Multicellular oxidant defense in unicellular organisms.

Abstract

Although catalase is thought to be a major defense against hydrogen peroxide (H202), the (17) were provided by P. C. Loewen (University ofManitoba). E. coli were grown statically in brain heart infusion broth or M9 minimal salts medium supplemented with 10 mM glucose (M9/glucose) (25) at 370C in room air overnight (18-20 hr) to early stationary-phase (OD600 = 2.2-2.4). Thiamin was not added to the growth medium because these strains of E. coli were phenotypically Thi+, probably from a spontaneous Thi--Thi+ reversion. Bacteria in midexponential growth were obtained by diluting these stationary-phase cultures 1:100 in prewarmed fresh brain heart infusion broth and incubated further until the OD6(o of the culture reached 0.5-1.0. Cells were harvested from the culture by centrifugation (10,000 x g for 10 min at 20C), washed thrice in cold phosphate-buffered saline (140 mM NaCI/10 mM Na2HPO4/10 mM KH2PO4, pH 7.2) or in M9/glucose before being resuspended in the indicated buffers, and the absorbance at 600 nm was measured. The correlation of OD6(, with bacterial concentration was affirmed by direct microscopic counts and by colonyforming units obtained by growth on LB agar. Bacterial stocks were maintained on LB agar plates, stored at 2-4C. The catalase status of colonies was constantly monitored by direct catalase assay and by H202 drop tests.Streptococcal strains were obtained from the American Type Culture Collection (Streptococcus sobrinus; 33478), G. Germaine (University of Minnesota) (Streptococcus mitis, 9811), and E. L. Thomas (University of Tennessee) (Streptococcus mutans OMZ176 and S. mutans GS5). Organisms were cultured in Todd-Hewitt broth statically at 370C either in room air (S. mutans) or in a candle extinction jar (S. sobrinus and S. mitis) and washed as described for E. coli.H202 King of E. cofi. Suspensions of E. coli were diluted to the concentration of 5 x 107 cells per ml (concentrated) or 500 cells per ml (dilute) in M9/glucose and prewarmed to 370C before being exposed to H202 at the indicated concentrations. Bacterial viability, or, more accurately, ability ofthe bacteria to replicate, was assayed by pour-plating diluted aliquots (in triplicate) of the H202/bacteria mixture in bovine catalase-supplemented (100 units per ml) LB agar at different times during incubation. Killing experiments were performed not only in pure cat(+) and pure cat(-) E. coli suspensions but also were carried out in mixtures of cat(+) and cat(-) E. coli as indicated. To differentiate cat(+) from cat(-) E. coli after H202 exposure of such mixtures, bacteria ofboth strains were made resistant to ampicillin by pUC118 (a derivation of Abbreviations: LB agar, Lennox L agar; M9, minimal salts medium; M9/glucose, M9/10 mM glucose; cat(-), catalase-deficient mutant; cat(+), wild-type strain CSH7. *To whom reprint requests should be addressed. 7924The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S...

Possible accumulation of non-active molecules of catalase and superoxide dismutase in S. cerevisiae cells under hydrogen peroxide induced stress

“…The latter are in accordance with the previous study reported that in E. coli strain, which does not generate an extracellular/intracellular gradient of hydrogen peroxide, catalase only protected high density or colonial bacteria and did not protect low density or individual cells [31]. Therefore, it can be suggested that cell density may influence the yeast response to oxidative stress.…”

Decrease of H2O2 Plasma Membrane Permeability during Adaptation to H2O2 in Saccharomyces cerevisiae

“…Our results conciliate the contradictory findings in the literature concerning whether catalase is important in the adaptive response of cells at low densities; it was shown in an E. coli strain that an increase in catalase activity does not protect low density or individual E. coli (7), which was attributed to the inability of this strain to form an extracellular/intracellular H 2 O 2 gradient; however, in Sc cells catalase was found to be important even at low density conditions, a discrepancy that was not explained (23). If the gradient concept is taken into account this behavior is expected because the imposition of a gradient is much more difficult in E. coli than in Sc cells because the surface to volume ratio is much higher in the relative smaller E. coli cells than in the relative larger Sc cells.…”

“…These results were later confirmed in an Escherichia coli strain by others (6). Furthermore, if an extracellular/intracellular gradient is not formed, the intracellular enzymes are not able to protect individual cells against H 2 O 2 , and it was shown in an E. coli strain that does not show a gradient that the increase in catalase activity only protects high density or colonial E. coli and does not protect low density or individual E. coli (7). Further supporting the importance of the plasma membrane is the observation that after a nonlethal dose of H 2 O 2 , the levels of mRNA that are more repressed are those corresponding to the enzyme HMG-CoA reductase (8).…”

“…Approximately between 2 and 3 billion years ago two events occurred: (a) the slow conversion of a small prokaryote ancestor into a large phagocytic cell possessing most of the characteristics of the modern eukaryotes and able to acquire endosymbionts (39, 40) and (b) massive generation of H 2 O 2 resulting from the oxidation of the abundant Fe 2ϩ ions to Fe 3ϩ by the O 2 produced by cyanobacteria, which lead to the formation of ferric oxide deposits (41). H 2 O 2 concentrations in the aqueous environment during this period have been estimated in the millimolar range (7). Taking into consideration that during this period cellular iron-chelating systems would not be yet fully optimized, H 2 O 2 would be extremely hazardous to cells, because upon diffusion into the cell it would easily react with Fe 2ϩ producing hydroxyl radicals.…”

The Role of Annexin 1 in Drought Stress in Arabidopsis

“…Survivability of DoxyR in liquid culture was decreased, and the large amounts of cellular debris encountered interfered with optical density measurements. Moreover, during the early stages of colony formation, when bacterial density is low, aerobic conditions result in a nonspecific decrease in plating efficiency (Ma and Eaton, 1992). Only after several attempts were we able to establish an experimental protocol to successfully perform quantitative measurements of AnnAt1 effects on H 2 O 2 survivability in the DoxyR strain.…”