintained in stationary phase for extended periods, is susceptible to environmental stresses, including exposure to methanol, oxygen radical generation by paraquat, high salt concentrations, and repeated heating to 42°C. The pcmmutation also results in a competitive disadvantage in stationary-phase cells. All of these phenotypes can be complemented by a functionalpcm gene integrated elsewhere in the chromosome. These data suggest that protein denaturation and isoaspartyl formation may act synergistically to the detriment of aging E. coli and that the repair methyltransferase can play a role in limiting the accumulation of the potentially disruptive isoaspartyl residues in vivo.